United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/540/R-94/509
March 1994
£EPA
A User's Guide to
Environmental
Immunochemical
Analysis
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/Margins for all text pages--
' same as originals.
EPA/540/R-94/509
March 1994
A USER'S GUIDE TO ENVIRONMENTAL IMMUNOCHEMICAL ANALYSIS
Shirley J. Gee
Bruce D. Hammock
Department of Entomology
University of California
Davis, CA95616
and
Jeanette M. Van Emon
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
EPA Cooperative Research Grant 891047
Project Officer
Jeanette M. Van Emon
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY-LAS VEGAS
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
Printed on Recycled Paper
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NOTICE
The information in this document has been funded in part by the United States
Environmental Protection Agency through its Office of Research and Development under
assistance agreement #CR819047-01 to the Department of Entomology, University of
California at Davis. It has been subject to the Agency's peer and administrative review, and it
has been approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
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ABSTRACT
Immunochemical methods for the analysis of environmental contaminants are relatively
new on the analytical chemistry scene. These methods are based on the use of a specific
antibody as a detector for the analyte of interest. Immunoassays are rapid, sensitive, and
selective, and are generally cost effective for large sample loads. They have been applied to
diverse chemical structures (i.e. triazines, sulfonylureas, organophosphates, polychlorinated
biphenyls, cyclodienes) and are adaptable to field use. These characteristics make
immunochemical analysis a valuable tool for use by the environmental analytical chemist.
This document is designed to facilitate the transfer of this valuable technology to the
environmental analytical chemistry laboratory. Field personnel who may need to employ a
measurement technology at a monitoring site may also find this manual helpful.
This document is a tutorial designed to instruct the reader in the use and application of
immunochemical methods of analysis for environmental contaminants. A brief introduction
describes basic principles and the advantages and disadvantages of the technology, and
gives a listing of references which supply more detail. Preparation of the laboratory for use of
this technology and the general scientific considerations prior to using the technology are
discussed. Detailed step-wise procedures are given for analysis of selected analytes, triazine
herbicides, carbaryl, paraquat, and p-nitrophenols in environmental samples as well as triazine
mercapturates in urine samples. In addition to the specific immunoassay methods, a series of
support techniques necessary to perform immunochemical methods are described. These
support techniques include pipetting, sample preparations, testing for matrix effects, optimizing
reagent concentrations, data analysis, recordkeeping, and equipment maintenance. A general
troubleshooting guide is provided to aid both the novice and experienced analyst.
This document provides specific instruction for certain analytes, but also serves to
familiarize the novice reader with many generic concepts needed to successfully utilize
immunochemistry technology including: applications, sampling, sample preparation,
extraction, cleanup, quality assurance, methods development and optimization, data handling
and troubleshooting. It is not necessary for the reader to actually perform the immunoassays
given in this User's Guide to obtain familiarity with these concepts. The Guide is written so
that the information presented can be applied to other immunoassays not given here. Thus,
the strength of the Guide is its universal applicability to immunoassay methods.
HI
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TABLE OF CONTENTS
Section Page
Notice ii
Abstract iii
List of Figures vi
List of Tables vi
Abbreviations and Acronyms vii
Acknowledgments viii
Executive Summary ix
1. Introduction
1.1 Brief History . . 1
1.2 Advantages/Disadvantages , 1
1.3 Principles , 2
1.4 Applications 4
1.5 Bibliography 6
2. Preparing the Laboratory
2.1 Laboratory Resources . 7
2.2 General Laboratory Supplies 7
2.3 Chemicals 7
2.4 Immunochemical Reagents 7
2.5 Immunochemical Supplies 7
2.6 Instrumentation 8
3. Laboratory Considerations
3.1 Assay Optimization . 9
3.2 Protocol Design . 9
3.3 Sample Preparation 10
3.4 Matrix Considerations 10
3.5 Data Handling 10
3.6 Quality Control 11
3.7 Pipetting Techniques 12
3.8 Troubleshooting 12
3.9 Safety Considerations 12
3.10 Waste Disposal 13
3.11 References 13
4. Immunoassay Tutorials for Selected Environmental Analytes 14
4.1. Analysis of Triazines in Environmental Samples
Utilizing a Double Antibody-Coated Microtiter
Plate ELISA Method 15
4.2. Analysis of Triazines in Environmental Samples
Using a Single Antibody-Coated Microtiter
Plate ELISA Method 22
IV
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TABLE OF CONTENTS (con't)
Section Page
4.3 ELISA Method for Analysis of Carbaryl in Environmental and
Biological Samples 29
4.4 ELISA Method for the Analysis of Paraquat in Environmental
Samples 35
4.5 ELISA Method for the Analysis of 4-Nitrophenols in Environmental
Samples 41
4.6 ELISA Method for the Analysis of Triazine Mercapturates
in Urine 48
5. Tutorials for Support Techniques 54
5.1 Pipetting Techniques 55
5.2 Considerations in Sampling and Sample Preparation
for Immunoassay Analysis 57
5.2.1 Solid Phase Extraction (SPE) of s-Triazine
Herbicides from Water 60
5.2.2 Solid Phase Extraction (SPE) of Atrazine
Mercapturate from Urine 61
5.3 Approaches to Testing for Matrix Effects 62
5.4 Data Analysis Guidelines 64
5.5 Optimization of Reagent Concentration by
Checkerboard Titration 67
5.6 Record Keeping . '. 73
5.7 Preparation of Buffers for Use in the ELISA 74
5.8 Preparation of Calibration Standards and
Samples Using the 8 X 12 Array 76
5.9 Outline for a Quality Assurance Document for
Using Immunoassay Methods 78
5.10 Guidelines for the Efficient Use of 96-Well
Microtiter Plates 79
5.11 General Troubleshooting Guidelines to Optimize the
Enzyme Immunoassay Method Performance 80
5.12 Maintenance and Performance Validation of a 96-Well
Microplate Reader 83
5.13 Performance Checks, Calibration and Maintenance
of Air Displacement Pipettors 85
6. Glossary of Commonly Used Terms in Immunoassay . 86
Appendices
Appendix I. Performance Assurance for Air Displacement Pipettes 1
Appendix II. Performance Log and Performance Test Worksheets for
Air Displacement Pipettors 1
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LIST OF FIGURES
Figure
Page
1. Schematic of the procedure for conducting an immobilized
antigen ELISA 3
2. Schematic of an immobilized antibody ELISA . 4
3. Typical ELISA template for placement of standards and samples 10
4. Model 4-parameter calibration curve 11
5. Schematic of double antibody-coated microtiter plate ELISA 15
6. Schematic of single antibody-coated microtiter plate ELISA 22
7. Schematic of the antigen-coated plate ELISA format 29
8. Schematic of the antigen-coated plate ELISA format for paraquat 35
9. Schematic of the antigen-coated plate ELISA format 41
10. Schematic of the double antibody-coated ELISA ~. 48
11. Representation of the forward and repetitive pipetting techniques 55
12. Comparison of diluted sample and diluted standard for the assessment of
matrix effects 63
13. Model 4-parameter calibration curve 65
14. Example protocol for titer determination: coating antigen or anti-analyte
antibody 67
15. Example protocol for titer determination: anti-analyte antibody
or enzyme-IabeLIed hapten 68
16. Plot of checkerboard titration data 70
17. Plot of checkerboard titration data 70
18. Schematic of the 8x12 array for sample preparation 77
19. Typical ELISA template 79
Appendix:
1. Normal distribution 2
2. Measured values vs. specification 4
LIST OF TABLES
Table
Page
1. Troubleshooting guidelines to optimize the enzyme immunoassay
method performance 80
2. Troubleshooting guidelines for the use of air displacement pipettors 85
Appendix:
1. Weight class standards for balance calibration 5
2. Z-factors 8
VI
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ABBREVIATIONS AND ACRONYMS
Ab
Ag
AP
ATR-N(C5)-HRP
BLK
BSA
BTX
CONA
DMSO
EIA
ELISA
EMSL
EPA
HPLC
HRP
'C50
IgG
KLH
MSDS
OVA
PBS
PBS-Tween
PBS-Tween/Azide
PGB
PQ
SIM-N(C2)-AP
SFE
SITE
SPE
TMB
UC
Antibody
Antigen
Alkaline phosphatase
Triazine hapten-labeled horseradish peroxidase conjugate
Blank
Bovine serum albumin
Benzene, toluene, xylene
Conalbumin
Dimethylsulfoxide
Enzyme immunoassay
Enzyme linked immunosorbent assay
Environmental Monitoring Systems Laboratory
Environmental Protection Agency
High performance liquid chromatography
Horseradish peroxidase
Concentration producing 50% inhibition
Immunoglobulin
Association constant
Dissociation constant
Keyhole limpet hemocyanin
Materials Safety Data Sheets
Ovalbumin
Phosphate buffered saline
Phosphate buffered saline containing Tween 20
Phosphate buffered saline containing
Tween 20 and sodium azide
Polychlorinated biphenyls
Paraquat
Triazine hapten-labeled alkaline phosphatase conjugate
Supercritical fluid extraction
Superfund Innovative Technology Evaluation
Solid phase extraction
3,3'5,5' -Tetramethylbenzidine
University of California
VII
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ACKNOWLEDGMENTS
The authors thank Don Gurka and Llewellyn Williams of, the Environmental Protection
Agency, Environmental Monitoring Systems Laboratory, Las Vegas, Nevada, Rosie Wong of
American Cyanamid, Princeton, New Jersey, and George Fong, Florida Department of Agricul-
ture, Tallahasee, Florida for helpful review and comments. The authors also thank Al Reed
and Virginia Kelliher, Senior Environmental Employment (SEE) Program enrollees assisting
the Environmental Protection Agency under a Cooperative Agreement with the National
Association for Hispanic Elderly for their helpful review and comments. The authors especially
thank Al Reed for his expert editorial assistance.
VIII
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EXECUTIVE SUMMARY
Immunochemistry has broad applications for a wide variety of environmental
contaminants. However, the potential for applying immunochemica! methods to environmental
measurements is just beginning to be realized. Immunochemical methods are based on
specific antibodies combining with their target analyte(s). Many specific antibodies have been
produced for targets of environmental and human health concern. Such antibodies can be
configured into various analytical methods. The most popular immunochemical technique in
environmental analyses today is immunoassay. Immunoassays have been shown to detect
and quantify many compounds of environmental interest such as pesticides, industrial
chemicals, and products of xenobiotic metabolism. Among the most important advantages of
immunoassays are their speed, sensitivity, selectivity and cost-effectiveness.
Immunoassays can be designed as rapid field-portable, semi-quantitative methods or
as standard quantitative laboratory procedures. They are well suited for the analysis of large
numbers of samples and often obviate lengthy sample preparations. Immunoassays can be
used as screening methods to identify samples needing further analysis by classical analytical
methods. Immunoassays are especially applicable in situations where analysis by
conventional methods is either not possible or is prohibitively expensive.
Environmental immunoassays have broad applications for monitoring studies. The
EPA has used immunoassay methods for monitoring groundwater and cleanup activities at
hazardous waste sites. Immunoassays can also be used as field screening tools to confirm
the absence and or presence of particular contaminants or classes of contaminants for special
surveys. Other federal and state agencies are employing immunoassay technology where
appropriate such as for extensive monitoring studies that generate a large sample load.
In addition to detection methods, other immunochemical procedures can be used for
environmental analysis. Immunoaffinity techniques now used extensively in pharmaceutical
and biotechnology applications can be adapted to extract, and cleanup environmental
samples. Selective and sensitive sample collection systems such as air and personal
exposure monitors can be designed based on the principal of immunoaffinity. Although
immunoaffinity procedures are not addressed in this tutorial, they are mentioned here to
illustrate to the reader that immunochemical methods can be adapted to a wide variety of
monitoring situations.
The U.S. EPA Environmental Monitoring Systems Laboratory at Las Vegas, Nevada
(EMSL-LV) has a program to develop and evaluate immunochemical methods for
environmental analysis. The EMSL-LV immunochemistry program consists of the following
major components: identification of need for an immunochemical method, identification of
existing technologies, development of new technologies, adaptations of existing technologies,
evaluations of existing technologies, field demonstration of portable technologies, and finally
technology transfer. Overall program goals, as well as prioritization of compounds for
methods development, are based upon input from client EPA Program Offices as well as the
EPA Regions. Analytical needs are defined as to target analytes, matrices, detection limits
and application of the method.
Methods and immunologic reagents have been developed for the polychlorinated
biphenyls (PCBs), BTX (benzene, toluene, xylene) and various pesticides and nitroaromatic
IX
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compounds through the EMSL-LV immunochemistry program. Additional methods are under
development for pyrethroid and organophosphorus pesticides.
The EMSL-LV immunochemistry program conducts laboratory and field evaluations to
assess method performance. The evaluation, characterization and testing of a particular
analytical method is necessary to ensure the intended use of the method is met. Evaluations
are conducted according to EPA guidelines requiring the determination of precision, within and
among laboratories bias, method detection limit, matrix effects, interferences, limit of reliable
measurement and ruggedness of the method. Demonstrations under the Superfund
Innovative Technology Evaluation (SITE) program have been used to document method
performance under real-world environmental conditions. SITE demonstrations of
Jmmunoassay methods for the PCBs, pentachlorophenol, and BTX have been completed,
other demonstrations are being planned. After a SITE demonstration the methods can be
submitted to the Superfund Field Screening Methods compendium for inclusion and
distribution.
Technology transfer activities include providing guidance and training to EPA regional,
EPA headquarters, and state personnel on the use of immunoassays. A computer animated
graphics program has been developed to provide instruction on the theory and applications of
immunoassays. This graphics program may be a useful training aid to the tutorials contained
in this document. Other instructional activities planned include the development of training
videos for performing immunoassays. A "hands on" workshop at the EMSL-LV is also being
considered. Individual training for EPA personnel has been conducted and will remain an
option for interested individuals.
Another vehicle to facilitate the implementation of immunochemical methods are
annual meetings of researchers, developers and end-users of immunochemical methods. The
EMSL-LV has sponsored two meetings to discuss the direction of immunochemical methods
research, development, application, and acceptance within the regulating and regulated
communities. The last Immunochemistry Summit Meeting was held in September 1993 and
included representatives from EPA and other federal and state agencies, large chemical
companies, biotechnology companies, and research institutes. It is anticipated that this type
of meeting will continue to be an annual forum for concerns and issues regarding
environmental immunochemical methods.
Considering the advantages and versatility of immunochemical methods, it is surprising
that the technology has not been more widely accepted by environmental analytical chemists.
Although many immunoassay methods have been reported in the literature, their potential has
not been practically realized. Part of the problem is misunderstanding and perhaps skepticism
on the part of analytical chemists. A thorough understanding of the advantages and
limitations of immunoassay methods is essential to applying the technology in situations where
they offer the most promise. It is the intent of this document to dispel the mystery in
understanding and performing an environmental immunoassay.
This document presents six specific immunoassay methods. The methods are based
on the same working principle but illustrate different applications of the technology for various
analytical situations. Two methods are presented to describe immunoassays for lipophilic
analytes using the triazine herbicides as examples. Although either method can be used for
environmental samples, both are presented to illustrate different formats for the same analyte.
The third method is for the insecticide carbaryl which is applicable for both environmental and
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biological samples. Methods for p-nitrophenol, paraquat and for various triazine mer-
capturates are examples of water soluble analytes. The triazine mercapturates method
illustrates the application of immunoassay for urine samples and hence exposure assessment
studies. Accompanying solid phase extraction procedures to extract triazines from water and
atrazine mercapturate from urine are also provided. All of the methods described are
intended to serve as examples of the utility of immunoassay technology.
In addition to the six specific immunoassay methods, this document also describes
analytical laboratory techniques necessary to perform immunoassays. Suggestions for
general laboratory considerations such as protocol design, sample preparation, data handling
and analysis, and safety precautions are also given. Examples of troubleshooting and quality
control practices are included which can be applied to assays not contained in this tutorial.
Protocols for preparing buffers, determining reagent integrity and for optimizing assay
conditions are also useful for immunoassays in general. An appendix of commonly used
terms in immunoassay should facilitate understanding of the technology.
Although immunoassays are now being employed for environmental analysis, there
may still be a need for training non-analysts in the use of immunoassay or updating the
experienced analytical chemist on an unfamiliar analytical format. It is hoped that the
methods and procedures found in this users guide will be beneficial and help to standardize
the immunochemical analysis of small molecules. Comments and written requests for
additional information may be directed to Jeanette M. Van Emon, U.S. Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, P. O. Box 93478,
Las Vegas, Nevada 89193-3478.
XI
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SECTION 1
INTRODUCTION
This manual is designed to introduce the analytical chemist to the general concepts
and use of immunoassays for the analysis of pesticides and other small molecules. By writing
this manual we hope to encourage the analytical chemist to consider this technology among
the repertoire of methods available for solving analytical problems. As with any other
analytical technique, it will be just as important for the analyst to be able to identify when
immunochemical technology is appropriate, as it is to learn how to conduct an
immunochemical analysis. Field personnel who may need to employ a measurement
technology in the field may also find this manual helpful.
The manual is organized first to provide some general information on the technology,
second to provide tutorials consisting of some specific examples of immunoassays and thirdly
to provide guidelines and information on those procedures specific to immunochemical tech-
niques which may not currently be in use in the typical analytical chemistry laboratory. All of
these procedures were developed in an academic laboratory and may need to be adjusted to
meet regulatory requirements of the various agencies within the government as to method
performance and their procedural guidelines. This caveat also can apply to a Contract Laboratory.
1.1 Brief history
The development of chromatographic instrumentation by pesticide analytical chemists
was closely paralleled by the development of immunoassay techniques by clinical analytical
chemists. Immunoassays are routinely used in clinical situations for the analysis of proteins,
hormones and drugs. The success that these immunochemical procedures has achieved in
the clinical area is being transferred to the area of pesticide analysis.
The first antibodies developed against pesticides were reported in the 1970's. Recently
this technology has been refined for use in the pesticide analytical chemist's laboratory to the
point that commercial test kits are now available. With the availability of kits, it is imperative
that the analyst understand the underlying principles of the methodology in order to evaluate
the strengths and limitations of any one "kit" for their specific application. Since many of these
easy-to-use test kits are designed for the non-analyst, it is important that these users also
have the same fundamental understanding.
1.2 Advantages/Disadvantages
Immunoassays are a useful complement to the analytical chemist's repertoire of
methods for the detection of pesticides and other environmental chemicals. Immunoassays
are rapid, sensitive and selective for the analyte of interest and generally cost effective for
large sample loads. Immunoassays have been applied to diverse chemical structures and are
adaptable to field use. As with any technology there are disadvantages. Antibodies may bind
to structural analogs of the analyte of interest (termed cross-reactivity). This technology is not
easily adapted to a multianalyte method, since each antibody binds primarily to a single
analyte or class of analytes. Reagent stability is often cited as a problem, but can be
overcome based on knowledge gained from the clinical field. This technology also requires a
large sample load to justify development of a new assay for an analyte of interest, due to the
expense of producing antibodies and establishing the procedure. For intermittent analysis, it
might be more cost effective to use existing commercially prepared test kits.
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1.3 Principles
Immunoassays rely on the reaction of an analyte or antigen (Ag) with a selective
antibody (Ab) to give a product (Ab Ag) that can be measured. This reaction is characterized
by the Law of Mass Action (shown below), thus immunoassays are physical assays.
Ab + Ag » *• Ab-Ag
In its most generic form, immunoassay is an analytical method dependent on the
specific binding of an antibody with its target analyte. This specific interaction can provide
quantitation of the target analyte. Many types of labels have been used for quantitating
immunoassays including radioactivity, enzymes, fluorescence, phosphorescence,
chemiluminescence, and bioluminescence. Each of these labels has its own particular
advantage. However, the use of enzymes and colorimetric substrates is probably the most
common for environmental analysis. Several different types of enzyme immunoassays (ElAs)
have been developed, the two broadest categories being heterogeneous and homogeneous
enzyme immunoassays. Heterogeneous immunoassays require a separation of bound and
free reagents throughout the assay. This is easily accomplished by simply washing the solid
phase (e.g. test tube, microtiter plate wells, cuvettes, etc) with buffer and surfactant.
Homogeneous immunoassays do not require separation or washing steps. However, in these
immunoassays the enzyme label is required to function in the sample matrix which often
poses difficulties. Due to this restriction, homogeneous immunoassays are popular in the
clinical field, while heterogeneous assays are used predominately for environmental matrices.
A common heterogeneous immunoassay is the enzyme-linked immunosorbent assay
(ELISA). The methods in this guide are based on the ELISA format schematically shown in
Figure 1 (adapted from Wie & Hammock, 1982). The following is a generic description for
preparing the microtiter plates and reagents for an ELISA. Preparation of microtiter plates: A
constant amount of the coating antigen is bound to the surface of polystyrene microtiter plate
wells by passive adsorption. After a pre-determined incubation period the wells are washed to
remove unbound coating antigen.
Preparation of ELISA reactants: 1) A constant amount of anti-analyte antibody (first
antibody) is incubated with increasing amounts of analyte in separate test tubes (tubes B, C,
and D, Figure 1). This incubation period enables the formation of analyte-antibody
complexes. The number of analyte-antibody complexes formed and the remaining amount of
free reactants is dependent upon the amount of analyte present in the samples or standards.
2) The incubation mixture is added to the prepared microtiter plate wells. The coating
antigen competes with remaining free analyte for available antibody. A washing step removes
all materials not bound to the microtiter well. 3) A second antibody covalently coupled to an
enzyme (the enzyme label) is next added which binds to the first antibody now bound to the
coating antigen. If the first antibody was developed in a rabbit, the appropriate second
antibody would be goat anti-rabbit IgG covalently coupled to alkaline phosphatase (or another
enzyme label). Excess second antibody is then washed out. 4) Finally substrate is added to
produce a color change. This ELISA is typically called an inhibition assay since a high
concentration of analyte in the samples or standards inhibits the first antibody from binding to
the coating antigen on the microtiter plate well. This is due to the number of analyte-antibody
complexes formed in the initial incubation (tubes A-D Figure 1). The amount of enzyme
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Log Concentration
•••• -•<" Polystyrene
C Coating Antigen
E Enzyme coupled to
R Second Antibody
n First antibody
1 Hapten/Analyte
1J Substrate to
Product
Figure 1. Schematic of the procedure for conducting an immobilized
antigen ELISA.
product formed is directly
proportional to the amount of first
antibody bound to the plate and
is inversely proportional to the
amount of analyte in the samples
and standards (tubes A-D, Figure
1). In this example, the
maximum color intensity is
observed in the wells containing
the contents from tube A (Figure
1) where all the available first
antibody is bound to the coating
antigen. As increasing amounts
of analyte are added, the color
intensify decreases leading to a
sigmoidal analyte dose response
curve similar to that shown in
Figure 1.
There are several
variations in this "format". For
example, the first antibody and
analyte may be added directly to
the coated microtiter plate well.
A second, commonly used,
format is the direct competition
assay. In this immunoasssay format, the antibody is immobilized on the solid phase. Analyte
in the sample competes with a known amount of enzyme-labelled hapten for binding sites on
the immoblized antibody (Figure 2). In step 1, the anti-analyte antibody is adsorbed to the
microtiter plate well. In the competition step, the analyte and a hapten-labelled enzyme are
added to the microtiter plate well. All unbound reagents are washed out. The final step is the
addition of substrate. As in the first format described above, the production of color is
inversely related to the concentration of analyte. This particular format is commonly employed
in the commercial immunoassay test kits.
Formats will vary and it would be useful to know the format being tested as it may be
important to the performance of the assay. For example in the second ELISA schematic, the
sample is in contact with the enzyme labelled hapten. If the enzyme is sensitive to a matrix
effect, then you will get inhibition of the enzyme activity which may then lead to a false
positive result. If this is the case, the format of choice, would be the first format in which.the
sample does not come in contact with the enzyme.
Another important thing to remember with regard to formatting, is that the same
immunoassay reagents can be formatted for a highly quantitative laboratory test, for a semi-
quantitative test, or for rapid yes/no field tests. In general, assays that are simple and very
rapid, tend to be less sensitive. Assays designed for laboratory use may perform less repro-
ducibly in the field. As with any analytical method, immunoassays are designed to perform to
certain specifications under the conditions given. An assay designed to measure an analyte
in a groundwater sample in the laboratory may not perform the same when analyzing ground-
water at a field workstation, or even analyzing surface, instead of groundwater, in the
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3).
2).
1).
Enzyme
substrate
Colored
product
Anti-
hapten
antibody
Figure 2. Schematic of an immobilized antibody ELISA.
laboratory. That is not to say the
assay is not performing correctly.
It is only to say that it will perform
differently, although the difference
should be consistent under given
conditions.
As a final note to for-
matting and optimization; the
tutorial methods given in this
user's manual use the reading of
absorbances after a fixed period
of time or until the zero control
sample attains a given absorb-
ance value as an endpoint. An
alternative to the endpoint mode
is to use a kinetic read mode. In
this case, the absorbances in the
wells of the microtiter plate are
read at fixed intervals (several
seconds) immediately after addi-
tion of the substrate. In this way,
the rate of the enzyme reaction is
monitored. A major benefit of
kinetics reading is the minimi-
zation of well-to-well variation for replicate analyses due to the time difference in the addition
of substrate. Other advantages to this method include a decrease in analysis time and a
decrease in the amount of reagent needed to obtain a useful signal. In addition, one can
check the linearity of the enzyme reaction, thus further verifying integrity of the assay.
1.4 Applications
An easy way to introduce immunoassay into the analytical laboratory is to use specific
Immunoassays as screening methods to determine dilution levels for routine instrumental
analysis. Used in this manner, immunoassays can minimize instrument down time by
protecting sensitive components such as electron capture detectors. The U.S. Environmental
Protection Agency has used immunoassay methods for monitoring cleanup activities at
hazardous waste sites. Many EPA Regions have expressed satisfaction in utilizing
immunoassay methods for these types of field monitoring activities. In some Regions
immunoassay methods are used for groundwater monitoring as a screening tool. For these
monitoring situations, a sample yielding a positive result, is confirmed by an alternative
method. The EPA, in conjunction with the state of Idaho, is currently evaluating the use of
immunoassay to monitor water in the vadose zone for pesticides, as a way of determining the
efficiency of irrigation management practices to prevent leaching. Additional studies are being
conducted by the EPA such as those through the-Superfund Innovative Technology Evaluation
program (Gerlach et al., 1993). Other state and federal agencies (e.g. the U.S. Army Corps of
Engineers, the U.S. Geological Survey, the U.S. Department of Agriculture, the National
Institute of Occupational Safety and Health, and the U.S. Food and Drug Administration) are
implementing or evaluating the use of immunochemical methods for their respective
monitoring programs.
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The California Department of Food and Agriculture Chemistry Laboratory is an
example of a State regulatory laboratory introducing immunoassay for part of their normal
operation. Their goal is to replace, in a cost effective manner, instrumental analysis for
specific compounds in their routine compliance monitoring program with immunoassay. This
includes considerations of protocol design, dealing with outliers, curve fitting techniques,
consideration for generating "defensible" data in terms of processing and analysis and having
rapid, real time access to quality assurance data.
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1.5 Bibliography
References by Voller and Tijssen provide information on general principles of enzyme
immunoassays. The other references listed are reviews prepared in this laboratory giving
overviews on the development of immunoassays.
Gerlach, R. W., White, R. J., O'Leary, N. F. D. and Van Emon, J. M. 1993. Superfund
Innovative Technology Evaluation (SITE) Program Evaluation Report for Antox BTX
Water Screen (BTX Immunoassay). EPA/540/R-93/518, U.S. Environmental Protection
Agency, Las Vegas, Nevada. 91 pp.
Hammock, B. D. and R. O. Mumma. 1980. Potential of immunochemical technology for
pesticide analysis. In Advances in Pesticide Analytical Methodology, pp. 321-352, (J.
Harvey Jr. and G. Zweig, eds.) American Chemical Society Symposium Series, ACS
Publications, Washington, D.C.
Hammock, B. D., S. J. Gee, R. O. Harrison, F. Jung, M. H. Goodrow, Q. X. Li, A. D. Lucas, A.
Szekacs, and K. M. S. Sundaram. 1990. Immunochemical technology in
environmental analysis: addressing critical problems. In: Immunochemical Methods
for Environmental Analysis, pp. 112-139 (J.M. Van Emon and R.O. Mumma, eds.),
ACS Symposium Series 442.
Jung, F., S. J. Gee, R. O. Harrison, M. H. Goodrow, A. E. Karu, A. L. Braun, Q. X. Li and B.D.
Hammock. 1989. Use of immunochemical techniques for the analysis of pesticides.
Pest. Sci. 26:303-317.
Tijssen, P. 1985. Practice and Theory of Enzyme Immunoassays. Elsevier, New York,
549 pp.
Van Emon, J. M., J. N. Seiber and B. D. Hammock. 1985. Applications of immunoassay to
paraquat and other pesticides. In: Bioregulators for Pest Control, pp. 307-316 (P.A.
Hedin, ed.), American Chemical Society Symposium Series 276, Washington D.C.
Van Emon, J. M., J. N. Seiber, and B. D. Hammock. 1989. Chapter 17: Immunoassay
techniques for pesticide analysis. In: Analytical Methods for Pesticide and Plant
Growth Regulators: Advanced Analytical Techniques, Vol. XVII, (J. Sherma, ed.),
Academic Press. New York. pp. 217-263.
Voller, A., A. Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31 :507-520.
Wie, S. I. and B. D. Hammock. 1982. Development of enzyme-linked immunosorbent assays
for residue analysis of Diflubenzuron and BAY SIR 8514. J. Agric. Food Chem.
30:949-957.
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SECTION 2
PREPARING THE LABORATORY
The materials listed below may be purchased from a number of commercial sources.
Examples of some items are given for the convenience of the reader.
2.1 Laboratory Resources
Very little additional space or resources are necessary to be able to analyze samples
using immunochemical techniques. The majority of space would be necessary for the
preparation of samples, similar to that already used for other analytical techniques. Electrical
outlets and a vacuum line or pump are the only services required.
2.2 General Laboratory Supplies
Magnetic stirrers/Magnetic stir bars
Vortex mixers
Weigh boats/paper/spatulas
Paper towels
Laboratory wipes (i.e Kimwipes)
Laboratory plastic film (i.e. Parafilm)
Assorted glassware (beakers, erlenmeyers, graduated cylinders)
Felt tipped markers
Benchtop absorbent paper
Assorted borosilicate glass test tubes
Assorted disposable gloves
Label tape
Soap, brushes, wash tubs
2.3 Chemicals
1) Assorted acids/bases and solvents (HCI, NaOH, methanol, acetonitrile, dimethyl
sulfoxide, dimethyl formamide, 2-propanol, etc.)
2) Buffer salts (sodium chloride; sodium phosphate, mono and dibasic; potassium
phosphate, mono and dibasic; sodium carbonate; sodium bicarbonate; diethanolamine;
sodium citrate; potassium chloride, etc.)
2.4 Immunochemical Reagents
The reagents described in the following procedures are examples. To facilitate the
transfer of technology to an environmental monitoring laboratory for routine monitoring studies,
similar reagents are available through commercial sources. The American Association for the
Advancement of Science publishes a yearly "Guide to Biotechnology Products and
Instruments" which includes sources of antibodies for environmental compounds. Similarly,
the American Chemical Society publishes a "Biotech Buyers' Guide."
2.5 Immunochemical Supplies
This supply list is designed to supplement the list of supplies that already exists in the
typical analytical laboratory. It is divided into two areas. In the first area, it assumes the
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analyst is evaluating a test kit which contains all the necessary tubes or plates and reagents.
In the second area, it assumes the analyst is evaluating component reagents and will have to
already have some standard reagents or chemicals. These supplies will vary depending on
the format of the assay. Either kit assays or component reagents should always come
supplied with a complete protocol, listing any unusual reagents that might be needed.
1) Pipet tips for multichannel pipettor
2) Pipet tips for single channel pipettors.
3) Multichannel pipet reservoirs.
4) Vacuum tubing and one 1 L and one 4 L vacuum flask for hand held plate washer
5) Several 8 L carboys for wash buffers, and stock buffers
6) Assorted test tubes or other tubes for diluting samples. Individual test tubes may be used
to make dilutions. Test tubes (2 mL) are available in a 8X12 array for use with the
multichannel pipettors. For very small volumes, dilutions may be made in 96 well
microtiter plates that are NOT designed for high binding or use in the ELISA (i.e. Item #2
listed below).
These are some of the supplies and reagents that may be necessary when evaluating
component reagent assays:
1) High binding fiat bottomed 96 well microtiter plates - "ELISA plates" (i.e. Nunc
Immunoplate II or equivalent)
2) Flat bottomed 96 well tissue culture plates - used only for diluting samples (i.e. Dynatech
plates or equivalent)
3) Acetate plate sealers
4) Sodium azide (used as a preservative in buffers)
5) Goat anti-rabbit IgG antibody conjugated to alkaline phosphatase or horseradish
peroxidase (second antibody, - depends on format and animal source of primary antibody)
6) Substrates (p-nitrophenylphosphate or 3,3',5,5'-tetramethylbenzidine) - depends on
enzyme label used.
7) Tween 20 detergent. Surfactant to prevent nonspecific binding.
8) Bovine serum albumin (Fraction V). Sometimes used as a "blocking" agent, i.e. to cover
up potential sites for nonspecific binding.
2.6 Instrumentation
1) Variable volume 12 or 8-channel pipettors (approximately $700)
2) Single channel pipettors (various volume ranges, approximately $200 each)
3) Plate washer (may be a simple as a wash bottle, or as complex as an automated plate
washer, approximately $3000).
4) For microplate-based assays a spectrophotometer designed as a 96-well microplate
reader or a strip reader will be necessary for quantitation. A plate reader may cost as
much as $20,000. There are also smaller hand held or benchtop spectrophotometers
which will read an 8-well strip. These are often used for laboratory-based assays that can
be conducted in the field. Most tube-based assays are semi-quantitative in that you might
determine the concentration by eye compared to a standard. These are also adaptable
though, to reading in a spectrophotometer for more quantitative data.
8
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SECTION 3
LABORATORY CONSIDERATIONS
3.1 Assay Optimization
When evaluating immunoassays, it is important to keep in mind that these are
governed by the Law of Mass Action. The reagents are thus in an equilibrium condition. The
assay then is subject to fluctuations due to temperature (of the reagents and of the laboratory
in which the assays are conducted) and length of incubation time. Since reactions are
occurring at the surface of the microtiter plate, shaking the plate to mix the contents of the
wells may affect the local concentration of reactants. Each of these factors should then be
controlled in order to improve the precision of the measurements. Typically assays are
conducted with reagents which have been equilibrated to room temperature. If room
temperature is not constant (within 3 - 5 degrees of variation), than assays should be
conducted using a forced-air incubator. Shaking the plates periodically during incubation
periods may also improve precision. For immunoassays utilizing 30 minutes or longer
incubation periods, the reactants have likely come nearly to equilibrium and thus conducting
assays with precise timing is unnecessary. However, for immunoassays utilizing shorter
incubation periods, precise timing will improve precision.
3.2 Protocol Design
The methods used most commonly in the analytical laboratory are based on the
96-well microtiter plate format. There are numerous permutations and combinations of ways
that samples and standards can be placed on a 96-well microtiter plate. The number of
calibration wells and the known concentrations used for the calibration curve affect the
precision of the determinations of the unknowns, as does the choice of the number of
replicates of each unknown. Within the framework of a 96-well microtiter plate, how does one
maximize the number of samples analyzed while maintaining the best possible accuracy and
precision. A statistically based method for determining the weight of these factors has been
presented by Rocke et al. (1990). In broad terms there is a tradeoff between efficiency in the
number of samples that can be run per plate vs the additional precision obtained by running
more replicates of each sample or standard. Samples are generally analyzed at several
dilutions. For example, 1:2, 1:4 and 1:8. Values obtained for at least one of the dilutions
should fall near to the center of the calibration curve. This approach is taken in the event that
a positive response is due to a matrix effect. If multiple dilutions are analyzed then
discrepancies among the calculated values may indicate an effect of matrix. If a single
dilution is analyzed then a matrix effect may not be revealed until the sample is confirmed by
an independent method. However, if the matrix is known to not interfere in the analysis, a
single dilution may be analyzed. If the result is too high, then further dilutions can be made.
Last, efficiency of analysis may dictate splitting replicates of unknowns between microtiter
plates. This allows the achievement of desired accuracy at the lowest cost. A typical layout
for a 96-well microtiter plate is shown in Figure 3.
A typical plate format should have a calibration curve with enough replicates as shown
in Figure 3. In fact, it is recommended that a calibration curve be run on every plate because
the reactants are governed by the Law of Mass Action, they are in a dynamic equilibrium. If a
given plate is subject to differences in manipulation time, temperature of incubation or other
factors which may effect the equilibrium, the samples on that plate can be compared to a
calibration curve subjected to those same variables. See also tutorial 5.10. Guidelines for the
Efficient Use of 96-Well Microtiter Plates.
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Figure 3. Typical ELISA template for placement of standards and samples.
3.3 Sample Preparation
The extraction and cleanup steps used for the more conventional detection methods
can be used as a starting point in devising sample preparation schemes for the corresponding
immunoassay. Since immunoassays are aqueous based analyses, this may result in a
reduction in sample preparation in general. Recommendations are given in each protocol for
sample preparation where information is known. General "rules of thumb" are presented in
tutorial 5.2. Consideration in Sampling and Sample Preparation for Immunoassay Analysis.
3.4 Matrix Considerations
The utility of any analytical method depends on the absence of interferences derived
from reagents and the matrix. The interference question must be addressed by running
appropriate blanks as controls prior to analysis. Due to the selectivity of the antibody for the
analyte, immunoassays usually do not require the rigorous extraction and cleanup methods
often used for other instrumental methods. In addition, the sensitivity of the antibody can be
exploited such that interferences may be "diluted out" while still maintaining the desired
detection limits. Interferences may vary with reagent batches and matrix sources and thus
must be checked frequently by a combination of running appropriate blanks, and confirming
positive samples by an alternate analytical method. The latter is crucial to using any assay
method, including ELISA, for monitoring samples of unknown origin when corresponding field
blanks are not available. Other commonly used methods for identifying and normalizing for
matrix effects on an analysis, such as the method of standard addition, can also be used
(Miller and Miller, 1984). If these approaches fail to adequately address the problem of
interference from the matrix, then some sample preparation may be necessary, for example,
solid phase extraction. Once the analyst begins to introduce sample preparation steps prior to
immunochemical analysis, than consideration must be given to whether this method is in fact
the most time saving and cost effective method available for this specific analytical problem.
See also tutorial 5.3. Approaches for Testing for Matrix Effects.
3.5 Data Handling
As with any analytical technique, the generation of a reproducible standard curve with
minimal error is critical. The standard curves generally resulting from immunoassays are
sigmoidal in shape, suggesting that the best fit curve could be log-logit or 4-parameter.
However, other curve fits such as linear, quadratric, semi-log or log-log can be used to find
the best fitting standard curve. A good reference pertaining to curve fitting appears in "Data
10
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Analysis and Quality Control of Assays: A Practical Primer", by R. P. Chenning Rogers in
Practical Immunoassay, editor Wifrid R. Butt; published by Marcel Dekker, Inc., N.Y. 1984. If
the choice of standards provides a complete definition of the shape of the curve, (i.e., the
curve has at least 2 to 3 points each defining the upper and lower asymptote and at least 4
points defining the linear region), the 4-parameter fit of Rodbard (1981) is the method of
choice for data analysis in the authors' laboratories. It is important that enough standard
concentrations are used to ensure that the curve is well defined and constant for these
concentrations. Without this information, the computer could force an improper fit (Gerlach et
al., 1993). The equation for the 4-parameter fit is:
y = (A-D)/(1 + (x/C)AB) + D
where y is the absorbance, x is the concentration of analyte, A and D are the upper and lower
asymptotes respectively, B is the slope and C is the central point of the linear portion of the
curve, also known as the \C50 (Figure 4).
cr
co -
o
3-
D)
O
Log Concentration
Figure 4. Model 4-parameter calibration curve.
The best quantitation of unknowns is carried out when unknown absorbances fall in the
central portion of the linear region of the calibration curve. The use of the 4-parameter fit
extends the usefulness of the upper and lower concentrations of the calibration curve.
However, the values calculated from these upper and lower concentrations have greater error
associated with them. To save on reagents, and to keep the error on the estimation of
concentrations of unknowns to a minimum, concentrations for standard curves should be
performed in the linear range after the complete standard curve has been defined with upper
and lower asymptotes. A semi-tog curve fit should then be used to fit the data to this
truncated calibration curve and the absorbance values for unknowns should fall in the central
portion of the linear region of this calibration curve. If a kit is being used, the package insert
should indicate the standard curve analysis method to use based on the range of standard
concentrations used for the calibration curve. See also tutorial 5.4. Data Analysis Guidelines.
3.6 Quality Control
There are several approaches to quality control for immunoassays. The first is to
monitor the parameters of the standard curve to ensure that these remain within the desired
coefficient of variation range. Second, it is important to establish relevant quality control
11
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standards (i.e. positive and negative controls). These too, should be monitored on a regular
basis for variations around a determined mean. This may be evaluated for example, by
construction of Shewhart charts (Wernimont & Spendley, 1985). See also tutorial 5.9. Issues
in Quality Control and Quality Assurance.
3.7 Pipetting Techniques
Pipetting is an integral part of this immunochemical technology. Assuming the error
derived from the specific assay design are fixed, the next largest source of error in analytical
data derived from immunoassay is from pipetting errors (Li et al,, 1989). Another important
aspect of pipetting error is related to the light path in the microplate reader. For some 96-well
microplate readers, the light path is through the bottom of the microtiter plate well, thus the
path length is directly related to the height of the solution in the microtiter plate well. See the
tutorial 5.1. Pipetting Techniques for full details.
3.8 Troubleshooting
Troubleshooting is probably the most useful skill that any analytical chemist can
develop. The most common problems in immunoassays are poor precision among microtiter
well replicates, spurious color development and no or low color development. Poor plate
washing and pipetting technique are the largest contributors to spurious color development.
No or low color development is most likely due to a reagent failure. The type of 96-well
microtiter plate used is also an important factor. Some plates will bind antigens differently,
and some have greater variability in binding capacity from well to well which would contribute
to variability. Generally selecting a manufacturer whose plate gives reproducible assay
performance parameters for a given assay is the best solution. Another significant factor is
temperature. The reactions that are occurring on the plate are based on the Law of Mass
Action. They are therefore equilibrium reactions and are sensitive to temperature. Reagents
should be used at room temperature, and during analysis, plates should be protected from
wide fluctuations in temperature (i.e. if the laboratory ambient temperature varies more than
3-5 degrees during the day or under field conditions). With the 96 well microtiter plates, the
tendency is for the outer wells to reach temperature sooner than the inner wells, which then
has an effect on the equilibrium reactions. Variations in final absorbances are generally
manifested in what is called an "edge effect." Conducting incubations in a forced-air incubator
may eliminate problems due to temperature fluctuations. Temperature-related effects on
equilibrium are more likely to be seen in assays whose incubation times are very short.
Problems specific to a given assay (for example, stability of standards) are addressed in the
individual tutorials. A comprehensive troubleshooting guide is currently beyond the scope of
this manual. When evaluating test kits or component assays, it is best to keep open lines of
communication with the supplier in order to obtain answers to questions and obtain assistance
in troubleshooting. See tutorial 5.11. General Guidelines for Troubleshooting.
3.9 Safety Considerations
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Specific safety considerations for the target
analytes and organic solvents that may be used in sample preparation are given with each
tutorial method where appropriate.
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3.10 Waste disposal
Disposal of hazardous wastes is given in each tutorial method. This technique utilizes
a number of disposable items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips,
sample diluent vessels). In general, the only hazard would be due to the presence of target
analyte in any of these items. Proper disposal may depend on the analyte and the regulations
in effect at your work site. Recycling is encouraged where appropriate.
3.11 References
Gerlach, R. W., White, R. J., Deming, S. N., Palasota, J. A. and Van Emon, J. M. 1993. An
evaluation of five commercial immunoassay data analysis software systems. Anal.
Biochem. 212:185-193.
Li, Q. X., Gee, S. J., McChesney, M. M., Hammock, B. D. and Seiber, J. N. 1989.
Comparison of an enzyme-linked immunosorbent assay and a gas chromatographic
procedure for the determination of molinate residues. Anal. Chem. 61:819-823.
Li, Q. X., Zhao, M. S., Gee, S. J., Kurth, M. J., Seiber, J. N. and Hammock, B. D. 1991.
Development of enzyme-linked immunosorbent assays for 4-nitrophenol and
substituted 4-nitrophenoIs. J. Agric. Food Chem. 39:1685-1692.
Miller, J. C. and Miller, J. N. 1984. Statistics for Analytical Chemistry, Ellis Horwood, Ltd.,
Chichester, England, pp. 100-102.
Rocke, D., Bunch, D. and Harrison, R. O. 1990. Statistical design of ELISA protocols. J.
Immunol. Meth. 132:247-254.
Rodbard, D. 1981. Mathematics and statistics of ligand assays. An illustrated guide. In
Ligand Assay, Langan, J., Clapp, J. J., eds.; Masson: New York; pp. 45-99.
Wernimont, G. T. and Spendley, W. 1985. Use of Statistics to Develop and Evaluate
Analytical Methods, Association of Official Analytical Chemists, Arlington, VA.
13
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SECTION 4
IMMUNOASSAY TUTORIALS FOR SELECTED ENVIRONMENTAL ANALYTES
Six specific immunoassay tutorials are given here (tutorials 4.1 - 4.6). The assay
principles are identical for all six, differing only in the analyte detected and the format of the
reagents. In general, the specific assays that are presented may be used for additional
matrices. However, other matrices may require optimization of the assay. The most
important consideration is the interference that may be a result of that matrix. The level of
interference will determine the amount of sample preparation required prior to analysis. For
example, with water soluble analytes, very little or no sample preparation is usually required.
For lipophilic analytes, it may be necessary to introduce water miscible co-solvents into the
assay. Further hints on preparing samples for analysis by ELISA appear in tutorial 5.2. The
first two protocols describe immunoassays for triazine herbicides as examples of lipophilic
anaiytes. The third protocol is for the insecticide carbaryl. The last three protocols are for
p-nitrophenol, paraquat, and triazine mercapturate and are used as examples of water soluble
analytes.
For further information regarding these tutorial methods contact Shirley J. Gee,
Department of Entomology, University of California, Davis, CA 95616, telephone
916-752-8465, telefax 916-752-1537, E-mail address: SJGEE@UCDAVIS.EDU.
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4.1 ANALYSIS OF TRIAZINES IN ENVIRONMENTAL SAMPLES UTILIZING A
DOUBLE ANTIBODY-COATED MICROTITER PLATE ELISA METHOD
Introduction:
The general assay design is shown in Figure 5. This assay is a competitive enzyme
immunoassay which utilizes a capture or trapping antibody for the first coating which binds the
triazine-specific antibody in a second coating step. A hapten-enzyme conjugate is used as
the label. This assay has been optimized for detection of atrazine. Due to the structural
similarity of triazines as a class, some cross reactivity with other triazines occurs. See Note 1.
This assay utilizes a horseradish peroxidase (HRP) enzyme label. Do not use sodium azide
in any of the buffers or wash solutions, as it inhibits HRP enzyme activity. All directions
for the preparations of buffers and other solutions used in this tutorial method are given In
tutorial 5.7.
Assay Protocol:
Coating the Microtiter Plate with Trapping
Antibody.
1. Coat the microtiter plate with the
trapping antibody. Make a solution
of goat anti-mouse IgG antibody that
is diluted 1/2000 in pH 9.6 carbonate
buffer (coating buffer). Add 100 (iL
to each well of a high binding ELISA
microtiter plate. See Note 2.
2. Cover the microtiter plate with a plate
sealer and incubate at 4°C overnight.
See Note 3.
3. Wash the microtiter plate 5X with
PBS-Tween and tap dry. The wash
procedure involves flooding each well
with buffer repeatedly to remove
unbound reagents.
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microtiter plate ELISA. Line indicates a wash step.
Numbers correspond to the steps in the assay
protocol.
4. Perform the second coating step.
Make a solution of anti-triazine
antibody (AM7B2.1) that is diluted
1/3200 in pH 9.6 carbonate buffer (coating buffer). Add 100 jo.L to each well of the
microtiter plate which has previously been coatsd with goat anti-mouse IgG antibody.
Cover the microtiter plate with a plate sealer and incubate at 4°C overnight.
5. Wash the double antibody-coated microtiter plate 5X with PBS-Tween and tap dry.
15
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Then freeze or use immediately in the ELISA step 7 below. This microtiter plate is
termed the "coated" plate. See Note 4.
Competitive Inhibition Steps.
6. Prepare standards, samples, and quality control samples (See Note 5.) in PBS-Tween.
This step can utilize the wells of a microtiter plate (of the type used for dilution only,
termed "mixing" plate, see Materials section). Using this technique several standard
curves can be prepared simultaneously (one per row) using a multichannel pipettor.
Multiple dilutions of samples may also be prepared in this manner. Samples can then be
transferred to the coated microtiter plate using the multichannel pipettor. See tutorial 5.8
for an example of this 8x12 array.
7. Add 50 pL of standard or sample from the mixing plate to each well of the coated plate.
8. Add 50 ^L of ATR-N(C5)-HRP (hapten-labeled enzyme conjugate) that has been diluted
1/3000 to 1/6000 in PBS-Tween to each well of the coated plate, except those wells that
serve as blanks. In the wells that serve as blanks, replace the hapten-labeled enzyme
conjugate with buffer. See Note 6.
9. Cover the coated plate containing standards, samples and enzyme label with a plate
sealer and incubate 15 minutes at room temperature.
10. Wash the coated plate 5X with PBS-Tween and tap dry.
11. Add 100 nL of substrate solution to each well of the coated plate and cover the plate
with a plate sealer. Incubate at room temperature for 15 minutes. (See tutorial 5.7 for
preparation of the substrate.)
12. Add 50 jxL of 4N sulfuric acid to each well of the coated plate to stop the enzyme
reaction.
13. Read at 450-650 nm. See Note 7. The maximum absorbance obtained is about 0.6-0.8
in the wells containing antibody, but no atrazine (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is about 1.0 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-1000 ng/mL The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine and dissolving in 2
mL of dimethylsulfoxide (DMSO). DMSO was chosen because it is water miscible, does not
interfere in the assay at the concentrations used and is not volatile. The primary stock is
diluted 1/100 in DMSO to make a working stock. The working stock is diluted 1/100 in PBS-
Tween to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of DMSO in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water.
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Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the sample indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect, than a simple cleanup step may be used (see Note 5). For example, tutorial
5.2.1 shows the sample preparation method for water in the analysis of triazines. Since
triazines are lipophilic and relatively nonvolatile, they are easily extracted from water using
solid phase extraction. The compounds are eluted from the column in ethyl acetate. Since
ethyl acetate is not a suitable solvent for immunoassay analysis, it is evaporated to dryness
and the residue taken up in PBS-Tween. If concentrations of triazines in the sample are very
high, some cosolvent may be necessary to solubilize the residue (i.e. methanol). Use as little
cosolvent as possible. If the cosolvent is found to interfere with the assay, running the
standard curve in the equivalent concentration of cosolvent can normalize for the interference.
This may compromise the parameters of the calibration curve compared to running the
calibration curve in buffer, but the change is reproducible. Another approach to avoiding
interference is to take advantage of the assay sensitivity. Many interferences can simply be
"diluted away." See tutorial 5.3 for approaches to evaluating matrix effects.
Notes:
1. Percent Cross Reactivity of AM7B2.1 (Atrazine = 100%)
Simazine 32 Hydroxyatrazine 2
Prometon 3 Hydroxysimazine 0
Terbutryn 19
(See Schneider et al., 1993 Table III, for a more complete list of compounds tested.)
2. The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
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3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these double antibody-coated microtiter plates can be stored
frozen for more than one month with no change in assay characteristics (i.e. \C50, slope
or maximum absorbance). We have also found that coating for more than overnight
before use or freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the no analyte control in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage. If strong
matrix effects occur, the protocol describe in tutorial 4.2 may be worth trying. There will
be about a 10 fold decrease in assay sensitivity, however in our hands this format
appeared more resistant to matrix and modifiers (such as cosolvents) in the sample
(Lucas etal., 1991).
6. Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in a
checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (ATR-N(CS)-HRP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials: '
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. ATR-N(C5)-HRP has the following structure and is
conjugated to horseradish peroxidase. It should be stored in the freezer. Periodically
tests of the enzyme activity should be run to assure no loss of activity on storage of the
stock solution. Working dilutions should be made up immediately before use and the
excess discarded. A change in'the assay performance parameters will be an indication
of possible degradation of the hapten-enzyme conjugate. Provided by Dr. Bruce
Hammock, Department of Entomology, University of California, Davis, CA 95616.
Revision 0
18 March 22, 1993
-------
CI
o
H H H
6-{{4-Chloro-6-[(1-methylethyl)amino]-
1,3,5-triazin-2-yl}arnino}hexanoic acid
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
o
II
CH2CH2CN—Protein
(CH3)2
H H
3-{{4-Ethylamino)-6-[(1-methylethyl)amino]-
1,3,5-triazin-2-yl}thio}propanoic acid
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
This antibody was provided by Dr. Alex Karu, Hybridoma Center, University of California at
Berkeley, 1050 San Pablo Avenue, Albany, CA 94706. Other triazine antibodies are
commercially available.
Revision 0
19 March 22, 1993
-------
Purchased Reagents:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
2) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
5) S.S'S.S'-Tetramethylbenzidine (Sigma Catalog No. T-2885 or equivalent. Use only the
highest purity.)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. 3,3'5,5'-Tetramethylbenzidine is an irritant; avoid breathing vapors.
This compound is used in dimethylsulfoxide, which may promote dermal absorption. Avoid
skin contact. It is assumed the analyst will have in place procedures for the safe handling of
organic solvents and samples containing the ;analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Revision 0
20 '. March 22, 1993
-------
Karu, A. E., R. O. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow,
A. Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M., L. H. Stanker, B. E. Watkins, and
D. W. Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
Lucas, A. D., P. Schneider, R. O. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and
D. E. Rolston. 1991. Determination of atrazine and simazine in water and soil using
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
Schneider, P. and B. D. Hammock. 1992. Influence of the ELISA format and the hapten-
enzyme conjugate on the sensitivity of an immunoassay for s-triazine herbicides using
monoclonal antibodies. J. Agric. Food Chem. 40:525-530.
Revision 0
21 March 22, 1993
-------
4.2 ANALYSIS OF TRIAZINES IN ENVIRONMENTAL SAMPLES
USING A SINGLE ANTIBODY-COATED MICROTITER PLATE ELISA METHOD
Introduction:
The general assay design is shown in Figure 6. This assay is a competitive enzyme
immunoassay which utilizes a capture or trapping antibody for the coating. The principle is
the same as shown for tutorial 4.1, except that the triazine-specific antibody is not
precaptured. Instead the triazine-specific antibody is reacted in free solution with the analyte
and is trapped by the adsorbed capture antibody. This assay is about 10X less sensitive than
the method described in tutuorial 4.1, bat Js more resistant to matrix and modifiers (such as
cosolvents) in the sample (Lucas et al.f 1991). This assay has been optimized for detection of
atrazine. Due to the structural similarity of triazines as a class, some cross reactivity with
other triazines occurs. See Note 1. All directions for the preparations of buffers and other
solutions used in this tutorial method are given in tutorial 5.7.
12).
9).
1).
Trapping
antibody
Y Y
Assay Protocol:
Coating the Microtiter Plate with Trapping
Antibody.
1. Coat the microtiter plate with the trapping
antibody. Make a solution of goat anti-
mouse IgG antibody that is diluted 1/2000 in
pH 9.6 carbonate buffer (coating buffer).
Add 100 (J.L to each well of a high binding
ELISA microtiter plate. See Note 2.
2. Cover the microtiter plate with a plate sealer
and incubate overnight at 4°C. See Note 3.
3. Wash the single antibody-coated microtiter
plate 5X with PBS-Tween/Azide and tap dry.
The wash procedure involves flooding each
well with buffer repeatedly to remove
unbound reagents. This plate is termed the
"coated" plate. Then freeze or use
immediately in ELISA step 9 below. See Note 4.
Competitive Inhibition Steps.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing plate"; see Materials section) for the preparation of dilutions
and for premixing reagents prior to their addition to the coated plate. Using this
technique several standard curves can be prepared simultaneously (one per row) using
a multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
Figure 6. Schematic of single antibody-coated
microtiter plate ELISA. Lines indicate a wash
step. Numbers correspond to the steps in the
assay protocol.
22
Revision 0
September 28, 1989
-------
5. Add 40 [il_ of standard or sample to each well of the mixing plate.
6. Add 100 (xL of SlM-N(C2)-AP (hapten-labeled enzyme conjugate) that has been diluted
1/3000 to 1/6000 in PBS-Tween/Azide to each well of the mixing plate, except those
wells that serve as no-enzyme-conjugate blanks. In the microtiter plate wells that serve
as blanks, replace the enzyme conjugate with buffer. See Note 6.
7. Add 100 jxL of anti-triazine antibody (AM7B2.1 medium) diluted 1/200 to 1/600 in PBS-
Tween/Azide to each well of the mixing plate,
8. Cover the mixing plate with a plate sealer and incubate 60 minutes at room temperature.
9. Transfer 50 JJ.L from each well of the mixing plate using a 12-channel pipettor to the
respective wells of the coated microtiter plate.
10. Cover the coated plate with a plate sealer and incubate 60 minutes at room temperature.
11. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
12. Add 100 M.L of 1 mg/rnL substrate solution (freshly made, one 5 mg tablet per 5 ml_ 10%
diethanolamine substrate buffer) to each well of the coated plate and cover with a plate
sealer. Incubate at room temperature for about 60 minutes (see Note 7).
13. Read at 405-650 nrn. See Note 8. The maximum absorbance obtained is about 0.5-0.6
in wells containing antibody, but no atrazine (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is 20 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-2000 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine and dissolving in 2
ml_ of dimethylsulfoxide (DMSO). DMSO was chosen because it is water miscible, does not
interfere in the assay at the concentrations used and is not volatile. The primary stock is
diluted 1/50 in DMSO to make a working stock. The working stock is diluted 1/100 in PBS-
Tween/Azide to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of DMSO in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the curve fit
model. For example, if a semi-log curve fit is utilized, then the calibration curve would only
utilize those standard concentrations which would yield a straight line.
23 Revision 0
September 28, 1989
-------
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the sample indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5). For example, tutorial
5.2.1 shows the sample preparation method for water in the analysis of triazines. Since
triazines are lipophilic and relatively nonvolatile, they are easily extracted from water using
solid phase extraction. The compounds are eluted from the column in ethyl acetate. Since
ethyl acetate is not a suitable solvent for immunoassay analysis, it is evaporated to dryness
and the residue taken up in PBS-Tween. If concentrations of triazines in the sample are very
high, some cosolvent may be necessary to solubilize the residue (i.e. methanol). Use as little
cosolvent as possible. If the cosolvent is found to interfere with the assay, running the
standard curve in the equivalent concentration of cosolvent can normalize for the interference.
This may compromise the parameters of the calibration curve compared to running the
calibration curve in buffer, but the change is reproducible. Another approach to avoiding
interference is to take advantage of the assay sensitivity. Many interferences can simply be
"diluted away." See tutorial 5.3 for approaches to evaluating matrix effects.
1. Percent Cross Reactivity of AM7B2.1 (Atrazine = 100%)
Simazine 40 Hydroxysimazine 3
Prometon 6 Both mono-N-dealkylated 1
Hydroxyatrazine 5 N,N'-di-dealkylated 0.1
2. The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these single antibody-coated plates can be stored frozen for more
than one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
24 Revision 0
September 28, 1989
-------
quality controls, assuming no degradation or loss of analyte on storage. If strong
matrix effects do not occur, the protocol given in tutorial 4.1 may be worth trying. The
advantage to method 4.1 is that the sensitivity is about 10X better than method 4.2, but
method 4.1 is more susceptible to matrix and modifiers (such as cosolvents) in the
sample (Lucas et al., 1991).
6. Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in
a checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (SIM-N(C2)-AP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
7. In order to facilitate the manual handling of several i.e., (10-25) coated plates in an
experiment, the length of incubation with the substrate has been optimized for 60
minutes. By adjusting reagent concentrations according to results obtained in the
checkerboard titration format, the assay may be optimized for shorter incubation times.
8. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. SIM-N(C2)-AP has the following structure and is
conjugated to alkaline phosphatase. It should be stored in the refrigerator.
Periodically tests of enzyme activity should be run to assure no loss of activity on
storage of the stock solution. Working dilutions should be made up immediately before
use and the extra discarded. DO NOT FREEZE - each freeze-thaw cycle will kill a
significant part of the conjugate-enzyme activity. A change in the assay performance
parameters will be an indication of possible degradation of the hapten-enzyme
conjugate. Provided by Dr. Bruce Hammock, Department of Entomology, University of
California, Davis, CA 95616.
25 Revision 0
September 28, 1989
-------
Cl
^
H H H
N-[4-Ch!oro-6-(ethyiarnino)-
1,3,5-triazin-2-vfl-p-alanine
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
This antibody was provided by Dr. Alex Karu, Hybridoma Center, University of California at
Berkeley, 1050 San Pablo Avenue, Albany, CA 94706. Other triazine antibodies are
commercially available.
o
SCH2C H2CN—Protein
1 H
H H
3-{{4-Ethylamino)-6-[(1-methylethyl)amino]-
113,5-triazin-2-yOthio}propanoic acid
Purchased Reagents:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
26 Revision 0
September 28, 1989
-------
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
2) 96 Well microtiter plates
a) High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b) Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; Sigma Catalog No. P-1379, or
equivalent)
5) p-Nitrophenyl phosphate substrate tablets (5 mg tablets, Sigma Catalog No. 104-105
or equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Karu, A. E., R. O. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow, A.
Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M., L. H. Stanker, B. E. Watkins, and D. W.
Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
27 Revision 0
September 28, 1989
-------
Lucas, A. D., P. Schneider, R. O. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and D.
E. Rolston. 1991. Determination of atrazine and simazine in water and soil using
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
Schneider, P. and B. D. Hammock. 1992. Influence of the ELISA format and the hapten-
enzyme conjugate on the sensitivity of an immunoassay for s-triazine herbicides using
monoclonal antibodies. J. Agric. Food Chem. 40:525-530.
28
Revision 0
September 28, 1989
-------
4.3 EL1SA METHOD FOR ANALYSIS OF CARBARYL
IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES
Introduction:
The general assay design is shown in Figure 7. This assay is a competitive enzyme
immunoassay which utilizes a carbaryl structural mimic covalently bound to a protein (termed
coating antigen) adsorbed to the microtiter plate surface. The sample containing carbaryl
competes with the carbaryl mimic on the coating antigen for a fixed amount of the anti-
carbaryl antibody. The amount of antibody bound is detected using a goat anti-rabbit IgG
antibody bound to alkaline phosphatase (termed second antibody). The analyst is referred to
Voller et al. (1976) for more details on this format. This assay has been optimized for the
detection of carbaryl. There is no cross reactivity with the major degradation product, 1 -
naphthol or naphthalene. Cross reactivity with other carbamate compounds is <5%. All
directions for the preparations of buffers and other solutions used in this tutorial method are
given in tutorial 5.7.
Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1. Coat the microtiter plate with the carbaryl
hapten conjugated to conalbumin (5-
CONA). (See Note 1.) Make a solution of
5-CONA that is 0.5 jig/mL in pH 9.6
carbonate buffer (coating buffer). Add 100
JJ.L to each well of a high binding ELISA
microtiter plate. See Notes 1 and 2.
2. Cover the microtiter plate with a plate
sealer and incubate overnight at 4°C. See
Note 3.
11.
5,6.
Enzyme
substrate
Rabbit
anti-H
antibody
Hapten-
protein
conjugate
Colored
product
Enzyme-labelled
anti-rabbit
antibody
H Competing
free
hapten
Well of polystyrene 96-well plate
Figure 7. Schematic of the antigen-coated plate
ELISA format. The lines represent wash steps.
The numbers correspond to the numbered steps
in the protocol.
3. Wash the coated microtiter plate 5X with
PBS-Tween/Azide and tap dry. The wash
procedure involves flooding each well with
buffer repeatedly to remove unbound
reagents. Then freeze or use immediately in ELISA step 5 below. This plate is termed
the "coated" plate. See Note 4.
Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
29
Revision 0
March 17, 1993
-------
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 50 p.L of standard or sample from the mixing plate to each well of the coated
plate.
6. Add 50 (J.L of Antibody #2114 diluted 1/40,000 in PBS-Tween/Azide to each well of the
coated plate, except those wells that serve as no-antibody blanks. (The final
concentration in the well is 1/80,000.) In the wells that serve as blanks, replace the
antibody with buffer. See Note 6.
7. Cover the coated plate with a plate sealer and incubate 1 hour at room temperature.
8. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
9. Prepare a solution of goat anti-rabbit IgG-alkaline phosphatase that is diluted 1/5000 in
PBS-Tween/Azide. Add 100 (xL of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
11. Add 100 jiL of substrate solution (1 mg/mL p-nitrophenylphosphate in 10%
diethanolamine buffer) to each well of the coated plate and cover with a plate sealer.
Incubate for 30 minutes at room temperature.
12. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is about 0.7-
0.8 in the wells containing antibody, but no carbaryl (zero analyte standard). The ICSO
(or midpoint of the calibration curve) for this assay is 2-5 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-100 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade carbaryl and dissolving in 2
mL of methanol. Methanol was chosen because it is water miscible, does not interfere in the
assay at the concentrations used and is not very volatile. The primary stock is diluted 1/100
in methanol to make a working stock. The working stock is diluted 1/1000 in PBS-
Tween/Azide to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of methanol in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water. For this protocol we prepare the stock solutions in methanol and stored
them at-20°C.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
30 Revision 0
Mtroh 17, 1993
-------
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the samples indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5).
A simple strategy to avoid interference is to take advantage of the assay sensitivity.
Many interferences can simply be "diluted away". Water, honey, milk and urine have been
tested for interferences following analysis of simple dilutions of these matrices. Water had no
effect on the assay performance. Honey had to be diluted 1/1000, milk 1/50,000 and urine
1/50 to eliminate influences on the standard curve. Soil was extracted with chloroform/
methanol. The solvent was evaporated and the residue taken up in methanol, then diluted
1/10 with PBS-Tween/Azide. This extract only needed to be diluted 1/25 to eliminate matrix
interferences. This assay can be used when samples contain up to 10% methanol. If other
solvents are used in sample preparation, their effects need to be tested (Marco et al., 1993).
Interference by a known media, such as 25% methanol can be corrected by running the
standard curve in the equivalent media. This may compromise the parameters of the
standard curve compared to running the standard curve in buffer, but the change is repro-
ducible. See tutorial 5.3 for approaches to evaluating matrix effects.
Notes:
1. The optimal amount of 5-CONA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (5-CONA) and
anti-carbaryl antibody (Rabbit #2114) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
2. We have tested other formats with these antibodies. Coating the plate with the
antibody (similar to tutorial 4.2) improved the sensitivity, however the maximum signal
to noise ratio was not as favorable as with the antigen coated plate format. In addition,
10X more antibody was required for the antibody-coated plate format.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. 1C50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
31 Revision 0
March 17, 1993
-------
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage. This antibody
can be used when samples contain up to 10% methanol. If other solvents are used in
sample preparation, their effects need to be tested. Matrix effects have been
demonstrated for soil, urine, honey and milk. Soil and urine have the least effect on
the assay performance, whereas milk has a significant effect even at dilutions of
1/25000.
6. The amount of anti-carbaryl antibody should be optimized in a checkerboard titration
where varying amounts of anti-carbaryl antibody and enzyme-labeled hapten are used
in the ELISA to optimize assay performance. Changes in assay performance may be
compensated for by reoptimizing reagents. See tutorial 5.5 for the checkerboard
titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Coating antigen. 5-CONA has the following structure and is conjugated to conalbumin.
A small aliquot of the stock may be stored in the refrigerator if used regularly. The
remainder should be stored frozen in small aliquots. Working dilutions should be made
up immediately before use and the extra discarded. Too many freeze-thaw cycles may
affect the integrity of the coating antigen. A change in the assay performance
parameters will be an indication of possible degradation of the coating antigen.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California,
Davis, CA95616.
32 Revision 0
March 17, 1993
-------
O H
N—(CH2)SCOOH
N-(2-Naphthoy1)6-aminohexanoic acid
2) Hapten specific antibody. Rabbit polyclonal antibody #2114 - final bleed directed
against the following hapten conjugated to keyhole limpet hemocyanin:
N-C-N-
(CH2)SCOOH
1 -(5-Carboxypentyl)-3-(1 -naphthyl)urea
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA 95616.
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
33 Revision 0
March 17, 1993
-------
1) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent) '
4) p-Nitrophenylphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent) ...... • -
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Marco, M. P., Gee, S. J., Cheng, H. M., Liang, Z. Y. and Hammock, B. D. 1993.
Development of an Enzyme Linked Immunosorbent Assay for Carbaryl. J. Agric. Food
Chem. 41:423-430.
Schneider, P. and Hammock, B. D. Influence of the ELISA Format and the Hapten-Enzyme
Conjugate on the Sensitivity of an Immunoassay for s-Triazine Herbicides Using
Monoclonal Antibodies. J. Agric. Food Chem. 40:525-530 (1992).
Voller, A., A. Bartlett, and D. E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
34
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March 17, 1993
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4.4 ELISA METHOD FOR THE ANALYSIS OF PARAQUAT
IN ENVIRONMENTAL SAMPLES
Introduction:
The general assay design is shown in Figure 8. This assay is a competitive enzyme
immunoassay which utilizes a paraquat structural mimic covalently bound to a protein (termed
coating antigen) adsorbed to the microtiter plate surface. The sample containing paraquat
competes with the paraquat mimic on the coating antigen for a fixed amount of the anti-
paraquat antibody. Quantitation of paraquat is determined by detecting the amount of anti-
paraquat antibody bound to the coating antigen. The bound antibody is detected using an
anti-rabbit IgG antibody labeled with alkaline phosphatase (termed second antibody). The
analyst is referred to Voller et al. (1976) for more details on this format. This assay has been
optimized for detection of paraquat. There is no cross reactivity with the major degradation
products, or other bipyridinium compounds such as diquat. All directions for the preparations
of buffers and other solutions used in this tutorial method are given in tutorial 5.7.
SPECIAL NOTE: Paraquat is known to bind to glass surfaces. Recovery studies indicate
that it does not bind to polystyrene (Van Emon et al., 1986). Avoid handling standards or
samples in glass containers. Polystyrene or polypropylene containers are highly
recommended.
Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1. Coat the microtiter plate with the
paraquat hapten PQ-C2 conjugated
to bovine serum albumin (PQ-C2-
BSA). (See Note 1.) Make a
solution of PQ-C2-BSA that is 1.25
M-g/mL in pH 9.6 carbonate buffer
(coating buffer). Add 100 (4.L to each
well of a high binding ELISA micro-
titer plate. See Note 1.
2. Cover the microtiter plate with a plate
sealer and incubate overnight at 4°C.
See Note 2.
11.
5,6.
Enzyme
substrate
Colored
product
Enzyme-labelled
anti-rabbit
antibody
Rabbit
antl-H
antibody
H Competing
free
hapten
Well of polystyrene 96-well plate
Figure 8. Schematic of the antigen-coated plate
ELISA format for paraquat. The lines represent wash
steps. The numbers correspond to the numbered
steps in the protocol.
Wash the coated microtiter plate 5X
with PBS-Tween/Azide and tap dry.
The wash procedure involves flood-
ing each well with buffer repeatedly
to remove unbound reagents. Then freeze or use immediately in ELISA step 5 below.
This is termed the "coated" plate. See Note 3.
35
Revision 0
March 17, 1993
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Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 4) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 50 (4.L of standard or sample from the mixing plate to each well of the coated
plate.
6. Add 50 |j.L of Antibody #21 diluted 1/5,000 in PBS-Tween/Azide to each well of the
coated plate, except those wells that serve as no-antibody blanks. (The final
concentration in the well is 1/10,000.) In the wells that serve as blanks, replace the
antibody with buffer. See Note 5.
7. Cover the coated plate with a plate sealer and incubate 30 minutes at room
temperature.
8. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
9. Prepare a solution of goat anti-rabbit IgG-alkaline phosphatase that is diluted 1/5000 in
PBS-Tween/Azide. Add 50 |xL of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
11. Add 100 pi- of substrate solution (1 mg/mL p-nitrophenyphosphate in 10%
diethanolamine buffer) to each well of the coated plate and cover with plate sealer.
Incubate for 30 minutes at room temperature.
12. Read at 405-650 nm. See Note 7. The absorbance obtained is between 0.4-0.5 in the
wells containing antibody, but no paraquat (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is 1 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-4 ng/mL. The primary stock
solution is prepared by weighing 20 mg of analytical grade paraquat and dissolving in 2 mL of
double distilled water in a polystyrene or polypropylene container and storing at room
temperature. Appropriate dilutions are made in double distilled water in order to prepare the
highest concentration to be tested.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
36 Revision 0
March 17, 1993
-------
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
As noted above, paraquat binds to glass. Do not use glass in sample preparation.
The amount of sample preparation needed will depend on the matrix. The first approach
would be to attempt to analyze the sample with little or no sample preparation. For example,
water samples may be buffered, then analyzed directly in the assay. For matrices which
require extraction, a simple sonication with 6N HCI has proven useful for food matrices (Van
Emon et al.,1987). The HCI is then evaporated to dryness. The residue is resuspended in
PBS-Tween/Azide, then analyzed. For lipid matrices, such as oils, extraction with water may
provide suitable recovery. The aqueous phase may then be analyzed directly in the
immunoassay. This assay has been used successfully to measure paraquat in serum and
lymph without any sample preparation. These antibodies have also been used to "extract"
paraquat from air filter samples (see Van Emon et al., 1986).
Notes:
1. The optimal amount of PQ-C2-BSA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (PQ-C2-BSA)
and anti-paraquat antibody (Rabbit #21) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
2. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
3. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
4. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
5. The amount of anti-paraquat antibody should be optimized in a checkerboard titration
where varying amounts of anti-paraquat antibody and coating antigen are used in the
ELISA to optimize assay performance. Changes in assay performance may be
compensated for by reoptimizing reagents. See tutorial 5.5 for the checkerboard
titration format.
37 Revision 0
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-------
6. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Coating antigen. PQ-C2-BSA has the following structure and is conjugated to bovine
serum albumin. A small aliquot of the stock may be stored in the refrigerator if used
regularly. The remainder should be stored frozen in small aliquots. Working dilutions
should be made up immediately before use and the extra discarded. Too many freeze
-thaw cycles may affect the integrity of the coating antigen. A change in the assay
performance parameters will be an indication of possible degradation of the coating
antigen. Provided by Dr. Bruce Hammock, Department of Entomology, University of
California, Davis, CA 95616.
2) Hapten specific antibody. Rabbit polyclonal antibody #21 - final bleed directed against
the following hapten conjugated to conalbumin:
N-(4-Caiboxyelh-1-yi>N'-methytt>ipyridilium
1} Coaling antigen.
^4-Carboxybut-1-y()-Nt-melhy(bipyridilium
2) Mapten specific antibody.
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA95616.
38
Revision 0
March 17, 1993
-------
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
1) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
4) p-Nitrophenyphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-weII microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
References:
Van Emon, J. M., J. N. Seiber and B. D. Hammock. 1985. Applications of immunoassay to
paraquat and other pesticides. In: Bioregulators for Pest Control, pp. 307-316 (P.A.
Hedin, ed.), American Chemical Society Symposium Series 276, Washington D. C.
Van Emon, J., B. Hammock, and J. N. Seiber. 1986. Enzyme-linked immunosorbent assay
for paraquat and its application to exposure analysis. Anal. Chem. 58:1866-1873.
39 Revision 0
March 17, 1993
-------
Van Emon, J., J. Seiber, and B. Hammock. 1987. Application of an enzyme-linked
immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and
potatoes. Bull. Environ. Contam. Toxicol. 39:490-497.
Voller, A., A. Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
40 Revision 0
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4.5 ELISA METHOD FOR THE ANALYSIS OF 4-NITROPHENOLS
IN ENVIRONMENTAL SAMPLES
Introduction:
The general assay design is shown in Figure 9. This assay is a competitive enzyme
immunoassay which utilizes a 4-nitrophenol structural mimic covalently bound to a protein
(termed coating antigen) adsorbed to the microtiter plate surface. The sample containing 4-
nitrophenol competes with the 4-nitrophenol mimic on the coating antigen for a fixed amount
of the anti-4-nitrophenol antibody. Quantitation of 4-nitrophenol is determined by detecting the
amount of anti-4-nitrophenol antibody bound to the coating antigen. The bound antibody is
detected using a goat anti-rabbit IgG antibody labeled to alkaline phosphatase (termed second
antibody). The analyst is referred to Voller et al. (1976) for more details on this format. This
assay has been optimized for detection of 4-nitrophenol and certain monosubstituted 4-
nitrophenols. There is very little cross reactivity (<2%) to substituted nitrobenzenes, 2- or 3-
nitrophenol, other substituted phenols and 4-nitropyridine-N-oxide. See Note 1 for an
indication of relative cross reactivity of nitrophenols. All directions for the preparations of
buffers and other solutions used in this tutorial method are given in tutorial 5.7.
SPECIAL NOTE: This assay is sensitive to changes in pH, as might be expected for
4-nitrophenol which may be ionized. Control of pH in antibody binding steps is critical to
precise assay performance.
Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1. Coat the microtiter plate with the
4-nitrophenol hapten conjugated
to ovalbumin (C-OVA). (See
Note 2.) Make a solution of C-
OVA that is 2.0 ^ig/mL in pH 9.6
carbonate buffer (coating buffer).
Add 100 |oL to each well of a
high binding ELISA microtiter
plate.
2. Cover the microtiter plate with a
plate sealer and incubate
overnight at 4°C. See Note 3.
3. Wash the coated microtiter plate
5X with PBS-Tween/Azide and
tap dry. The wash procedure
involves flooding each well with
11.
9.
5,6.
1.
Enzyme
substrate
Colored
product
Rabbit
anti-H
antibody
•i —
Hapten-
protein
conjugate
Enzyme-labelled
anti-rabbit
antibody
H Competing
free
hapten
Well of polystyrene 96-well plate
Figure 9. Schematic of the anitgen-coated plate ELISA
format. The lines represent wash steps. The numbers
correspond to the numbered steps in the protocol.
41
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March 17, 1993
-------
buffer repeatedly to remove unbound reagents. Then freeze or use immediately in
ELISA step 8 below. This plate is termed the "coated" plate. See Note 4.
Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 120 ILL of standard or sample to the wells of a mixing plate.
6. Add 120 jxL of Antibody #1812 diluted 1/1000 in PBS-Tween/Azide to each well of the
mixing plate, except those wells that serve as no-antibody blanks, (The final
concentration in the well is 1/2000.) In the wells that serve as Wanks, replace the
antibody with buffer., Se.e; Note 6.
7. Cover the mixing plate with a plate sealer and incubate overnight at room temperature.
8. Add 50 \iL from each well of; the mixing plate to the respective wells of the coated
plate. Cover the coated plate with a plate sealer and incubate at room temperature for
3 hours.
ST. Wasrr the coated plate^SX with PBSrTween/Azide., Tap; dry.
10. Prepare a solution of goat; anti-rabbit IgG-alkaline phosphatase that is diluted 1/2500 in
PBS-Tween/Azide. Add 50 |j,L of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room: temperature.
11. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry,
12. Add 100_ y.L of substrate solution (1 mg/mL p-nitrophenylphosphate> in 10%
diethanolamine buffer) tcreach weir of the coated plate and cover with plate sealer.
Incubate for 30 minutes at room temperature.
13. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is between
0.4-0.5 in the wells containing antibody, but no 4-nitrophenol (zero analyte standard).
The IC50 (or midpoint of the calibration curve) of this assay is 8-10 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-2000 ng/mL. 4-NitrophenoI is
highly water soluble. Standard solutions can be prepared in distilled water and stored: at room
temperature. Methanol is an alternative solvent. Solutions may be kept in a sealed container
and stored in a refrigerator. The primary stock solution is prepared by weighing 20 mg of
42 Revision 0
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-------
analytical grade 4-nitrophenol and dissolving in 2 ml_ of double distilled water. This primary
stock is diluted appropriately in double distilled water to give the highest concentration to be
tested. Stocks that have been alternatively prepared in methanol, should also be diluted in
double distilled water. This assures that the concentration of methanol in the assay is quite
low. If other solvents are used to make the stock solution, care should be taken to insure that
this solvent does not interfere in the assay and that the solvent is miscible with water.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the samples indicate that there is no matrix effect,
then the sample is buffered, and placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5). This assay has also
been used to analyze soil containing parathion. Parathion, extracted from soil using
supercritical fluid extraction was analyzed as 4-nitrophenol by ELISA after oxidation to
paraoxon using dimethyldioxirane followed by hydrolysis (Wong et al., 1991). This is a good
example of extraction and derivatization techniques which demonstrate the general principle in
immunoassay of using volatile extraction solvents and derivatizing agents to minimize
interferences with the subsequent immunoassay. This assay will tolerate up to 25% methanol
or 5% ethyl acetate. Several other solvents were tested at 5% (ethanol, acetonitrile, and
dimethyl formamide) and showed no effect on the calibration curve. Thus samples can be
prepared and analyzed in these solvents, if the concentration remains below 5%. Another
simple strategy to avoid interference is to take advantage of the assay sensitivity. Many
interferences can simply be "diluted away".
Notes:
1. Relative Cross Reactivity for Rabbit #1812
Compound Percent Cross-Reactivity
4-Nitrophenol 100
2-Chloro-4-nitrophenol 190
2-Amino-4-nitrophenol 104
3-Methyl-4-nitrophenol 92
2,4-Dinitrophenol 48
43 Revision 0
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2. The optimal amount of C-OVA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (C-OVA) and
anti-4-nitrophenol antibody (Rabbit #1812) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
6. The amount of anti-4-nitrophenol antibody should be optimized in a checkerboard
titration where varying amounts of anti-4-nitrophenol. antibody and coating antigen are
used in the ELISA to optimize assay performance. Changes in assay performance
may be compensated for by reoptimizing reagents. See tutorial 5.5 for the
checkerboard titration format,
7. Absorbance variability is decreased by shaking the plate before, reading to mix the
contents of the microtiter plate wells. Reading in two wavelengths can eliminate
absorbance discrepancies due to; flaws> in the plate.
Materials:
Specialized Reagents::
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available, commercially.,
1) Coating antigen. C-OVA has the following structure and is conjugated to ovalbumin. A
small aliquot or the stock may be stored in the refrigerator if used regularly. The
remainder should be stored frozen in small aliquots. Working dilutions should be made
up immediately before use and the extra discarded. Too many freeze-thaw cycles may
affect the integrity of the coating antigen. A change in the assay performance
44 Revision 0
March 17, 1993
-------
parameters will be an indication of possible degradation of the coating antigen.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California,
Davis, CA 95616.
2) Hapten specific antibody. Rabbit polyclonal antibody #1812 - final bleed directed
against the following haptep conjugated to keyhole limpet hemocyanin at the 2-
position:
NO2
4-Nitrophenyt acetic acid
1) Coating antigen. C-OVA
2-Hydroxy-5-rttrobenzyl bromide
2) Haplen specific antibody.
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA95616.
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may yield satisfactory results, however the authors have
not evaluated the performance of these alternative materials.
1) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc immunoplate II (Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
45
Revision 0
March 17, 1993
-------
2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equfvalent)
4) p-Nitrophenylphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the anafyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
46 Revision 0
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References:
Li, Q.X., Zhao, M.S., Gee, S.J., Kurth, M.J., Seiber, J.N. and Hammock, B.D. 1991.
Development of enzyme-linked immunosorbent assays for 4-nitrophenol and
substituted 4-nitrophenols. J. Agric. Food Chem. 39:1685-1692.
Voller, A., A. Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
Wong, J.M., Li, Q.X., Hammock, B.D. and Seiber, J.N. 1991. Method for the analysis of 4-
nitrophenol and parathion in soil using supercritical fluid extraction and immunoassay.
J. Agric. Food Chem. 39:1802-1807.
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4.6 ELISA METHOD FOR THE ANALYSIS OF
TRIAZINE MERCAPTURATES IN URINE
Introduction:
The general assay design is shown in Figure 10. This assay is a competitive enzyme
fmmunoassay which utilizes a capture or trapping antibody for the first coating and which
binds the triazine mercapturate-specific antibody in a second coating step. A hapten-enzyme
conjugate is used as the label. This assay has been optimized for detection of atrazine
mercapturate. Due to the structural similarity of triazines as a class, some cross reactivity
with other triazines occurs. See Note 1. All directions for the preparations of buffers and
other solutions used in this tutorial method are given in tutorial 5.7.
Assay Protocol:
Coating the Microtiter Plate with
Trapping Antibody.
1.
4.
Coat the microtiter plate with
the trapping antibody. Make a
solution of goat anti-mouse
antibody that is diluted 1/2000
in pH 9.6 carbonate buffer
(coating buffer). Add 100 |j,L to
each well of a high binding
ELISA microtiter plate. See
Note 2.
Cover the microtiter plate with
a plate sealer and incubate
overnight at 4°C. See Note 3.
Wash the coated microtiter
plate 5X with PBS-Tween/Azide
and tap dry. The wash
procedure involves flooding
each well with buffer repeatedfy
to remove unbound reagents.
ID.
7.8),
D.
Enzyme
substrate
Colored
product
Antl-
hapten
antibody
Anti-
mouse
antibody
Figure 10. Schematic of a double antibody-coated ELISA.
Line indicates a wash step. Numbers refer to steps in
protocol.
Perform the second coating step. Make a solution of anti-triazine antibody (AM7B2.1)
that is diluted 1/3200 in pH 9.6 carbonate buffer (coating buffer). Add 100 pL to each
well of the mfcrotiter pfate which has previously been coated with goat anti-mouse IgG
antibody. Cover the microtiter plate with a plate seafer and incubate overnight at 4°C
overnight.
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5. Wash the double antibody-coated microtiter plate 5X with PBS-Tween/Azide and tap
dry. Then freeze or use immediately in EL1SA step 7 below. This microtiter plate is
termed the "coated" plate. See Note 4.
Competitive Inhibition Steps.
6. Prepare the standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
7. Add 50 JJ.L of standard or sample from the mixing plate to each well of the coated plate.
8. Add 50 |o,L/well of SIM-N(C2)-AP (hapten-labeled enzyme conjugate) that has been
diluted 1/10000 in PBS-Tween/Azide to each well of the coated plate, except those
wells that serve as blanks. In the wells that serve as blanks, replace the hapten-
labeled enzyme conjugate with buffer. See Note 6.
9. Cover the coated plate containing standards, samples and enzyme label with a plate
sealer and incubate 30 minutes at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
11. Add 100 H.L of 1 mg/mL substrate solution (freshly made, one 5 mg tablet per 5 ml_
10% diethanolamine substrate buffer) to each well of the coated plate and cover with
plate sealer. Incubate at room temperature for 15-30 minutes.
12. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is about 0.3-
0.4 in the wells containing antibody, but no atrazine mercapturate (zero analyte
standard). The IC50 (or midpoint of the calibration curve) for this assay is 1 ng/mL
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-200 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine mercapturate and
dissolving in 2 mL of dimethylsulfoxide (DMSO). DMSO was chosen because it is water
miscible, does not interfere in the assay at the concentrations used and is not volatile. The
primary stock is diluted 1/100 in DMSO to make a working stock. The working stock is diluted
1/100 in PBST to make the highest concentration to be tested. This assures that a
reasonable amount of analytical standard is weighed and ultimately, the concentration of
DMSO in the assay is quite low. If other solvents are used to make the stock solution, care
should be taken to insure that this solvent does not interfere in the assay and that the solvent
is miscible with water.
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Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation.
Another approach to avoid intereference would be to take advantage of the assay sensitivity.
Many interferences can simply be "diluted away." In this tutorial we found urine samples vary
widely in composition. For example some may be more dilute, more concentrated with salts
or protein, etc., more or less colored, and may depend on diet, etc. We have found for
measuring atrazine mercapturate in urine, a dilution of the urine in PBS-Tween/Azide to 25%
is adequate to remove interferences in the samples we tested. In the event that the matrix
effects cannot be diluted out, to quantitate the sample, a solid phase extraction using a phenyl
column may be used. (See tutorial 5.2.2). See tutorial 5.3 for approaches to evaluating
matrix effects.
Notes:
Percent Cross Reactivity of AM7B2.1 (Atrazine mercapturate = 100%)
Simazine
Atrazine
Prometon
Hydroxyatrazine
9 Hydroxysimazine <0.1
30 Both mono-N-dealkylated <0.1
2 N,N'-di-dealkylated <0.1
2 Cyanazine 32
3.
4.
5.
The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
We have found that these double antibody-coated plates can be stored frozen for more
than one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight, before use or
freezing, results in an increase in well to well variability.
CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in urine samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
50
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6.
7.
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in
a checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (SIM-N(C2)-AP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. SIM-N(C2)-AP has the following structure and is
conjugated to alkaline phosphatase. It should be stored in the refrigerator. Run periodic tests
of enzyme activity to assure no loss
of activity on storage of the working
solution. Working dilutions should be
made up immediately before use and
the extra discarded. DO NOT
FREEZE - each freeze-thaw cycle
will kill a significant part of the
conjugate-enzyme activity. A change
in the assay performance parameters
will be an indication of possible
degradation of the coating antigen.
Provided by Dr. Bruce Hammock,
Department of Entomology,
University of California, Davis, CA
95616.
Cl
CH3CH2
A.
N
O
NCH2CH2CN—AP
H H
N-[4-Chloro-6-(ethylamino)-
1,3,5-triazin-2-y1]-p-alanine
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
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o
SCH2CH2CN-Protein
(CH3)2—
T if
'^v
H
H
3-{{4-Ettrylamino)-6-[(1-melhytethyl)aminol-
1,3,5-triazin-2-y)}thio}propanoic acid
Antibodies should be stored
frozen in small aliquots to minimize
freeze-thaw cycles. This antibody
was provided by Dr. Alex Karu,
Hybridoma Center, University of
California at Berkeley, 1050 San
Pablo Avenue, Albany, CA 94706.
Purchased Reagents:
The following materials are
listed for the convenience of the
reader. Similar products are available
from other vendors and may yield
satisfactory results, however the authors have not evaluated the performance of these
alternative materials.
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
2) Microtiter plates
a) High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b) Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; Sigma Catalog No. P-1379, or
equivalent)
5) p-Nitrophenyl phosphate substrate tablets (5 mg tablets, Sigma Catalog No. 104-105 or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
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items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Karu, A. E., R. O. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow, A.
Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M., L. H. Stanker, B. E. Watkins, and D. W.
Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
Lucas, A. D., A. D. Jones, M. H. Goodrow, S. G. Saiz, C. Blewett, J. N. Seiber, and B. D.
Hammock. 1993. Determination of atrazine metabolites in human urine: Development
of a biomarker of exposure. Chem. Res. Toxicol. 6:107-116.
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SECTION 5
TUTORIALS FOR SUPPORT TECHNIQUES
The following tutorials describe techniques that the analyst will likely use while
conducting immunoassays. Some techniques will be familiar as these are common to
analytical chemistry. Other techniques will be new to the analyst.
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5.1 Pipetting Techniques
The following techniques generally apply to all positive air displacement pipettes.
General:
Never set the volume selector to volumes above or below the specified range for the
pipettor, or it will require recalibration. Always operate the plunger button slowly and smoothly
at all times. Never let the plunger button snap back. Ensure that clean pipette tips are firmly
pushed onto the tip cones of the pipette and that there are no foreign bodies inside the tips.
Wet the newly attached pipette tips with the solution being pipetted before any actual pipetting
takes place. This is done by filling and emptying the pipette tips 2-3 times. Hold the pipette
vertically during liquid intake. Pipettors should be stored in an upright position.
Pipetting Techniques:
Forward technique: (See Figure 11.)
1)
2)
3)
4)
Depress the operating button
to the first stop.
Dip the tips just under .the
surface of the liquid in the
reservoir and slowly release
the operating button. This
action will fill the tips.
Withdraw the tips from the
liquid, touching them against
the edge of the reservoir to
remove excess liquid.
Deliver the liquid by gently
depressing the operating
button to the first stop. After a
delay of about a second,
continue to depress the
operating button all the way
down to the second stop. The
action will empty the tips.
Forward Technique
fill dispense
II I
Ready position
First stop
Second stop
Repetitive Technique
dispense refill
I
set
volume
dispensed
Figure 11. Representation of the forward and repetitive
pipetting techniques.
Release the operating button to the ready position. If necessary, change the tips and
continue with the pipetting.
55
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Repetitive Technique: (See Figure 11.)
This technique is suitable for dispensing liquids having a high viscosity or which have a
tendency to foam easily. The technique is also recommended for dispensing very small
volumes and is the method of choice for the authors' laboratories.
1) Depress the operating button all the way down to the second stop.
2) Dip the tips just under the surface of the liquid in the reservoir and slowly release the
operating button. This action will fill the tips. Withdraw the tips from the liquid,
touching them against the edge of the reservoir to remove excess liquid.
3) Deliver the preset volume by gently depressing the operating button to the first stop.
Hold the operating button at the first stop. Some liquid will remain in the tip and
should not be included in the delivery. (For a multichannel pipettor, a quick visual
scan of the remaining liquid will show you whether the channels are delivering the
same volume as the menisci should all be lined up evenly.)
4) Dip the tips just under the surface of the liquid in the reservoir and slowly release the
operating button. This action will refill the tips. Continue pipetting by repeating the last
two steps. The remaining liquid is either discarded with the tips or pipetted back into
the container.
Maintenance/Calibration
Follow all maintenance and calibration procedures as outlined by the manufacturer.
(See tutorial 5.13. Performance checks, calibration and maintenance of air displacement
pipettors.)
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5.2 CONSIDERATIONS IN SAMPLING AND SAMPLE PREPARATION
FOR IMMUNOASSAY ANALYSIS
Sampling Considerations for Immunoassay Methods:
It is important to recognize that immunoassays require a much smaller sample size.
Advantages to this are that more samples may be taken for analysis in the field as less room
for storage is necessary and shipping costs are reduced for smaller sampling containers.
However, smaller sample sizes present unique problems for sampling. For example, it is
important to assure that the original sample is homogeneous prior to subsampling. For more
discussion on sampling and subsampling see van Ee et al. (1990) and EPA (1992).
Importance of the Analvte:
"Every type of material that is to be prepared for analysis presents its own practical
difficulties. The requirements for suitable sample preparation are dictated by the consistency
and the chemical characteristics of the analyte and the matrix, and by the distribution of the
analyte in the sample."
This statement by Garfield (1991) is applicable to any analytical technique, including
immunoassay. When forming an approach to sample preparation, the analyst in general is
familiar with the chemical properties of the analyte in question. Thus, the volatility, polarity,
relative stability to acid, base, heat, etc. are known. The general approach is to use as little
sample preparation as possible to get the analyte into a form suitable for analysis by a chosen
method. In the case of immunoassay, the analyte needs to basically be in a water miscible
media.
Begin by using the most simple approach. For example, if the matrix is aqueous, try
analyzing the sample directly. If the matrix is causing some interference, try checking the pH
and adjusting to neutrality. Next try diluting the matrix effect. If analyte signal is lost, then a
sample concentration/purification step will be necessary. Standard methods for sample
preparation of the compound are a good starting place for the design of your sample
preparation for immunoassay. The following guidelines should be considered in developing
approaches to sample preparation.
1). Immunoassays are conducted in aqueous media, thus samples should be prepared
with this in mind. Aqueous solubility of a lipophilic compound may not be a problem in
the concentration ranges being tested.
2). Whenever possible make use of the sensitivity of the assay. Often times matrix
interferences can be eliminated by diluting the sample.
3). Use water miscible organic solvents whenever possible. Methanol and acetonitrile
seem to be the most desirable.
4). Use as few steps as possible. Often times the immunoassay analysis method is
quicker simply because fewer sample preparation steps are necessary.
57
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5). Exploit the chemical properties of the compound in order to design simple partition
methods.
6). Explore the volatility of the compound. Can it first be extracted into organic solvent,
followed by solvent evaporation without loss of analyte? Can the residue be taken up
in a water miscible solvent. Even for relatively volatile compounds, a trapping solvent
may be used. For example the compound could be extracted in ethyl acetate with a
small amount of propylene glycol added. After evaporation of the ethyl acetate, the
analyte now trapped in propylene glycol, is brought to a reasonable volume with water
or a water miscible solvent.
7). Use simple solid phase extraction methods. A wide variety of bonded phases are now
available in small columns (including reverse phase, normal phase and ion exchange).
Two example solid phase extraction methods are given in tutorials 5.2.1 and 5.2.2.
8). Consider supercritical fluid extraction (SFE) for solid samples. Samples are extracted
and concentrated in one step. The extraction media is easily removed and water
miscible trapping solvent can be used. (See Wong et al., 1991 for an example).
9). Most immunoassays can tolerate methanol or acetonitrile up to 10%. Many assays
can tolerate more, however, assay performance parameters may be altered. If you
find that you need to use more solvent to get adequate cleanup of the sample, the
assays can be run using this concentration of solvent in the calibration curve. This will
normalize for the effects of the solvent. In most cases this will mean a decrease in the
signal/noise ratio. There may also be a shift in 1C50.
10). Consider using the immunoassay as a supplemental method. For example, as a
downstream detector for an HPLC, as described in a review by de Frutos & Regnier
(1993).
Importance of the Matrix:
Knowing as much as possible about the matrix with which you are working is always a
valuable asset when analyzing the sample. For soils, humic acid is known to effect
immunoassays. In addition there are some 2000 soil types in the United States alone, thus
optimization on each soil type analyzed would be important. In water samples, pH, the
presence of metals, ions or bacteria may have an effect on the assay. Optimization of the
assay for the water sample, or preparation of the sample by simple procedures such as
buffering, filtering, addition of chelate, etc. may help. With urine, variability in matrix effects
may occur due to diet, amount of liquid consumed, health, etc. Since every matrix may have
an effect, it is critical to include a procedure for the evaluation of potential matrix effects. (See
Tutorial 5.3. Approaches to Testing for Matrix Effects.)
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References:
de Frutos, M. and Regnier, F.E. 1993. Tandem chromatographic-immunological analysis.
Anal. Chem. 65:17A-25A.
EPA. 1992. Characterizing Heterogeneous Wastes: Methods and Recommendations. EPA
Report, EPA/600/R-92/033, February, 1992.
Garfield, F.M. 1991. Quality Assurance Principles for Analytical Laboratories. Association of
Official Analytical Chemists, Arlington, VA. pp 70.
van Ee, J.J. Blume, LJ. and Starks, T.H. 1990. A Rationale for the Assessment of Errors in
the Sampling of Soils. EPA Report, EPA/600/4-90/013.
Wong, J.M., Li. Q.X., Hammock, B.D. and Seiber, J.N. 1991. Method for the analysis of 4-
nitrophenol and parathion in soil using supercritical fluid extraction and immunoassay.
J. Agric. Food Chem. 39:1802-1807.
59
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5.2.1 SOLID PHASE EXTRACTION (SPE) OF
S-TRIAZINE HERBICIDES FROM WATER
Using this extraction system, recovery of atrazine from water was >98% for the
concentration range 0.1 ng/L to 1 mg/L using a 75 mL sample size. At <0.1 ng/L, the signal
was not distinguishable from background. At 10 mg/L, the recovery dropped significantly, likely
due to the lack of solubility of atrazine in the ELISA system. Although atrazine is reportedly
soluble to 33 mg/L in water, the presence of salts in the ELISA assays buffers significantly
decreased the solubility of atrazine. A white precipitate could be seen when attempting to
resolubilize the residue from the ethyl acetate evaporation in assay buffer. Adding about 10%
methanol helped the solubilization, and the effects of methanol could be diluted out, as this
concentration of atrazine is 1000 times larger than the IC50 of the assay.
Equipment and Supplies:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
vacuum manifold
C18 cartridges, 2.8 mL, 500 mg (i.e. Analytichem or equivalent)
reservoirs and adapters
pesticide grade hexane, ethyl acetate, acetone, methanol
double distilled water
Procedure:
1. Preclean all glassware and plasticware with acetone (except C18 cartridges).
2. Set manifold flow rate at 5 to 10 mL/min.
3. Place cartridge in manifold.
4. Wash the cartridge with the following, in order:
2 column volumes of hexane
2 column volumes of ethyl acetate
2 column volumes of methanol
2 column volumes of water
5. Attach adapter and add sample (1 liter maximum).
6. Wash cartridge with 1 column volume of water.
7. Air dry under vacuum for 5 to 15 minutes.
8. Elute cartridge with 2 mL ethyl acetate.
9. Evaporate eluate to dryness under nitrogen stream.
10. Reconstitute with PBS-Tween/Azide buffer, assay or store.
(See tutorial 5.7 for preparation of PBS-Tween/Azide buffer.)
Reference:
Lucas, A. D., P. Schneider, R. O. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and
D. E. Rolston. 1991. Determination of atrazine and simazine in water and soil using
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
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5.2.2 SOLID PHASE EXTRACTION (SPE) OF
ATRAZINE MERCAPTURATE FROM URINE
Using this phenyl SPE system, 99% of atrazine, 70% of either of the mono
N-dealkylated products, 50% of hydroxyatrazine, and no measurable didealkylated atrazine
were retained using this SPE system. This immunoassay does cross react with atrazine,
however in urine, the mercapturate accounts for the majority of the immunoreactivity and
parent atrazine is found at levels 500-1000 times less. If, however, the presence of parent
atrazine is of concern, it may be eliminated by partitioning the urine with chloroform (1:1) and
then analyzing the aqueous phase (or subjecting it to the phenyl column for further cleanup)
for the mercapturate.
Equipment and Supplies:
The following materials are listed for the convenience of the reader. Similar products are
available from other vendors and may yield satisfactory results, however the authors have not
evaluated the performance of these alternative materials.
vacuum manifold
phenyl cartridges, 2.8 ml_, 500 mg (i.e. Analytichem or equivalent)
reservoirs and adapters
pesticide grade acetone
double distilled water
hydrochloric acid
Procedure:
1. Preclean all glassware and plasticware with acetone (except phenyl cartridges).
2. Set manifold flow rate at 5 to 10 mL/min.
3. Place cartridge in manifold.
4. Wash the cartridge with the following, in order:
2 column volumes of acetone
2 column volumes of acidified water (pH 2.5-3)
5. Attach adapter and add sample (10 ml, acidified to pH 2.5-3).
6. Wash cartridge with 1 column volume of acidified water.
7. Air dry under vacuum for 5 to 15 minutes.
8. Elute cartridge with 2 ml_ acetone.
9. Evaporate eluate to dryness under nitrogen stream.
10. Reconstitute with PBS-Tween/Azide buffer, analyze or store. (See tutorial 5.7 for the
preparation of PBS-Tween/Azide buffer.)
Reference:
Lucas, A. D., Jones, A.D., Goodrow, M.H., Saiz, S.G., Blewett, C., Seiber, J.N. and Hammock,
B.D. 1993. Determination of atrazine metabolites in human urine: development of a
biomarker of exposure. Chem. Res. Toxicol. 6:107-116.
Revision 0
61 March 23, 1992
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5.3 APPROACHES TO TESTING FOR MATRIX EFFECTS
There is potential for any sample or components within the sample to interfere with
proper quantitatiori of the analyte of interest with any analytical method. When presented with
a new matrix, the analyst can save a lot of time if a few key experiments are run first.
1). Ideally the analyst will have available a "blank" sample matrix that is identical to the
matrix within which the analyte of interest will be measured. The blank and the sample
are prepared identically. Prepare the standard calibration curve in this matrix and
determine if the parameters are significantly different from the standard curve in buffer.
If the curve is not different, then it can be assumed that all samples can be run in this
manner. If the curve is different, then try repeating the experiment with several
dilutions of the matrix to determine if the effect can be diluted out. As long as the
dilution is considered reasonable, such that the limit of detection in the sample is still
acceptable, than this dilution can be applied to all unknowns to be analyzed, if the
matrix cannot be diluted out appropriately, then a cleanup will be necessary. After
employing the cleanup method, the extract should be tested as above.
2). If an appropriate "blank" sample is not available, a sample can be used, preferably one
in which the analyte is present in very low levels. The method of standard additions is
then recommended for evaluating the effect of matrix (Miller & Miller, 1984). Briefly, in
this method the sample is split and one split is fortified with a known concentration of
anaiyte. If the concentration determined for the unknown in one split is subtracted
from the concentration determined in the split that was fortified, then the result should
be the level of the fortification. If it is not, then a matrix effect may be assumed, and a
cleanup may be necessary.
3). An alternative to cleanup in each of the above cases, would be to run the standard
curve in the matrix of interest, so that the influence on quantitation would be
normalized. For example, if methanol is needed at 30% in order to solubilize the
compound, the standard curve should be run in the presence of 30% methanol to
normalize for its effects.
4). Another way to verify the integrity of the data and determine if there is a matrix effect
is to analyze the sample at several dilutions. The dilutions chosen should result in
absorbances that lie on the linear portion of the standard curve. A plot of the
absorbance obtained from each dilution should be parallel to the slope of the
calibration curve in the absence of matrix effects (Figure 12).
62
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0.8
-------
5.4 DATA ANALYSIS GUIDELINES
The data resulting from the protocols given in this manual are in the form of absorbance
values. Each sample is assayed at several dilutions. There are usually four well replicates of
a given dilution of a sample. Each concentration of standard is also assayed in four well
replicates. The following is one way to systematically evaluate the data.
Calibration curve and duality control samples:
1). Is the variation in absorbance among well replicates acceptable? Check the mean and
coefficient of variation, if the coefficient of variation is above an established value,
evaluate each well absorbance. Record outliers elsewhere.
2). Are the parameters of the standard curve and the shape of the curve acceptable?
3). Do the parameters of the quality control standards fall within acceptable standards?
Samples (unknowns):
1). Are the variation in absorbance among well replicates for a sample acceptable?
Check the mean and coefficient of variation, if the coefficient of variation is above an
established value, evaluate each well absorbance. Record outliers. The coefficient of
variation may be larger for samples than standards.
2). Do the dilutions of the sample show less inhibition with increasing amount of dilution?
3). Are the absorbances within the linear portion of the standard curve?
4). If the absorbance values fall near the upper (lower limit of detectability) or lower
(complete or near complete inhibition) asymptotes of the 4-parameter fit, there will be
more variation in the calculated values. The closer the absorbance is to the
absorbance of the concentration at the IC50, the more reliable the resulting value.
Assay performance parameters:
The precision, accuracy and sensitivity should be described in the protocol that
accompanies the method.
Outliers are often a result of pipetting errors or poor plate performance. There are a
number of methods for assessing outliers that can be found in any statistical manual. For
example Dixon's Q test (Miller & Miller, 1984). As a first pass, rapid assessment, a typical
rule would be to identify absorbances which lie outside two standard deviations of the mean.
In dealing with a new assay or matrix, it will be important to verify a portion of positives
and negatives by a second independent method, until the analyst has full confidence in the
method.
False positives - more likely because any perturbation of the system usually results in
decreased signal, resulting in a false positive.
64
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False negatives - rare if the characteristics of the method are well known, i.e. ICSO and
lower limit of detectability. Realize that these will change from matrix to matrix as it does with
any analytical technique.
Limit of detection - The limit of the detection for the assay may vary from matrix to
matrix. A detection range should be given in the protocol.
When analyzing a sample near the detection limit, one may find that the final
concentration value does not agree from dilution to dilution. As the dilution factor increases,
the apparent concentration of analyte increases. This is likely an affect of the dilution factor
multiplication. It is important to look at the absorbances and assure that they are in the linear
portion of the standard curve.
Notes on the 4-parameter curve fitting model:
As with any analytical technique, the generation of a reproducible standard curve with
minimal error is critical. The standard curves generally resulting from immunoassays are
sigmoidal in shape. If the choice of standards provides a complete definition of the shape of
the curve, (i.e., the curve has at least 2 to 3 points each defining the upper and lower
asymptote and at least 4 points defining the linear region), the 4-parameter fit of Rodbard
(1981) is the method of choice for data analysis in the authors' laboratories. It is important
that enough standard concentrations are used to ensure that the curve is well defined and
constant for these concentrations. Without this information, the computer cduld force an
improper fit (Gerlach et al., 1993). The equation for the 4-parameter fit is:
y = (A-D)/(1 + (x/C)AB) + D
where y is the absorbance, x is the concentration of analyte, A and D are the upper and lower
asymptotes respectively, B is the slope and C is the central point of the linear portion of the
curve, also known as the ICSO (Figure 13).
cr
w
o
5-
0)
o
(D -
Log Concentration
Figure 13. Model 4-parameter calibration curve.
65
-------
The best quantitation of unknowns is carried out when unknown absorbances fall in the
central portion of the linear region of the calibration curve. The use of the 4-parameter fit
extends the usefulness of the upper and lower concentrations of the calibration curve.
However, the values calculated from these upper and lower concentrations have greater error
associated with them. To save on reagents, and to keep the error on the estimation of
concentrations of unknowns to a minimum, concentrations for standard curves should be
performed in the linear range after the complete standard curve has been defined with upper
and lower asymptotes. A semi-log curve fit should then be used to fit the data to this
truncated calibration curve and the absorbance values for unknowns should fall in the central
portion of the linear region of this calibration curve. If a kit is being used, the package insert
should indicate the standard curve analysis method to use based on the range of standard
concentrations used for the calibration curve.
66
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5.5 OPTIMIZATION OF REAGENT CONCENTRATION
BY CHECKERBOARD TITRATION
The titer of the reagent refers to the dilution required to give a reasonable signal (i.e.
0.5-1.0 absorbance units) in your assay system. This procedure is recommended when
evaluating "component reagents" that have not specifically been designed to be delivered to
the user in a kit format. The specific reagent to titer will depend on the assay format. In
general, when you receive reagents for evaluation, a suggested dilution or concentration will
be given for each reagent. Due to the potential for degradation during shipping or storage, it
is usually recommended that a checkerboard titration experiment be performed to assure that
the reagent concentrations suggested are adequate. The checkerboard titration described
here tests two reagents simultaneously. Checkerboard titrations are also therefore called two
dimensional titrations. The steps in the checkerboard titration are performed identically to that
of the immunoassay tutorial for the specific compound, except that no inhibitor is used. For
the checkerboard titration the reagents are added at varying concentrations.
For the antigen-coated plate format such as tutorial 4.3 or 4.4, the reagents to titer
would be the coating antigen and the anti-analyte antibody. One could also titer the "second
antibody". This usually is unnecessary as the amount used is in excess. However, to save
on reagent, one could titer the "second antibody" to reduce the amount to the minimum
needed to assure proper assay performance.
For the antibody-coated plate formats such as tutorials 4.1 and 4.2, the reagents to
titer would be the anti-analyte antibody and the hapten-enzyme tracer. One could also titer
the "trapping antibody". This is not usually necessary as the amount used is in excess.
However, to save on reagent, one could titer the "trapping antibody" to reduce the amount to
the minimum needed to assure proper assay performance.
A
B
C
D
E
F
G
H
12345676 8 10 11 12
20 u
10 u
5ug
2.5 1
1.25
0.62
0.31
Oug
1/mL
1/mL
nl_
g/mL
ug/mL
>ug/ml
ug/ml
mL
Figure 14. Example protocol for titer determination: coating antigen (antigen-coated plate format) or
anti-analyte antibody (antibody-coated plate format). Each concentration is added to the 12 wells in the
row.
67
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Procedure:
Antigen-coated plate format:
1.
Coat the plate. Each well of the microtiter plate has added to it a fixed amount of
coating antigen. To test the range of the titer, the coating antigen is applied to the
plate at several different concentrations. (See Figure 14). The reagent dilutions are
made in coating buffer and added to each well of the microtiter plate as specified in the
specific assay procedure. Cover the microtiter plate with a plate sealer and place in
the refrigerator overnight.
Prepare the anti-analyte antibody. Anti-analyte antibody will also be added to the plate
at several concentrations (Figure 15). Make a dilution of anti-analyte antibody in PBS-
Tween/Azide. These solutions may be prepared the day of the titration or the night
before and left at room temperature while the coated plates are incubating in the
refrigerator.
A
B
C
D
E
F
G
H
12345678 9 10 11 12
0
1
2
I
\
1
-*
1
r*
1
i
?
%
\
§
\
s
*
\
i
j
i
8
i
\
&
-
i
•
§"*
i
Figure 15. Example protocol for titer determination: anti-analyte antibody (antigen-coated plate format)
or enzyme labelled hapten (antibody-coated plate format). Each dilution is added to the 8 wells in the
column.
3. On the following day, remove the microtiter plate from the refrigerator. Wash the
microtiter plate 3-5 times with PBS-Tween/Azide and tap dry. This is termed the
"coated" plate. The wash procedure involves flooding each well with buffer repeatedly
to remove unbound reagents.
4. Add 50 nL of the appropriate dilution of anti-analyte antibody solution to each well of
the coated microtiter plate as shown in Figure 15. Cover the microtiter plate with a
plate sealer.
5. Incubate for 1-2 hr. at room temperature. (Use the same incubation time as used in
the tutorial for the specific method.)
68
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6. At the end of the incubation, remove plate sealer and wash the coated plate 3-5 times
with PBS-Tween/Azide. Dry thoroughly by tapping on a paper towel.
7. Add 50 |4,L of goat anti-rabbit IgG conjugated to alkaline phosphatase to each well of
the coated plate. This solution is prepared by diluting goat anti-rabbit IgG conjugated
to alkaline phosphatase with PBS-Tween/Azide to 1/2500 or 1/5000 as recommended
by the supplier. (Use the same concentration recommended in the specific procedure.)
Note: If the source of the anti-analyte antibody is a mouse, use goat anti-mouse IgG
conjugated to alkaline phosphatase.
8. Cover the microtiter plate with a plate sealer and incubate for 1 -2 hr at room
temperature. (Use the same incubation time as used in the tutorial for the specific
method.)
9. Remove an aliquot of the substrate buffer, 10% diethanolamine, pH 9.8 from the
refrigerator to allow equilibration to room temperature. Do not add the p-nitrophenyl
phosphate until just before use in step 11. Protect from light prior to use.
10. After the incubation, remove the plate sealer and wash the coated plate 3-5 times with
PBS-Tween/Azide. Dry thoroughly as above.
11. Add 100 (xL of substrate solution to each well of the coated microtiter plate. Substrate
solution is 1 mg p-nitrophenyl phosphate/ml 10% diethanolamine buffer, pH 9.8 for the
enzyme label alkaline phosphatase.
12. Incubate at room temperature about 30 minutes or until the desired color is obtained.
13. Read on the plate reader (spectrophotometer) at 405-650 nm (for alkaline
phosphatase) as indicated in the specific immunoassay tutorial.
Data analysis.
1. Plot the absorbance on the Y axis and the concentration of coating antigen on the X
axis for each concentration of antibody. You will obtain a family of curves such as the
ones shown in Figure 16. The absorbances should increase linearly for any one curve
until reaching saturation.
2. Select the coating antigen concentration at which the absorbance no longer increases.
This is the concentration of coating antigen that will trap all of the antibody added to
the well. From Figure 16 this would be about 2.5 ug/mL. Select the antibody dilution
for the above coating antigen concentration that gives a reasonable absorbance,
preferably between 0.5-1.0.
69
-------
3.
Absorbance
0.8
234
Coating Antigen Concentration (ug/mL)
Figure 16. Plot of checkerboard titration data.
In Figure 16 this
would be the 1/1000
antibody dilution.
Larger absorbances
may give a larger
signal to noise ratio.
If increased
sensitivity is desired,
the coating antigen
concentration may be
decreased into the
linear area. As long
as the signal is
strong and the assay
conditions (time,
temperature) are held
constant, this often
results in more
sensitive assays.
Monitoring the enzyme
reaction over time
(kinetic readings) rather
than an endpoint mode should
be used under these conditions.
Alternatively, plot the absorbance
on the Y axis and the
concentration of antibody on the X
axis for each concentration of
coating antigen. You will obtain a
family of curves such as the ones
shown here (Figure 17). The
absorbances decline as the
antibody concentration decreases.
This is representative of what
would happen in the presence of
inhibitor. That is, if inhibitor is
present, there would be less
antibody in the well, thus the
absorbance would decrease. Ideal
data would show a steep slope.
Thus for small changes in the
amount of antibody inhibited, there
would be a significant change in
absorbance. From Figure 17 using
a coating antigen concentration of
1.25 ug/mL and an antibody dilution of 1/3000 would be ideal.
Select the coating antigen concentration that gives a strong signal and the antibody
dilution with a steep slope.
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
10
Reciprocal Antibody Dilution X 1000
Figure 17. Plot of checkerboard titration data.
70
-------
5. These are the concentrations that will be used for the ELISA when it is used to
measure a target compound in a competitive assay.
Antibody coated plate format:
1. Coat the plate. Each well of the microtiter plate has added to it a fixed amount of
trapping antibody (i.e. for tutorial 4.1, the trapping antibody is coated at 1/2000). Cover
the microtiter plate with a plate sealer and place in the refrigerator overnight.
2. For the single antibody-coated method, prepare the anti-analyte antibody. Anti-analyte
antibody will be added to the plate at several concentrations. Make a dilution of anti-
analyte antibody in PBS-Tween/Azide (in coating buffer if this a double antibody-coated
format). These solutions may be prepared the next day or the night before and left at
room temperature overnight while the coated plates are incubating in the refrigerator.
3. On the following day, remove the microtiter plate from the refrigerator. Wash 3-5X with
PBS Tween/Azide and tap dry.
4. Add 50 jo! of the anti-analyte antibody solution to each well of the microtiter plate as
shown in Figure 14. Cover the microtiter plate with a plate sealer.
5. Incubate for 1 -2 hr. at room temperature. (Use the same incubation time as used in
the tutorial for the specific method.) For the double antibody-coated method, solutions
are prepared in coating buffer and incubated in the refrigerator overnight.
6. At the end of the incubation, remove the plate sealer and wash the microtiter plate 3-5
times with PBS-Tween/Azide. Dry thoroughly by tapping on a paper towel.
7. Add 50 jxL of enzyme labeled hapten to each well of the microtiter plate in various
dilutions in PBS-Tween/Azide. (See Figure 15).
8. Cover the microtiter plate with a plate sealer and incubate for 1 -2 hr at room
temperature. (Use the same incubation time as used in the tutorial for the specific
method.)
9. Take the substrate buffer (10% diethanolamine, pH 9.8) out of the refrigerator to allow
equilibration to room temperature.
10. After the incubation, remove the plate sealer and wash the microtiter plate 3-5 times
with PBS-Tween/Azide. Dry thoroughly as above.
11. Add 100 yL of substrate solution to each well of the microtiter plate. Substrate solution
is 1 mg p-nitrophenyl phosphate/ml 10% diethanolamine buffer, pH 9.8 if the enzyme
label is alkaline phosphatase.
12. Incubate at room temperature about 30 minutes or until the desired color is obtained.
13. Read on the plate reader at 405-650 nm for alkaline phosphatase.
71
-------
Data analysis.
1. Plot the absorbance on the Y axis and the concentration of enzyme-labeled hapten on
the X axis for each concentration of antibody. You will obtain a family of curves that
looks like Figure 17.
2. Examine the antibody dilution curves with the steepest slopes.
3. Select the enzyme-labeled hapten dilution for the antibody dilution that gives a
reasonable absorbance, preferably between 0.5-1.0. One should also evaluate the
signal to noise ratio (i.e. the absorbance of the zero analyte standard/absorbance of
the blank). Since background or the absorbance of the blank is usually <0.10, larger
absorbances are desirable due to the larger signal to noise ratio.
4. The concentrations chosen from the checkerboard titrations will be used for the ELISA
when it is used to measure the target compound in a competitive assay.
72
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5.6 RECORD KEEPING
The following is a guideline for the keeping of records generated from immunochemical
analysis in an academic laboratory. Procedures may be altered to comply with regulations
pertaining to your particular laboratory.
All raw data, whether generated by hand or by a software program designed for
specific output, are recorded as hard copies, referenced to a raw data file name and placed in
the notebook. Raw data files are also kept on floppy disks which are catalogued in binders to
include the individual filenames. All processed data are recorded as hard copies and placed
in the notebook. All experiments will be of a quality acceptable for publication in a referred
journal in the appropriate field, i.e. Analytical Chemistry, Journal of Agriculture and Food
Chemistry, etc.
Typically, software programs associated with data collection and analysis for
immunoassay generate a large number of pages of output. To maximize efficient use of the
notebook the following suggestion is made for data output management: —
1). Hard copy data: The hard copy should be given a unique page number and placed in
a looseleaf binder. The notebook should refer to the hard copy data by looseleaf
binder number and page number and the filename (if available). The hard copy in turn
should have a reference to the page in the notebook. Periodically the loose pages
should be permanently bound.
2). Electronic data: Spreadsheet data, microtiter plate reader software data, etc. can be
archived on diskette. The file should have a descriptor attached which identifies the
notebook page. The notebook should have the filename.
3). Reagents (i.e. antibodies, haptens, etc.): All reagents are labeled with a notebook
number and page number, providing a unique identifier.
Guidelines for Entries:
1). Make entires in permanent ink.
2). Use consecutive pages.
3). Date entries.
4). Identify subject matter.
5). Include sketches, diagrams, etc.
6). Explain sketches, etc.
7). Photos, drawings, etc., should be identified and permanently attached.
8). Avoid erasures.
9). Do not change entry; make new entry.
10). Periodically have quality assurance officer, laboratory manager or project leader look
73
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5.7 PREPARATION OF BUFFERS FOR USE IN ELISA
AH buffers are prepared with double distilled water. These procedures are given with
sodium azide added as a preservative. If the buffers are used rapidly, or stored in the
refrigerator, sodium azide may be omitted. If utilizing horseradish peroxidase as the enzyme
label, it is best to omit the sodium azide, as it is inhibitory to the enzyme. In this case sodium
ethylmercurithiosalicylate may be substituted (0.005%). PBS-Tween is recommended as the
wash buffer and for making all dilutions involving antibody or sample. It may be possible to
substitute plain water containing Tween 20 for the wash step. Tween 20 is mandatory in the
buffers to minimize non-specific binding. The following materials are given as examples.
Similar reagents from other vendors may be appropriate. Sources are listed here as a
convenience to the reader.
1) Substrate Buffer for Alkaline Phosphatase (STORE REFRIGERATED).
97 mL diethanolamine (Aldrich)
0.2 g NaN3 (Adrich)
0.1 g MgCI2.6H2O (Fisher), is a cofactor for the enzyme
Bring to 800 mL with distilled water, adjust pH to 9.8 with 6N HCI.
Bring to final volume of 1L. Check pH after prolonged storage.
2) 10OX Tween/Azide (STORE AT ROOM TEMPERATURE)
2%NaN3(10g)
5% Tween 20 (25 mL) (Sigma, polyoxyethylene-sorbitan monolaurate)
Bring to 500 mL with distilled water (Tween 20 is a detergent; add water slowly to
limit foaming.).
3) Coating Buffer (STORE REFRIGERATED)
0.795 g NaaCOg
1.465g NaHCO3
0.1 g NaN3
Dilute to almost 500 mL with distilled water, adjust pH to 9.6 and bring to final volume
of 500 mL. Check pH after prolonged storage.
4) 10X PBS (Phosphate Buffered Saline) (STORE AT ROOM TEMP)
640 g NaCI
16gKH2PO4
91.96g Na2HPO4
(Add this slowly while stirring to prevent clumping of salts.)
16gKCI
Bring to approximately 7L with distilled water. Stir well until all salts dissolve.
Adjust pH to 7.5 and bring to final volume of 8L.
5) 1X PBS-Tween/Azide (STORE AT ROOM TEMPERATURE)
800 mL 10X PBS (#4 above)
Bring to approximately 7L with distilled water.
80 mL 100X Tween/Azide (#2 above, add after most of water has been added to avoid
foaming due to Tween 20), adjust pH to 7.5 if necessary, then bring to final volume of
8L
74
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6) Substrate Buffer for Horseradish Peroxidase
Citrate-acetate buffer
13.61 g Sodium citrate (100 mM)
Bring to approximately 1L with distilled water.
Adjust pH to 5.5 with acetic acid.
1% hydrogen peroxide
1 mi_ 30% H2O2 in 29 ml distilled water
Store in plastic container in the refrigerator.
0.6% 3,3'5,5'-Tetramethylbenzidine (TMB)
60 mg in 10 ml_ dimethylsulfoxide
Store at room temperature in the dark.
Just prior to use prepare the final substrate buffer by mixing:
0.4 ml_ 0.6% TMB in DMSO
0.1 mi_ 1% hydrogen peroxide
in 25 mL citrate-acetate buffer
Make sure buffer and TMB are at room temperature before mixing to avoid
precipitation.
75
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5.8 Preparation of Calibration Standards
and Samples Using the 8X12 Array
To facilitate the preparation of calibration standards and sample dilutions as well as
their transfer to the coated plate, the 8 X 12 microtiter plate array is a useful tool. In most
protocols, the analyst will prepare all the standards and samples prior to adding them to the
coated plate. Since adding reagents to the coated plate begins the equilibrium reactions, it is
desirable to add all the reagents as rapidly and uniformly (from plate to plate) as possible.
Using this array, the 8 or 12-multichannel pipettor can be used to prepare dilutions and rapidly
transfer these to the coated microtiter plate. A layout for a typical array is shown in Figure 18.
In this design, a single 8X12 array preparation of standards and samples is enough for four
coated plates (seen in step 2). This array is also useful in protocols which require a
preincubation step of the analyte and antibody prior to addition to the coated plate. In the
example below the 8x12 array is used not only for the transfer of reagents to the coated
microtiter plates, but also to prepare dilutions of the standards and samples. Alternatively,
standards and samples can be prepared volumetrically and transferred to the 8x12 array for
rapid transfer to the coated plates.
Step 1: Blanks (BLK), calibration standards (S01-S08) and samples (1A-20C) are added to a
sample preparation array. If serial dilutions of the sample or standards are used, these may
be done directly in the sample preparation array. For example, to prepare sample 1 in
dilutions of 2, 4 and 8 (1A, 1B, 1C, respectively), a volume of assay buffer is added to wells
A10, A11 and A12 of the sample preparation array. An equal volume of sample 1 is placed in
A10. After mixing, the same volume is transferred to well A11. This step is repeated for A12.
Using a multichannel pipettor, similar serial dilutions could be made simultaneously for other
samples in the column. This technique then can be expanded to the whole array.
Step 2: These four drawings, represent four coated microtiter plates. The standards and
samples from the sample preparation array (A1-A12) are transferred simultaneously using the
multichannel pipettor to rows A1-A12 on the Coated EIA Plate 1. This step is repeated for
rows B1-D12 on the coated plate resulting in four well replicates from a single standard or
sample dilution. Similarly, Row B1-B12 is added to the bottom half of Coated EIA Plate 1.
76
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9 10 11 12
A
B
C
D
E
F
G
H
12345
BIX
2A
BLK
7A
BLK
12A
BLK
17A
S01
2B
S01
78
SO1
12B
SO1
17B
S02
2C
S02
7C
302
12C
S02
17C
303
3A
SOS
8A
S03
13A
S03
18A
S04
3B
S04
8B
S04
13B
S04
18B
SOS
3C
SOS
80
SOS
13C
SOS
180
S06
4A
SOS
9A
SOS
14A
SOS
19A
SO?
4B
S07
9B
S07
14B
S07
19B
SOS
40
SOS
9C
SOS
140
SOS
19C
1A
5A
6A
10A
11A
15A
16A
20A
1B
SB
6B
10B
11B
158
16B
20B
1C
SO
6C
10C
11C
15C
160
20C
COMPETITIVE INHIBITION PLATE
10 11 12
1234587
9 10 11 12
RQW
RTQW
COATED EIA PLATE 1
12345373 9 10 11 12
COATED EIA PLATE 3
1 2 3 4 5 6 7 S 9 10 11 12
DVV
O 1-
COATED EIA PLATE 2
COATED EIA PLATE 4
Figure 18. Schematic of the 8X12 array for sample preparation.
77
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5.9 OUTLINE FOR A QUALITY ASSURANCE DOCUMENT
FOR USING IMMUNOASSAY METHODS
The following outline is to provide guidance to the reader in the development of a
quality assurance document once immunoassay methods have been added to the laboratory
methodology. It should be noted that many of the elements of the quality assurance
document are the same as may already exist for other analytical methods.
Contents:
Section 1. Introduction
General overview of the project
Objectives of the project
Section 2. Project Description
Project schedule chart
Section 3. Project Organization
Management structure
Responsibilities of participants
Section 4. Quality Assurance Objectives
Seven elements of data quality
Section 5. Quality Assurance and Quality Control Protocols
Quality control protocols for the immunoassay method
Quality control protocols for the confirmatory method
Quality control protocols for the extraction/cleanup procedures
Analysis of key samples
Section 6. Laboratory Operations
Laboratory quality assurance policies
Instrument maintenance and calibration
Standard operating procedures for cleanup, detection, and analysis
Sample handling
Section 7. Data Handling
Section 8. Assessment of Data Quality
Audits and review of data quality
Statistical evaluation of the data
Section 9. Quality Assurance Reports to Management
Briefings and status reports
Final project reports
78
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5.10 GUIDELINES FOR THE EFFICIENT USE OF
96-WELL MICROTITER PLATES
A
B
C
D
E
F
G
H
12345678 9 10 11 12
g
(1)
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XL
c
m
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-------
5.11 GENERAL TROUBLESHOOTING GUIDELINES TO OPTIMIZE
THE ENZYME IMMUNOASSAY METHOD PERFORMANCE
Troubleshooting is probably the most useful skill that any analytical chemist can
develop. It is beyond the scope of this manual to provide a complete troubleshooting guide.
However, we can list the most common symptoms and best responses. When evaluating test
kits or component assays, it is best to keep open lines of communication with the supplier in
order to answer questions and provide assistance in troubleshooting.
Table 1. Troubleshooting Guidelines to Optimize the Enzyme Immunoassay Method
Performance.
Symptom
Poor well to well replication3
Low or no color development0
Color development too high
Cause
Poor pipetting technique
Poor binding platesb
Coating antigen or antibody is
degrading
Coated plates stored too long
Poor washing
Uneven temperature in the
wells
Sample carryover
Loss of reagent integrity
Incubation temperature too
coldd
Sample matrix effect
Incubation too long or
temperature too high
Remedy
Check instrument, see tutorial
on pipetting, practice, calibrate
pipettor
Check new lot, change
manufacturers
Use new lot of coating reagent
Discard plates, coat a new set,
decrease storage time
Wash plates more, or more
carefully, remake buffer
Deliver reagents at room
temperature, avoid large
temperature fluctuations in the
room
Watch for potential carryover in
pipetting and washing steps
Systematically replace or check
reagents, including buffers
beginning with the enzyme
label
Lengthen incubation time or
increase temperature by using
a circulating air-temperature
controlled incubator (particularly
a problem if working in the
field)
Dilute matrix if possible, check
pH of matrix, increase the ionic
strength of the buffer
Decrease incubation time or
temperature
80
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Table I (con't)
Symptom
Change in calibration curve
parameters
High background
High plate to plate variation
Cause
Matrix effect
Degradation of reagents
For short assay times,
incubation too long
Incubation too long, favored
nonspecific binding
Used too high reagent
concentrations, favored
nonspecific binding
Poor uniformity of coating
Non uniform binding plates
Poor pipetting technique
Remedy
Dilute matrix or re-evaluate
matrix effects
Systematically check or replace
reagents, including buffers
Monitor incubation times
carefully
Monitor incubation times
carefully
Make sure the correct reagent
concentrations are used
Use new aliquot of coating
antigen or antibody
Choose new lot of plates
Check instrument, see SOP on
pipetting, practice
aA common problem in immunoassay is poor coefficient of variation on well replicates,
or spurious color development. Plate washing and pipetting are the largest contributors to
spurious color development.
The characteristics of the 96 well plate used is an important factor. Some plates will
bind antigens differently, some have greater variability in binding capacity from well to well
which would contribute to variability. Generally, the best solution is choosing a manufacturer
whose plate performance characteristics for that assay are constant.
°No or low color development is most likely due to a reagent failure. The most
common reagent to fail is the enzyme label. There are two potential problems; first that the
enzyme has lost activity; second that the conjugate has degraded and can no longer bind
efficiently to the antibody. The first case is easy to check. Dilute the conjugate about 2-5X
more than used for the assay. For example, if the method calls for a 1/2500 dilution of the
enzyme label, then make dilutions of 1/5,000 or 1/10,000 or greater. Add the substrate
solution directly to the enzyme dilution and incubate for the period of time indicated in the
method. The color development should be similar to that obtained in the assay. If the color
development is lower or there is any doubt, the enzyme label reagent should be replaced with
a new aliquot. The second case can only be remedied by replacing the reagent.
dAnother significant factor is temperature. The reactions that are occurring on the plate
are based on the Law of Mass Action. They are therefore equilibrium reactions, and are
sensitive to temperature. Reagents should be used at room temperature and during analysis -
plates should be protected from wide fluctuations in temperature (i.e. try to perform all steps of
the assay at the same ambient temperature each time). If an incubator is used or the ambient
temperature is high, there may be a problem with uneven heating in the wells. With the 96-
well plates, the tendency is for the outer wells to reach temperature sooner than the inner
81
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wells, which then has an effect on the equilibrium reactions. Variations in final absorbances
are generally manifested in what is called an "edge effect". Use of a forced-air incubator can
reduce this problem. Temperature related effects on equilibrium are more likely to be seen in
assays whose incubation times are very short.
"Blocking refers to the process of covering active sites in the microtiter plate well that
may not have reacted with the coating protein (f.e. coating antigen or coating antibody). To
conduct a blocking step after the normal coating procedure, the plate is washed and
100|j.L per well of a protein such as bovine serum albumin (BSA) is added. The amount of
BSA to add to the well will depend on the degree of blocking needed. Several experiments
may need to be run in which the concentration of BSA is varied. The lowest concentration of
BSA at which the background remains low and constant should be used. A commonly used
concentration is 0.1% BSA.
82
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5.12 MAINTENANCE AND PERFORMANCE VALIDATION
OF A 96-WELL MICROPLATE READER
The analyst should be familiar with the manufacturer's recommendations for operation
and maintenance of the reader. The manufacturer will often describe procedures for
calibration and validating performance. Tests such as checking the linearity of the response
to the chromophore, and repeatability of readings are most commonly used. The "reversed
wet plate test" (Harrison et al., 1988) can also be used to test for bias in the reader design.
General Maintenance Tips
1). Instrument should be kept clean and as dust free as possible. If a spill occurs on the
exterior, wipe off immediately. If a spill occurs on the interior, refer to the maintenance
manual for cleaning procedure.
2). Keep the drawer or platform which holds the microtiter plate closed and the instrument
covered to protect from dust when it is not in use.
3). Do not move or obstruct the automatic movement of the microtiter plate holder.
4). Before a reading, make sure the bottom of the microplate is clean and dry. The
bottom of the plate must be clean and dry to prevent distortion of the reading and to
prevent contamination of the optic device.
5). Determine the direction of the light path for the reader. For some readers the light
path is from the bottom of the plate through the solution. Thus the distance of the light
path is the distance from the bottom of the well to the meniscus of the solution. In this
case, it is very important that pipettors be calibrated, as the volume of the solution is
the critical determinant of the light path.
6). Analyst should be familiar with error messages which may be encountered as a quick
response may be necessary in order to prevent loss of the data.
Performance Validation:
All laboratory instruments and other equipment used in an immunoassay analysis
should be maintained in proper working order. For the spectrophotometer used to read the
96-well microtiter plates, performance and calibration checks should be done on a semi-
annual basis. As an extra precaution, the performance of key instruments should be tested by
spot checks prior to initiating a large project. In most cases, the protocols for performance
checks and calibrations are contained in the user manual for a particular instrument. If these
are not available, performance protocols must be developed to ensure adequate instrument
performance. For example, the "reverse wet plate test" of Harrison et al. (1988). These
protocols should be kept in a 3-ring binder or folder along with the instrument manual.
Records of instrument calibration, maintenance and repair should also be kept in 3-ring
binders and stored in a secure location with the instrument manuals. If necessary a schedule
for more routine performance checks or calibrations should be developed and kept with the
instrument records.
83
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References
Harrison, R.O. and Hammock, B.D. 1988. Location dependent biases in 96 well microplate
readers. J. Assoc. Off. Anal. Chem. 71:981-987.
84
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5.13 PERFORMANCE CHECKS, CALIBRATION AND MAINTENANCE
OF AIR DISPLACEMENT PIPETTORS
This section refers to both the single channel and multichannel air displacement pipettors.
Calibration
Micropipettors should be checked for accuracy and precision using the gravimetric method.
Most manufacturers provide a description in the instruction manual. Briefly, the pipettor is set to
the test volume. Distilled water is pipetted into a pre-weighed beaker. The weight of the water is
recorded. This procedure is repeated at least 5 times and the readings averaged. The mean and
standard deviation should match the performance criteria given by the manufacturer. If the
performance criteria are not met, the procedure for calibration will be specified by the
manufacturer. When necessary, pipets should be returned to the manufacturer's service
department for cleaning, recalibration and replacement of worn parts. See Appendix I for details
on principles of performance assurance for air displacement pipettes and Appendix II for blank
sample performance assurance log and worksheets.
General maintenance
Pipettes should be checked daily for dust or other contamination on the outside surface of
the pipettor as well as for splits, cracks or chips in the surface. For procedures for general
maintenance or cleaning of the interior of the pipette, see the manufacturer's instruction manual.
Troubleshooting:
Table 2. Troubleshooting Guidelines for the Use of Air Displacement Pipettors
Symptom
Sample leaks from the tip
Inaccurate dispensing
Inaccurate dispensings with
certain liquids
Liquid is an organic solvent
Cause
Tips not attached correctly
Pipet tip is dirty. Interior of
pipet shaft is dirty due to
sample splash.
Incorrect operation by user
Pipet tips have not been pre-
wetted
Tips not attached correctly
Not in calibration
Interior parts of pipet dirty,
worn or damaged
Unsuitable calibration
Remedy
Attach tips firmly
Replace pipet tip, clean interior of
pipettor if instructions are given by
the manufacturer. See also tutorial
5.1.
See manufacturer's manual and
tutorial 5.1
Pre-wet tips by drawing up and
dispensing solution 2 or 3 times
Attach tips firmly
Recalibrate according to instructions
See manufacturer's instructions for
cleaning or replacement of parts
Some viscous liquids require that
pipettor be recalibrated
Change to a positive displacement
pipettor
General Note: These pipettors should not be used to deliver organic solvents. Positive
displacement pipettors should be used for this purpose.
85
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SECTION 6
Glossary of Commonly Used Terms In Immunoassay
Accuracy - the proximity of the value obtained to the "true" value.
Adjuvant - a substance administered with the antigen to promote the immune response and
provide a carrier which depots the antigen for slow release. Examples: Freund's
adjuvant, RIBI adjuvant.
Affinity - the strength of the antibody recognition of the target molecule.
Amplification - a procedure which increases the signal of the assay so as to increase
detectability of the amount of bound antibody.
Analyte - the compound of interest for analysis.
Syn. target molecule.
Antibody (Ab) - refers to a group of immunoglobulins which will bind to an antigen.
Antigen (Ag) - a substance, usually a protein, which will elicit the production of specific
antibodies that will react with that substance.
Antigenic determinant - the smallest entity which can be recognized by an antibody.
Syn: epitope.
Antiserum - serum from an animal containing a group of immunoglobulins which will bind to an
antigen.
B
Bias - refers to data which varies in a predictable manner from the "true" value.
C
Carrier protein - the protein which has been covalently linked to the hapten. If the resulting
antigen is for immunizing, a protein foreign to the organism is useful. For example, we
use keyhole limpet hemocyanin as a protein to link to a hapten for an immunizing
antigen for mammals. For coating, we try to link to a protein which will have minimal
background binding to the antibody, smaller proteins such as bovine serum albumin
seem useful in this regard.
Checkerboard titration - an experimental design used in 96-well microtiter plates to optimize
for coating antigen and antibody concentrations. The coating antigen concentration is
varied by row and the antibody dilution is varied by column. The result is a plate which
gives increasing absorbance as you proceed diagonally across the plate, where more
absorbance is an indication of larger coating concentrations and/or smaller antibody
dilutions.
86
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Coating - the process of passively adsorbing the antigen, antibody, etc. to the solid phase
usually by noncovalent interactions at high pH.
Coating antigen - the antigen used in an ELISA which is bound to a solid phase such as a 96
well microtiter plate by noncovalent interactions.
Competitive inhibition - the process in which the target compound competes for specific
antibody.
Conjugation - the procedure which covalently binds the hapten to the carrier protein.
Syn: couple.
Couple - the procedure which covalently binds the hapten to the carrier protein.
Syn: conjugation.
Cross reactivity - the ability of compounds, structurally related to the target molecule, to bind
to the specific antibody.
Direct - an immunoassay method in which the primary antibody or an analyte mimic is labeled
so that the amount of label bound to the solid phase is directly measured.
Double antibody coated - assay format in which a trapping antibody is adsorbed ("coated") to
the solid phase, which subsequently binds or traps the analyte specific antibody.
Edge effects - the phenomenon of variability noted along the outer edges of a 96 well
microtiter plate. Most often due to uneven temperature during incubations or to poor
quality control of the microtiter plate manufacturer.
Enzyme immunoassay (EIA) - immunoassays which employ an enzyme as the label.
Enzyme linked immunosorbent assay (ELISA) - an immunoassay format in which the hapten
and coating antigen compete for the specific antibody and the amount of bound
antibody is detected by an enzyme labeled second antibody.
Syn: indirect EIA.
Enzyme tracer - the hapten covalently linked to the enzyme.
Epitope - the smallest entity which can be recognized by an antibody.
Syn: antigenic determinants.
First antibody - the antibody specific for the target analyte.
Syn: specific antibody, primary antibody
87
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Format - the manner in which the specific antibody, hapten, and target molecule are put
together for the resulting immunoassay.
Handle - the part of the antigen which binds the hapten to the carrier protein.
Syn: spacer, linker.
Hapten - a small molecule which cannot, by itself, elicit an antibody response but will
specifically react with an antibody.
Hapten density - the amount of hapten covalently bound to the carrier protein.
Syn: hapten loading.
Hapten loading - the amount of hapten covalently bound to the carrier protein.
Syn: hapten density.
Heterogeneous - refers to the immunoassay technique, where a separation step is required
between the bound and unbound antibody.
Heterologous - an immunoarray format in which one or all of the component reagents, e.g.,
hapten, linker, protein, differ between the coating antigen and immunizing antigen.
Homogeneous - refers to the immunoassay technique, where a step is not required to
separate bound and free antibody.
Homologous - refers to the immunoassay format, where the components are the same. For
example, an ELISA assay in which the hapten-linker combination is the same used for
coating and immunizing.
Hybridoma - the result of fusing an antibody producing spleen cell with an immortal myeloma.
The resulting hybridoma cells secrete specific antibody into the medium and can be
cultured in vitro due the properties conferred upon it by the myeloma.
i
IgG - immunoglobulin G, the most common subclass of immunoglobulins used, in
immunoassay.
Immunization protocol - the procedure used to inject animals with the goal of raising
antibodies.
Immunizing antigen - the antigen used when immunizing animals for the production of
antibodies.
Syn: immunogen.
Immunogen - synonym for antigen when emphasizing the ability of a compound to induce an
immune response.
88
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Indirect - an immunoassay method in which the amount of primary antibody bound is detected
by using anti-IgG antibodies which are usually labeled.
Label - is the technique used to visualize the bound antibody or to quantify the antibody-
antigen complex. May be an enzyme, radiolabel, fluorescent compound, etc.
Law of Mass Action - the physical law which governs immunoassay, i.e. that the antigen-
antibody interaction is a reversible one that comes to an equilibrium.
Linker- the part of the antigen which binds the hapten to the carrier protein.
Syn: spacer, handle.
M
Matrix - the substance which contains the target molecule for analysis.
Microtiter plate - polystyrene plates usually arranged in a 96-well format. Variability of the
assay may depend on the plate manufacturer, and even the particular lot.
Monoclonal antibodies- antibodies obtained from a specific clone of cells.
O
Optimization - systematic studies to select the most useful antibody, antigen, enzyme tracer,
etc. concentrations.
Plate reader - a spectrophotometer modified to read the absorbance in the wells of a 96-well
microtiter plate.
Polyclonal antibodies - antibodies obtained from the serum of an animal which contain
antibodies produced by many different cells.
Precision - the proximity of a value to the mean of a series of values obtained from repeated
measurement of the same sample.
Primary antibody - the antibody specific for the target molecule.
Syn: specific antibody, first antibody.
Radioimmunoassay (RIA) - an immunoassay in which the label is a radiotracer. Usually the
target molecule competes with labeled target for the antibody.
89
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Second antibody - in ELISA, it is the antibody against the IgG of the primary antibody and is
generally conjugated to an enzyme.
Serum - the fraction of blood obtained from the animal containing antibodies.
Single antibody coated - format in which a trapping antibody is adsorbed (coated) to the solid
phase.
Solid phase - Any support to which antigens or antibodies may be bound, for instance, 96 well
microtiter plates (polystyrene), polystyrene test tubes or nitrocellulose membranes.
Spacer - the part of the antigen which binds the hapten to the carrier protein.
Syn: handle, linker.
Specific antibody - the antibody which recognizes the target analyte.
Syrt: first antibody, primary antibody.
Target molecule - the compound for which the immunoassay is being developed.
Syn: analyte.
Titer - a description of the affinity of the antibody that can be variously defined. A good
definition for ELISA work is the greatest dilution that still gives an absorbance of about
0.3 at a defined coating antigen concentration and incubation conditions.
Tracer - the label used to detect bound materials.
Trapping antibody - an antibody directed against the IgG of the analyte specific antibody. For
example, an anti-mouse IgG would be the trapping antibody for a triazine antibody
raised in a mouse.
90
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APPENDIX I
PERFORMANCE ASSURANCE FOR AIR DISPLACEMENT PIPETTES
The use of the following material in no way implies endorsement of the manufacturer by
the authors or-the U.S. EPA for the products mentioned. This document is excerpted here as
there are many general concepts regarding performance measurements for air displacement
pipettes that are applicable regardless of the manufacturer. The performance specifications
given for certain pipettes are used as examples of the type of performance specifications that
should be available for any air displacement pipette.
The following material has been excerpted with permission from "Performance Assur-
ance for Air Displacement Pipettes," AB-1, October 1988, prepared by Liquid Measurement
Quality Control, Rainin Instrument Co., Inc., Mack Road, Woburn, MA 01801-4628.
Performance Assurance
Performance assurance provides a means to monitor the performance of air displace-
ment pipetting instruments throughout their life, whether they are old or new. Each pipet
should be given a unique identifier, for example a serial number, to record their performance
history. A service record tells how long the instrument has been in use. A performance
logbook indicates the reliability of the instrument. Statistical data will aid in error analysis of
any test. With an accurate record of performance, the studies become more accurate and
more efficient.
Optimizing Liquid Measurement Performance
Optimum performance can be achieved by four easy steps:
1). Use pipet tips that have been designated for your specific pipette.
2). Reviewing instruction manuals and ensuring implementation of recommended operating
procedures can enhance pipette performance.
3). Regular maintenance and cleaning will aid in keeping instruments trouble-free.
Instructions for proper care and troubleshooting are found in each manual and provide
information on keeping the pipette in order.
4). Utilize manufacturer's service departments for cleaning, calibration and replacement of
worn or broken parts when appropriate.
Measuring Performance
Accuracy in liquid measurement is the closeness of a measured volume to the true volume
specified by the setting of the instrument.
The accuracy of a measurement is expressed in terms of its error, the difference between the
measured volume and the true volume. A small error indicates an accurate measurement.
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Two types of errors occur in liquid measurement:
Systematic error. This type of error is consistent. It represents the effect of instru-
ment calibration, operating characteristics, and conditions that are constant or that change
only in a consistent, predictable way during a series of liquid measurements. Systematic error
normally biases all measurements at a given volume setting toward volumes that are either
higher or lower than the true value.
Random error. This type of error is inconsistent and unpredictable, except for the
frequency with which errors of a given magnitude are likely to occur. Random error is
normally seen as scatter, a distribution of measured values around a most probable or mean
value. It represents the effect of uncontrolled short-term variables in the operation of a liquid
measurement system.
68.2%
. 95.4% .
99.7%
Figure 1. Normal Distribution
The performance of a liquid measurement system at a given volume can be assessed
by performing a series of 10 to 30 measurements and determining the volume measured each
time by a method of much greater accuracy. The method most often used is weighing the
measured liquid on a properly calibrated electronic microgram balance with appropriate
corrections for liquid density and evaporation. If the frequency of occurrence is plotted vs.
measured volume for a large number of samples, a distribution similar to that in Figure 1
should be obtained.
-------
Two calculated statistical values are useful in summarizing and interpreting this type of
Qdtcil
Mean Volume. This is calculated from individual volume measurements as follows:
_
mean volume =V =-^ —
i.e., "
1 . Add up the individual measurements.
2. Divide the total by the number of measurements.
Standard Deviation. This is calculated as follows:
standard deviation = o =
V n-1
i.e.,
1. Subtract the mean volume from each individual volume measurement and square each
result.
2. Add up the squared values.
3. Divide the total by one less than the number of measurements.
4. Take the square root of the result.
NOTE: Many pocket calculators and personal computer spreadsheet programs have
statistical functions that will perform both the mean volume and standard deviation calculations
automatically, once data values are entered. Using a calculator or a personal computer can
greatly simplify these calculations.
The statistical values have meanings, illustrated by Figure 1.
The mean volume is the high-probability point of the normal distribution of measured
volumes. In the scatter of volume measurements due to random error, volumes close to the
mean volume are much more likely to occur and will occur with much higher frequency than
volumes farther from the mean volume.
The standard deviation quantifies the magnitude of scatter due to random error. As
shown by Figure 1, the probability that an individual volume measurement is within one
standard deviation from the mean volume is 68.2%; the probability that an individual measure-
ment is within two standard deviations is 95.4%. Thus, in using the liquid measurement
-------
system at this volume setting, two out of three measurements are expected to fall within one
standard deviation of the mean volume, and 19 out of 20 are expected to fall within two
standard deviations of the mean volume.
Figure 2 illustrates the relationship between the normal distribution of measured
volumes and the specifications of the liquid measurement system at a given volume setting.
F
R
Q
U'
E\
N
C
Y
true
value
_mean_
"error"
accuracy
specification
Figure 2. Measured Values vs. Specification
Mean error is the difference between the mean volume of actual measurements and
the true value of the volume set on the instrument. The accuracy specification provides an
upper limit to this mean error. Mean error is a measure of the systematic error component
of individual liquid measurements. The accuracy specification indicates that the instrument, as
delivered, is calibrated so that the mean volume will fall within the limit when the pipetting
system is used according to the instructions in the manual.
The precision specification is an upper limit on the standard deviation of measure-
ments performed at a given volume setting. It provides a limit to the amount of random error
that should be seen in individual liquid measurements as long as the pipetting system is
properly maintained and used in accordance with the recommendations of the manual.
As shown in Figure 2, the precision specification is normally much smaller than the
accuracy specification. This provides good assurance that the majority of individual measured
volumes will fall within the specified accuracy range. It also provides a high degree of
-------
assurance that accuracy checks based on four individual volume measurements can verify
proper instrument calibration. Without narrow limits on the scatter of individual measure-
ments, i.e., on standard deviation, many more data points would be necessary to check
performance.
Both accuracy and precision may be expressed as percent figures. When expressed
as a percent of nominal or set volume, the mean error figure is called percent error. When
expressed as a percent of the mean volume for a series of measurements, standard deviation
is called the coefficient of variation.
Methodology
In order to measure performance, a standard method is necessary. Standards
organizations such as the International Organization for Standardization (ISO) or the National
Committee for Clinical Laboratory Standards (NCCLS) provide standard guidelines to assess
the performance of a pipette. Here are some suggestions to develop your own program.
Calibrated Tips can easily be used on a daily basis to check the performance of an
instrument.
Set the instrument to one of the volumes of the calibration lines and aspirate a sample.
If the liquid level corresponds to the line on the tip, the system is operating properly. If the
liquid level fails to come close to the line, the instrument should be further tested on a
balance. The calibration marks on the tips are not accurate enough to be used for calibration
purposes, but they do provide some assurance of proper performance on a consistent basis.
The Gravimetric Method measures performance using an analytical balance with
distilled water as the standard. This primary method of analysis is recognized by ISO and
other organizations.
Balance. A regularly maintained microgram balance assures a good testing method
provided that the balance is calibrated with traceable weights. The following table gives
suggested weight classes from the National Bureau of Standards (NBS). Traceable weights
can usually be obtained through an authorized balance dealer or service representative.
Table 1. Weight Class Standards for Balance Calibration.
Test
Volume
1 (jiL)
10
100
1000
Balance
Sensitivity
0.001 (mg)
0.01
0.1
0.1
Standard
Deviation
0.002 (mg)
0.02
0.1
0.2
Weight Class
NBS Certified
M or J
S
S
SorS1
Weighing Accessories. The weighing vessel should not exceed 50 times the volume of
any sample to be measured. The vessel should be cylindrical so that liquid surface area will,
stay fairly constant as it fills. A cover will be useful to minimize evaporative effects when
measuring smaller volumes. The cover should be loose-fitting for easy manipulation.
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The weighing vessel should be handled only with clean forceps or tweezers to prevent
errors due to deposition of moisture, dirt, oil and also to prevent heating. The balance should
be placed on a weighing table to control vibration.
Environment. The room in which testing occurs should be controlled for factors that
affect pipetting system performance and this method of measurement. This includes a draft-
free, dust-free environment with proper lighting (avoid direct sunlight). A constant temperature
(19 - 23°C / 66-73°F) should be maintained (± 1°C). The relative humidity should remain
between 45-75% in order to reduce evaporation and the build up of electrostatic potentials.
Testing Medium. Water is used as the standard in gravimetric analysis. The water
used should be distilled and gas free. It should be placed in the sampling reservoir at least
one hour ahead of time and allowed to come to a steady-state temperature in the test
environment. A thermometer (0.1 °C) will be needed to measure the temperature of the water.
Technique. Pipetting technique should follow the recommendations in the instrument
manual. In order to correct for evaporation during the weighing procedure, an amount of
water sufficient to fill the weighing vessel to a depth of at least 3 - 4 mm should be present at
the start of testing. Timing of all operations should be consistent between samples. Evapora-
tion blanks should be determined by performing the identical sequence of manipulations
without sample aspiration.
Evaporative cooling effects will create minor water-temperature differences depending
on depth and radial position in the sampling reservoir. A temperature difference of 0.5°C can
introduce additional variation on the order of 0.01% in sample measurements. For this
reason, in very rigorous testing to determine pipetting system precision, all samples should be
drawn at approximately the same depth and position in the sampling reservoir.
Calculations. To convert measured sample weight to volume, a density correction must
be applied. This correction depends on both the density of water and the density of the air in
the test area. (The density of air is used to adjust for the buoyancy of water in air, which acts
to decrease the apparent density of the sample.) The density correction is achieved by
multiplying the sample weight by a quantity called the "Z-factor." The Z-factor is the reciprocal
of the apparent density of water under the test conditions.
Because of evaporative cooling, the steady-state temperature of water in the sampling
reservoir will be lower than the air temperature, usually by two or three degrees. Also, the
density of air will vary slightly with barometric pressure and humidity.
These differences may be taken into consideration when calculating Z-factors.
However, the combined effects of these factors on Z-factor values are significant only at the
0.01% level, at least one order of magnitude below the level of discrimination needed to verify
accuracy of an air-displacement pipetting system. The Z-factor table provided ignores these
minor differences, assuming that the air temperature is the same as that of water in the
sampling reservoir and that barometric pressure is one atmosphere. The values in this table
are sufficiently accurate for routine performance assurance purposes.
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Correction for evaporation will also increase the accuracy of the data and is especially
important for small volume measurements. An evaporation blank is used to estimate the
amount of water lost during the test. This amount is added to the mean sample weight.
Table 2. Z- Factors
Water Temperature
(°C)
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
Z-Factor
(uL/mg)
1.0020
1.0020
1.0021
1 .0022
1 .0023
1.0024
1.0025
1.0026
1.0027
Water Temperature
(°C)
19.5
20.0
20.5
21.0
21.5
22.0
22.5
23.0
Z-Factor
(uL/mg)
1.0028
1.0029
1 .0030
1 .0031
1.0032
1 .0033
1.0034
1.0035
(Values from ISO Standard.)
The extent of an appropriate performance assurance program should be determined by
your needs. Organizations such as ISO, NCCLS, and ASTM all provide in-depth procedures
for volume analysis. Further documentation is available from those organizations upon
request.
Routine Testing
A routine test program is recommended for all air-displacement pipetting systems.
Frequency
The frequency of instrument testing should be based upon:
• Frequency of use
• Number of operators using each instrument
• Type of samples used
• Need for accurate error analysis
Levels of Checking
Level 1. Daily checks using calibrated tips. Use for easy verification during pipettings.
Problems can be detected visually by verifying levels during pipetting.
Level 2. Gravimetric method - four weighings. Use as a routine quick check on accuracy for
volumes commonly being pipetted.
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Level 3. Gravimetric method - ten weighings. This level of check is probably the most
comprehensive check necessary. Use to determine accuracy with confidence,
particularly when introducing a new pipette to the program. Minor service e.g.,
cleaning a partially clogged tip, may need to be performed by user or manufacturer.
Accuracy and precision determinations can be done using ten weighings at three
different volume levels to obtain the best confidence. Use this check level after
major repair, e.g., realignment of settings, or when performing critical work. Manu-
facturer's certificates are usually based on the Level 3 check.
Level 4. Gravimetric method - thirty weighings. This is the most comprehensive check to
determine accuracy and precision. Not cost effective for user to routinely return
pipette to manufacturer for this level of checking. Suggestive use would be for
checking operator technique.
Lops and Worksheets
Performance Log. The performance log is a record for each instrument that tells when
the last test was performed on the instrument and a summary of the results. This information
can help you easily assess the condition of the instrument you are about to use. This is
especially useful in a laboratory where instruments may have multiple users.
Performance Worksheets. Worksheets aid gravimetric analysis by providing simple
charts to check liquid measurement instruments.
Here are some hints for their use:
Z-Factor conversions are found on page 7 and can be used to convert pure water to
volume.
Whether to use a new tip for each weighing or the same pre-rinsed tip for all weighings
is up to you. This procedure should reflect your method in daily use.
A frequency schedule of performance tests is desirable to keep the information up to
date.
Example Log Sheets
Examples of completed instrument logs and performance test worksheets are provided at
the end of this appendix. Forms for constructing performance logs and worksheets are given
in Appendix II.
8
-------
Example Performance Specifications
MODEL TEST VOLUME (jiL) ACCURACY RANGE (\iL) % ERROR
PRECISION
%CV
P-20
P-100
P-200
P-1000
P-5000
P-10
P-2
P-10ML
2
10
20
20
50
100
50
100
200
200
500
1000
1000
2500
5000
1
5
10
0.2
1.0
8.0
1000
5000
10000
1.9-
9.9-
19.8-
19.65 -
49.6-
99.2-
49.5-
99.2-
198.4-
197-
496-
992-
988-
2485-
4970-
0.975 -
4.925 -
9.9-
0.176 -
0.973 -
1.97-
950-
4950-
9920-
2.1
10.1
20.2
20.35
50.4
100.8
50.5
100.8
201.6
203
504
1008
1012
2515
5030
1.025
5.075
10.1
0.224
1.027
2.03
1050
5050
10080
5.0
1.0
1.0
1.5
0.8
0.8
1.0
0.8
0.8
1.5
0.8
0.6
1.2
0.6
0.6
2.5
1.5
1.0
1.2
2.7
1.5
5
1
0.8
0.04
0.05
0.06
0.10
0.12
0.15
0.20
0.25
0.30
0.6
1.0
1.3
3.0
5.0
8.0
0.012
0.03
0.04
0.012
0.013
0.014
6
,10
16
2.0
0.5
0.3
0.50
0.24
0.15
0.40
0.25
0.15
0.30
0.20
0.13
0.30
0.20
0.16
1.2
0.6
0.4
6
1.3
0.7
0.6
0.2
0.16
9
-------
P-1000
PERFORMANCE WORKSHEET
Operator J/§
Date lo/2X/!f
Model: P-1000
Serial: £.270.1
Test
Volumes: 200
191-7 I11-I
-70H-I 199-5
iqjf. 5 ZtoO-3.
|9
-------
P-1000
PERFORMANCE LOG
Instrument Model: \- IQ&O
Serial Number:
Purchase Date:
Date Initia
Inspection Level
# of Samples
4 10 30
Comments
fo/st,
ulfeb
(5-UJ
A c^,^^^
m
fi^^&e/- a>0«~s*c*.~*-oif' £L-f/~t2
-------
P-200
QUICK CHECK WORKSHEET
Operator
Date
H20 Temp
Z Factor
/.OO3"2-
Model
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
20
IV.U
It, SI
It.SI
1 19.5-20.5l
Serial Number:
100
200
.3*
.33
If*? .
I 99.2-100.8l
1 198.4-201.61
Model : 1 P-200 \
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
20
20. II
I 19.5-20.Sl
Serial Number:
100
ICX7.C7?
-£5733
200
12
1 99.2-100.8l
I 198.4-201.6|
Model :
I P-200 1
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
[
20
J
17.73
79.73
t 7:7*
I 19.5-20.5l
Serial Number: £ - % 9~3>OO A
L
100
J
L
200
J
. "21
/71./7
100. l~7
I 99.2-100.8l
/«¥•?./?
177,77
1 198.4-201.61
Model
1 P-200 I
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
L
20
]
-2.0.03
•zo.pc?
I 19.5-2O5I
Serial Number:
100
J
\oo.o-j
200
•2.C7C7-
99.2-100.8l
198. 4-201. 6
Comments
12
-------
APPENDIX II
PERFORMANCE LOG AND PERFORMANCE TEST WORKSHEETS
FOR AIR DISPLACEMENT PIPETTORS
The use of the following material in no way implies endorsement of the manufacturer by
the authors or the U.S.E.P.A. for the products mentioned. This document is excerpted here
as there are many general concepts regarding performance measurements for air displace-
ment pipettes that are applicable regardless of the manufacturer. The performance specifica-
tions given for certain pipettes are used as examples of the type of performance specifications
that should be available for any air displacement pipette.
The following material has been excerpted with permission from "Performance Assurance
for Air Displacement Pipettes," AB-1, October 1988, prepared by Liquid Measurement Quality
Control, Rainin Instrument Co., Inc., Mack Road, Woburn, MA 01801-4628.
The performance log and performance test worksheets provided on the following pages
can help in keeping good records. The worksheets are designed to allow serial numbers,
standard test volumes, and acceptable performance ranges, usually the instrument specifica-
tion, to be entered in the appropriate spaces. Photocopies can then be made for routine use
in recording data. Wide left margins are provided to allow records to be kept in a looseleaf
binder, if desired.
-------
PERFORMANCE LOG
Instrument Model:
Serial Number:
Purchase Date:
Date Initial
Inspection Level
# of Samples
4 10 30
Comments
-------
QUICK CHECK WORKSHEET
Operator
Date
Model:
Test Volume
Sample
Mean (mg)
Mean (\i\)
Specs
Model :
Test Volume
Sample
Mean (mg)
Mean (u.1)
Specs
Model :
Test Volume
Sample
\
Mean (mg)
Mean (u.1)
Specs
Model :
Test Volume
Sample
Mean (mg)
Mean (u.1)
Specs
H?0 Temp
Z Factor
,_ , Serial Number:
J 1 | [
2
3
4
v.
III 1
Serial Number:
! l l 1
1 — '
2
3
4
III |
Serial Number:
I I I I I
1 i 1
2
3
4
1 1 1 II
Serial Number;
1 1 I 1 1
2
3
4
L i r i i 1
-------
PERFORMANCE WORKSHEET
o
i
pi
z
z
o
o
1
fi
Operator
Date
Model-:
Serial :
Test Volume:
Samples:
Mean (mg)
Mean (\i\)
Specs [
% Error:
Specs- [
Std Dev:
Specs [
%CV:
Specs
Pass
Comments
Air Temp
Z Factor
Accuracy
Precision
Tips Used:
L
n
D
Approved
Date ,
-------
-------
------- |