September 2004
Environmental Technology
Verification Report
TETRACORE, INC.
ANTHRAX, BOTULINUM TOXIN, AND RICIN
ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA)
Prepared by
Battelle
Battelle
I he Business oj Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ ElV ElV
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September 2004
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Tetracore, Inc.
Anthrax, Botulinum Toxin, and Ricin
Enzyme-Linked Immunosorbent Assay (ELISA)
by
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
11
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development provides data and science support that
can be used to solve environmental problems and to build the scientific knowledge base needed
to manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six verification centers. Information about
each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received funding to plan, coordinate,
and conduct such verification tests for "Advanced Monitoring Systems for Air, Water, and Soil"
and report the results to the community at large. Information concerning this specific
environmental technology area can be found on the Internet at
http ://www. epa.gov/etv/centers/center 1 .html.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We sincerely appreciate the
contribution of drinking water samples from the New York City Department of Environmental
Protection (Paul Bennett), the City of Orlando (Terri Slifko), and the Metropolitan Water
District of Southern California (Paul Rochelle). Also, thank you to the Metropolitan Water
District of Southern California for concentrating each drinking water sample. We would also
like to thank Karen Bradham, U.S. EPA National Exposure Research Laboratory (NERL); Steve
Allgeier, U.S. EPA Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern
California; and Stanley States, Pittsburgh Water and Sewer Authority, for their careful review of
the test/QA plan and this verification report. Thanks go to Linda Sheldon, U.S. EPA NERL, for
her review of the verification reports and statements.
IV
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
1 Background 1
2 Technology Description 2
3 Test Design and Procedures 4
3.1 Introduction 4
3.2 Test Samples 5
3.2.1 Performance Test Samples 6
3.2.2 Drinking Water Samples 8
3.2.3 Quality Control Samples 8
3.3 Test Procedure 8
3.3.1 Laboratory Testing 8
3.3.2 Non-Laboratory Testing 9
3.3.3 Drinking Water Characterization 9
4 Quality Assurance/Quality Control 11
4.1 Sample Chain-of-Custody Procedures 11
4.2 Equipment/Calibration 11
4.3 Characterization of Contaminant Stock Solutions 11
4.3.1 Characterization of Botulinum Toxin and Ricin 11
4.3.2 Characterization of Anthrax Spores 12
4.3.3 Anthrax Enumeration Data 13
4.4 Technical Systems Audit 16
4.5 Audit of Data Quality 16
4.6 QA/QC Reporting 17
4.7 Data Review 17
5 Statistical Methods and Reported Parameters 18
5.1 Qualitative Contaminant Presence/Absence 18
5.2 False Positive/Negative Responses 18
5.3 Consistency 18
5.4 Lowest Detectable Concentration 19
5.5 Other Performance Factors 19
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6 Test Results 20
6.1 Qualitative Contaminant Presence/Absence 20
6.1.1 Anthrax 20
6.1.2 Botulinum Toxin 22
6.1.3 Ricin 23
6.2 False Positive/Negative Responses 23
6.2.1 Interfered PT Samples 24
6.2.2 DW Samples 25
6.2.3 Cross-Reactivity PT Samples 26
6.3 Consistency 26
6.4 Lowest Detectable Concentration 27
6.5 Other Performance Factors 27
7 Performance Summary 28
8 References 32
Figures
Figure 2-1. Tetracore ELISA 2
Tables
Table 3-1. Lethal Dose and Source of Contaminants 5
Table 3-2. Performance Test Samples 6
Table 3-3. Drinking Water Samples 7
Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples 10
Table 4-1. Characterization Information for Battelle Preparation of Anthrax Spores 13
Table 4-2. Anthrax Enumeration Data for PT Samples 14
Table 4-3. Anthrax Enumeration Results for Fortified Interferent and
Drinking Water Samples 15
Table 4-4. Summary of Data Recording Process 17
Table 6-la. Anthrax Contaminant-Only PT Sample Results 21
Table 6-lb. Botulinum Toxin Contaminant-Only PT Sample Results 23
Table 6-lc. Ricin Contaminant-Only PT Sample Results 23
Table 6-2. Interferent PT Sample Results 24
Table 6-3. DW Sample Results 25
Table 6-4. Potentially Cross-Reactive PT Sample Results 26
Table 7-1. Anthrax Summary Table 28
Table 7-2. Botulinum Toxin Summary Table 30
Table 7-3. Ricin Summary Table 31
VI
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List of Abbreviations
AMS Advanced Monitoring Systems
ATEL Aqua Tech Environmental Laboratories, Inc.
Ca calcium
CDC Centers for Disease Control and Prevention
cfu colony-forming units
COA certificate of analysis
DI deionized
DW drinking water
ELISA enzyme-linked immunosorbent assay
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
L liter
LOD limit of detection
MB method blank
Mg magnesium
mg/L milligram per liter
jiL microliter
mL milliliter
PT performance test
QA quality assurance
QC quality control
QMP quality management plan
RPD relative percent difference
SOP standard operating procedure
TSA technical systems audit
vn
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative tech-
nologies by developing test plans that are responsive to the needs of stakeholders, conducting
field or laboratory tests (as appropriate), collecting and analyzing data, and preparing peer-
reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance
(QA) protocols to ensure that data of known and adequate quality are generated and that the
results are defensible.
The EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the Tetracore, Inc., anthrax, botulinum toxin, and ricin
enzyme-linked immunosorbent assays (ELISA). Immunoassay detection technologies were
identified as a priority technology category for verification through the AMS Center stakeholder
process.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of environ-
mental monitoring technologies for air, water, and soil. This verification report provides results
for the verification testing of the Tetracore ELISA (Figure 2-1), including kits for detecting
anthrax, botulinum toxin, and ricin. The following is a description of the Tetracore ELISA based
on information provided by the vendor. The information provided below was not subjected to
verification in this test.
The antigen-capture Tetracore ELISA detects antigens in samples by capturing them between a
sandwich of antibodies. The immunosorbent assay uses immunological reagents to identify
antibodies. The Tetracore ELISA can be read qualitatively (visually) and recorded by hand or
quantitatively (using a photometer that measures and prints out the optical density of fluid
samples in the microplate). Readings were made qualitatively during this verification test.
To perform a test, positive and negative capture antibody reagents are applied to alternating
wells of a 96-well plate, where they are passively adsorbed. If the target antigen is present in the
sample, it will bind to the reagent. A detector antibody forms the top of the sandwich and binds
to any antigen in the sample after it is captured. The conjugate, to which the enzyme is
covalently bound, is the third reagent added; and it binds to the detector antibody. The substrate,
added after the conjugate, contains 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), which, in
the presence of horseradish peroxidase, changes to a bright green. The amount of color change is
directly proportional to the amount of
horseradish peroxidase present, which
correlates to the amount of antigen (target
contaminant) bound in the sandwich. The
color change confirms the "capture" of
antigen by the antibody reagents. For 48
samples, the process takes approximately 5
hours.
* _
I
e
Figure 2-1. Tetracore ELISA
The Tetracore ELISA kit includes two 96-
well plates in which the odd-numbered
rows had been pre-plated with the
antibodies specific for each target
contaminant and the even -numbered rows
had been pre-plated with antibodies not
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specific to the target contaminants. The inclusion of the non-target antibodies provides
assurance that a color change is due to an antigen-antibody interaction and is not due to a
reaction with one of the other reagents. Also provided are dilution buffer, wash buffer, and the
appropriate reagents needed for the analysis. The 96-well microplate is 12.5 centimeters (cm) by
8 cm. One Tetracore ELISA kit (positive and negative coated wells) costs $400.
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Chapter 3
Test Design and Procedures
3.1 Introduction
The objective of this verification test of immunoassay test kits was to evaluate their ability to
detect specific biological toxins and agents in water samples and to determine their
susceptibility to specific interferents added to pure water and to interferents inherently present in
several drinking water (DW) samples. The single-use Tetracore ELISA plates detect only one
contaminant at a time. For Tetracore ELISA analyses, the presence of contaminants is indicated
by a change in color of a solution within a sample well. When a water sample is added, this
color change occurs during the last step of a procedure that takes approximately 5 hours. With
Tetracore ELISA, usually a large number of samples (i.e., 48 samples) can be analyzed
simultaneously. Tetracore ELISA kits can be used in conjunction with an electronic reader to
determine the presence or absence of a contaminant or can be read visually. In this verification
test, the Tetracore ELISA 96-well plates for anthrax, botulinum toxin, and ricin were read
visually, without the aid of a plate reader.
During this verification test, the Tetracore ELISA was subjected to various concentrations of
anthrax spores, botulinum toxin, and ricin in American Society for Testing and Materials Type n
deionized (DI) water. Table 3-1 shows the contaminants and information about their detection,
including the vendor-stated limit of detection (LOD), the lethal dose concentrations, and the
source of the contaminant. The Tetracore ELISA also was used to analyze contaminant-fortified
DW samples that were collected from four water utilities that use a variety of treatment methods.
The effect of interferents was evaluated by analyzing individual solutions of organic acids
(humic and fulvic), magnesium (Mg) and calcium (Ca) in DI water both with and without the
addition of the contaminants with the Tetracore ELISA. In addition, specificity was evaluated by
exposing the Tetracore ELISA to a potentially cross-reactive compound or spore for each target
contaminant.
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Table 3-1. Lethal Dose and Source of Contaminants
Contaminant
Bacillus anthracis
Ames Strain (anthrax)
Botulinum toxin
Types A and B
Ricinus communis
Agglutinin n (ricin)
Vendor-Stated
LOD
2 x 104
spores/mL
0.004 mg/L
0.001 5 mg/L
Lethal Dose
Concentration^
200 spores/mL(1)
0.3 mg/L(2)
15 mg/L(3)
Source of Contaminant
BattelleandU.S. Army
Dugway Proving Ground
Metabiologics, Inc.
(Madison, Wisconsin)
Vector Laboratories, Inc.
(Burlingame, California)
^ The lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water
would probably cause the death of a 154-pound person based on human mortality data.
mL = milliliter
mg/L = milligrams per liter
The verification test for the Tetracore ELISA was conducted January 14 through April 23, 2004,
according to procedures specified in the Test/QA Plan for Verification of Immunoassay Test
Kits.m This test was conducted at Battelle laboratories in Columbus and West Jefferson, Ohio.
Aqua Tech Environmental Laboratories, Inc. (ATEL) of Marion, Ohio, performed physico-
chemical characterization for each DW sample to determine the following parameters: turbidity;
concentration of dissolved and total organic carbon; specific conductivity; alkalinity; concentra-
tion of Mg and Ca; pH; hardness; and concentration of total organic halides, trihalomethanes,
and haloacetic acids. Battelle confirmed the presence of anthrax spores using plate enumeration.
The Tetracore ELISA were evaluated for the following parameters:
• Qualitative contaminant presence/absence
• False positive/false negative response
- Interferents
- DW matrix effects
- Cross-reactivity
• Consistency
• Lowest detectable concentration
• Other performance factors
- Field portability
- Ease of use
- Sample throughput.
3.2 Test Samples
Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the
Tetracore ELISA to individually detect various concentrations of anthrax spores, botulinum
toxin, and ricin was evaluated by analyzing performance test (PT) and DW samples. PT samples
included DI water fortified with either the target contaminant, an interferent, both, or only a
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cross-reactive species. DW samples were analyzed using the Tetracore ELISA with the addition
of each target contaminant. All the samples listed in the test/QA plan were initially analyzed. As
discussed below, additional concentration levels were analyzed to more thoroughly evaluate the
performance of the Tetracore ELISA.
Table 3-2. Performance Test Samples
Type of PT
Sample
Contaminant-only
Interferent
Potentially
Cross-reactive
Sample Characteristics
Anthrax spores
Botulinum toxin Types A and B
Ricin
Contaminants in 46 mg/L Ca
and 1 8 mg/L Mg
Contaminants in 230 mg/L Ca
and 90 mg/L Mg
Contaminants in 0.5 mg/Lhumic
acid and 0.5 mg/L fill vie acid
Contaminants in 2.5 mg/L humic
acid and 2.5 mg/L fill vie acids
Bacillus thuringiensis (anthrax
analogue)
Lipopolysaccharide
(botulinum toxin analogue)
Lectin from soybean
(ricin analogue)
Approximate Concentrations
200tol010spores/mL(a)
0.004 to 0.3 mg/L
0.0015 to 15 mg/L
Anthrax - 106 spore s/mL
Botulinum toxin (Type B only) - 0.04
Ricin -0.0 15 mg/L
Anthrax - 106 and 10s spores/mL
Botulinum toxin (Types A and B) - 0
Ricin -0.0 15 mg/L
Anthrax - 106 spore s/mL
Botulinum toxin (Type B only) - 0.04
Ricin -0.0 15 mg/L
Anthrax - 106 and 10s spores/mL
Botulinum toxin (Types A and B) - 0
Ricin -0.0 15 mg/L
mg/L
04 mg/L
mg/L
04 mg/L
104 spore s/mL
0.04 mg/L
0.0 15 mg/L
This concentration range includes all samples analyzed, including spores preserved with and without phenol,
spores prepared at Battelle and at Dugway Proving Ground, and vegetative anthrax cells.
3.2.1 Performance Test Samples
The contaminant-only PT samples were prepared in DI water using certified standards of ricin
and botulinum toxin. Reference methods were not available for quantitative confirmation of the
botulinum toxin and ricin test solutions so certificates of analysis (COA) and QA oversight of
solution preparation were used to confirm their concentrations. Anthrax PT samples also were
prepared in DI water using anthrax spores prepared and characterized by Battelle using standard
methods. All test samples were prepared from the standards or stock solutions on the day of
analysis. Spores also were obtained from Dugway Proving Ground and enumerated by Battelle
during this verification test.
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Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Water Utility
Metropolitan
Water District of
California (CA)
New York City,
New York (NY)
Metropolitan
Water District of
California (CA)
New York City,
New York (NY)
Columbus, Ohio
(OH)
Orlando, Florida
(FL)
Water
Treatment
filtered
chloraminated
unfiltered
chlorinated
filtered
chloraminated
unfiltered
chlorinated
filtered
chlorinated
filtered
chlorinated
Source
Type
surface
surface
surface
surface
surface
ground
Cone. /
Unconc.
cone.
cone.
unconc.
unconc.
both
both
Approximate
Contaminant Concentrations
Anthrax
(spores/mL)
unspiked
106
10s
unspiked
106
10s
unspiked
106
unspiked
106
unspiked
106
unspiked
106
Botulinum
Toxin (mg/L)
unspiked
0.04 (Type B)
0.04 (Type A)
unspiked
0.04 (Type B)
0.04 (Type A)
unspiked
0.04 (Type B)
unspiked
0.04 (Type B)
unspiked
0.04 (Type B)
unspiked
0.04 (Type B)
Ricin (mg/L)
unspiked
0.015
unspiked
0.015
unspiked
0.015
unspiked
0.015
unspiked
0.015
unspiked
0.015
Initially, the test/QA plan called for the analysis of PT samples with concentrations including the
lethal dose; the vendor-stated LOD; and approximately 5, 10, and 50 times the LOD. These
samples were analyzed using the Tetracore ELISA. Preliminary results indicated that anthrax was
not detectable; therefore, the original test/QA plan was amended to include the analysis of higher
concentration levels of anthrax, as well as a second source of anthrax spores that were never
preserved in phenol and vegetative anthrax cells. This testing and the subsequent results are fully
described in Section 6.1.
The interferent PT samples consisted of samples of humic and fulvic acids isolated from the
Elliott River (obtained from the International Humic Substances Society) and Ca and Mg
(prepared from their chlorides), each spiked into DI water at two concentration levels. These
solutions were analyzed both with the addition of each target contaminant at one concentration
level and without the addition of any target contaminant. To be able to evaluate the susceptibility
of the ELISA test kit to false negative results due to interferents, the test/QA plan was amended to
include the fortification of detectable concentrations of anthrax spores into interferent solutions.
The last type of PT sample was a cross-reactivity check sample to determine whether the plates
produce false positive results in response to similar analytes. Bacillus thuringiemis (for anthrax),
lectin from soybean (for ricin), and lipopolysaccharide (for botulinum toxin) are chemically or
biologically similar to the specified targets. Solutions of these were prepared in DI water at
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concentrations similar to the vendor-stated LOD of the test kits for the specified targets and
analyzed using the Tetracore ELISA.
Three replicates of each PT sample were analyzed. A total of 162 PT samples was analyzed by
the Tetracore ELISA for this test. The results provided information about how well the Tetracore
ELISA detected the presence of each contaminant at several concentration levels, the consistency
of the responses, and the susceptibility of the Tetracore ELISA to some selected interferents and
possibly cross-reactive species.
3.2.2 Drinking Water Samples
Table 3-3 lists the DW samples collected from four geographically distributed municipal sources
to evaluate the performance of the Tetracore ELISA with various sample matrices. These samples
were unique in terms of their source and treatment and disinfection process. All collected samples
were finished DW either ready for the distribution system or from within the distribution system.
Approximately 120 L of each of the DW samples were collected in pre-cleaned high-density
polyethylene containers. All but 20 L of the DW samples were shipped to the Metropolitan Water
District of Southern California, dechlorinated with sodium thiosulfate, and then concentrated
through ultra-filtration techniques to a final volume of 250 mL. This concentration factor was
selected because it is the goal of an EPA onsite ultra-filtration method which is currently being
developed. The remaining 20 L of each DW sample was shipped to ATEL for water quality
analysis. Each DW sample (non-concentrated and concentrated) was analyzed without adding any
contaminant, as well as after fortification with individual contaminants at a single concentration
level. A total of 156 DW samples was analyzed by the Tetracore ELISA for this test.
3.2.3 Quality Control Samples
In addition to the 318 PT and DW samples analyzed, 33 method blank (MB) samples consisting
of DI water also were analyzed to confirm negative responses in the absence of any contaminant
and to ensure that no sources of contamination were introduced during the analysis procedures.
With each set of samples, 12 concentration levels (one replicate each) of the target contaminant
were analyzed to develop a calibration curve for use if a reader was used. In addition to serving as
a calibration curve, those samples served as positive control samples to confirm the function of
the Tetracore ELISA plate. Because of this control procedure, other positive control samples were
not analyzed.
3.3 Test Procedure
3.3.1 Laboratory Testing
The scope of this verification test required that most of the test samples be analyzed within
Battelle laboratories staffed with technicians trained to safely handle anthrax, botulinum toxin,
and ricin. Each day, fresh samples were prepared from standards or stock solutions in either DI
water, an interferent matrix, or a DW matrix. Each sample was prepared in its own container and
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labeled only with a sample identification number that also was recorded in a laboratory record
book along with details of the sample preparation. Prior to the analysis of each sample, the
verification staff recorded the sample identification number on a sample data sheet; then, after the
analysis was complete, the result was recorded on the sample data sheet. Three replicates of each
test sample were analyzed. The testing procedure included the following steps for analyzing liquid
samples for the presence of anthrax spores, botulinum toxin, or ricin: (1) 50 |^L of dilution buffer
were added to all wells that would contain a sample; (2) 50 |^L of test sample were added to the
dilution buffer, and the plates were incubated for one hour at room temperature; (3) plates were
washed three times with 200 |j,L of Tetracore ELISA wash buffer per well; (4) 100 |j,L of detector
antibody were added to each sample well, and plates were incubated for one hour at room
temperature; (5) plates were washed three times with 200 |j,L of Tetracore ELISA wash buffer per
well; (6) 100 |^L of conjugate were added to each sample well, and the plates were incubated for
one hour at room temperature; (7) the plates were washed three times with 200 |j,L of Tetracore
ELISA wash buffer per well; (8) 100 |^L of substrate solution were added to each sample well, and
the plates were incubated for 30 minutes at room temperature; and (9) the results were read
visually. Wells that changed to a color different than the negative control were recorded as having
given positive results. The procedure took approximately 5 hours.
3.3.2 Non-Laboratory Testing
The test/QA plan called for testing the Tetracore ELISA in a non-laboratory setting with both a
trained operator and an operator that had not been trained to operate the Tetracore ELISA and had
no laboratory experience. This approach was designed to test the performance of the Tetracore
ELISA in a non-laboratory setting, not to thoroughly evaluate the effect of changing conditions
such as temperature and humidity on the Tetracore ELISA. However, two characteristics of the
Tetracore ELISA prompted amending the test/QA plan to omit field portable verification
parameters for both the technically trained and untrained operators. First, the Tetracore ELISA
requires the use of a multichannel micropipettor to precisely manipulate solutions. Using a
multichannel pipettor is a skill that requires training and practice. The objective of this portion of
the testing was to evaluate the effectiveness of the Tetracore ELISA as a detection tool for
untrained operators. Therefore, the test/QA plan was amended to omit this testing parameter for
technologies such as the Tetracore ELISA that cannot be operated without some acquired
technical skill. Second, the testing procedure does not change at all when performing tests outside
the laboratory. The Tetracore ELISA plates and reagents could be packaged into a shoebox-sized
container and transported to a non-laboratory location. In the manner that the Tetracore ELISA
was tested, there was no requirement for electrical power. The only items required were a waste
container and a flat surface. Because observing the analyses in the laboratory was adequate for
determining the feasibility of using the Tetracore ELISA outside the laboratory, the non-
laboratory testing including the trained operator was omitted from the test/QA plan.
3.3.3 Drinking Water Characterization
An aliquot of each DW sample, collected as described in Section 3.2.2, was sent to ATEL prior to
concentration to determine the following water quality parameters: turbidity; concentration of
dissolved and total organic carbon; conductivity; alkalinity; pH; concentration of Ca and Mg;
hardness; and concentration of total organic halides, trihalomethanes, and haloacetic acids.
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Table 3-4 lists the methods used to characterize the DW samples, as well as the characterization
data from the four water samples collected as part of this verification test. Water samples were
collected and water quality parameters were measured by ATEL in January. Samples were then
transported and test strips were analyzed from January through March. Because of this, some of
the water quality parameters may have changed from the time of analysis by ATEL until testing
with the Tetracore ELISA test strips.
Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples
Sources of Drinking Water Samples
Parameter
Turbidity
Dissolved
organic carbon
Total organic
carbon
Specific
conductivity
Alkalinity
pH
Calcium
Magnesium
Hardness
Total organic
halides
Trihalomethanes
Haloacetic acids
Unit
NTU
mg/L
mg/L
|iS/cm2
mg/L
mg/L
mg/L
mg/L
Hg/L
Hg/L/
analyte
Hg/L/
analyte
Method
EPA180.1(5)
SM5310(6)
SM5310(6)
SM2510(6)
SM 2320(6)
EPA150.1(7)
EPA 200.8(8)
EPA 200.8(8)
EPA 130.2(7)
SM5320(6)
EPA 524.2(9)
EPA 552.2(10)
Columbus,
Ohio
(OH DW)
0.2
2
2
357
55
7.33
42
5.9
125
360
26.9
23.2
Orlando,
Florida
(FL DW)
0.5
2
2
325
124
7.93
41
8.4
137
370
80.9
41.1
New York City,
New York
(NY DW)
1.3
2
2
85
4
6.80
5.7
19
28
310
38.4
40.3
MWD,
California
(CA DW)
0.1
2
2
740
90
7.91
35
1.5
161
370
79.7
17.6
NTU = nephelometric turbidity unit
MWD = Metropolitan Water District
|lS/cm = micro Siemen per square centimeter
10
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Chapter 4
Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the quality
management plan (QMP) for the AMS Center(11) and the test/QA plan(4) for this verification test.
4.1 Sample Chain-of-Custody Procedures
Sample custody was documented throughout collection, shipping, and analysis of the samples.
Sample chain-of-custody procedures were in accordance with ASAT n-007, Standard Operating
Procedure for Chain of Custody for Dioxin/Furan Analysis. The chain-of-custody forms
summarized the samples collected and analyses requested and were signed by the person
relinquishing samples once that person had verified that the custody forms were accurate. The
original sample custody forms accompanied the samples; the shipper kept a copy. Upon receipt at
the sample destination, sample custody forms were signed by the person receiving the samples
once that person had verified that all samples identified on the custody forms were present in the
shipping container.
4.2 Equipment/Calibration
The Tetracore ELISA and all appropriate reagents and supplies specific for the detection of
anthrax, botulinum toxin, and ricin were provided to Battelle by the vendor. Because no plate
reader was used, the Tetracore ELISA test results were read visually; and, therefore, no calibration
was required. For DW characterization and confirmation of the possible interferents, analytical
equipment was calibrated by ATEL according to the procedures specified in the appropriate
standard methods. Pipettes used during the verification test were calibrated according to Battelle
Standard Operating Procedure (SOP) VI-025, Operation, Calibration, and Maintaining Fixed
and Adjustable Volume Pipettes.
4.3 Characterization of Contaminant Stock Solutions
4.3.1 Characterization of Botulinum Toxin and Ricin
Certificates of analysis for botulinum toxin and ricin were provided by the supplier. Because
standard reference methods do not exist, the concentration of botulinum toxin and ricin were not
11
-------
independently confirmed. The COAs stated that the ricin standard (Vector Laboratories, Inc.,
Burlingame, California) had a concentration of 1,000 mg/L and the botulinum toxin standards
(Metabiologics, Inc., Madison, Wisconsin) had concentrations of 2,000 mg/L for Type B and
1,000 mg/L for Type A. Test samples containing these contaminants were prepared by diluting
aliquots of these stock solutions with DI water.
4.3.2 Characterization of A nthrax Spores
Multiple sources of the Ames strain of Bacillus anthracis (anthrax) were evaluated during this
verification test. The primary source was a lot of spores prepared by Battelle and stored in a
1% stock solution of phenol in water as a means to prevent vegetative cell growth. This lot of
spores is referred to in this report as Battelle-prepared, phenol-preserved. Prior to testing, an
aliquot of the stock solution described above was centrifuged, the phenol/water solution was
removed, and the spores were reconstituted with DI water. This process was repeated two times to
ensure that the spores were suspended only in DI water. This lot of spores was characterized with
an 11-step characterization process prior to use in the verification test. For confidentiality reasons,
Table 4-1 gives the outcome of only five of the characterization parameters, as well as the location
at which each step was performed. These characterization steps were performed when this lot of
spores was prepared in September 2003. It should be noted that, once a stock solution of spores is
characterized, less concentrated solutions of spores can be prepared from the stock solution
without questioning the integrity of the spores. This lot of spores met all 11 acceptance criteria.
Two parts of the characterization process—DNA sequencing and gene identification—were
performed by Dr. Alex Hoffmaster at the Epidemiologic Investigations Laboratory, Meningitis
and Special Pathogens Branch of the Centers for Disease Control and Prevention (CDC). The
CDC analyses confirmed that the spores were Ames strain anthrax spores, and the guinea pig LD50
study confirmed their virulence. The stock solution of spores was enumerated after preparation to
determine its original concentration. In addition, a vegetative cell analysis showed that the stock
solution was 99.94% anthrax spores. Because at least one spore is needed to spur the growth of a
colony during an enumeration, the concentrations determined represented a minimum
concentration of spores. Care was taken to spread the samples to avoid clumping; but, if clumping
occurred, the spore concentrations would only be higher than shown in the data tables.
Another lot of anthrax spores from Dugway Proving Ground was obtained and used to investigate
the sensitivity of Tetracore ELIS A to a different spore preparation (referred to as Dugway-
prepared in this report). No characterization data except for enumerations were performed on this
lot of spores. Vegetative Bacillus anthracis was also analyzed during this verification test.
Vegetative cells from an enumeration of the Battelle-prepared, phenol-preserved spores were
collected, placed in solution, and then enumerated to determine the concentration of vegetative
Bacillus anthracis in the solution. No further characterization was performed on these vegetative
anthrax cells. Solutions of these cells were used to determine the sensitivity of the Tetracore
ELISA to vegetative cells.
12
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Table 4-1. Characterization Information for Battelle Preparation of Anthrax Spores
Characterization Outcome Analysis Performed By
% vegetative cells 0.06% Battelle
Viable spore count 5.26xio9 Battelle
Guinea pig 10 day LD50 10 spores Battelle
DNA fingerprinting MLVA Genotype 62 CDC
PA gene sequencing Protective Antigen Type I CDC
Regardless of the source and type of anthrax stock solution used to make test samples, its
concentration was confirmed by a plate enumeration method. This was done within 24 hours of
any stock solution being used for test sample preparation and is described in Battelle SOP MREF
X-054, Enumeration ofBL-2 andBL-3 Bacteria Samples Via the Spread Plate Technique. In
addition, four times during the verification test the serial dilution method was validated by
enumerating the PT samples. For example, for a 109 spores/mL sample to be enumerated, the
method requires that it be diluted to at least 103 spores/mL so 100 |^L of sample will provide a
countable number of spores on a culture plate. Therefore, if 100 |j,L of the 103 spores/mL solution
provided the correct number of spores to the plate, the concentration of every serial dilution made
to obtain that concentration was confirmed.
4.3.3 Anthrax Enumeration Data
Table 4-2 gives the results of all plate enumerations performed throughout the verification test on
anthrax solutions prepared in DI water. The data from enumerations to validate the serial dilution
method are also given in Table 4-2. The expected concentration, as determined from a previous
enumeration (if available), the actual concentration, and the relative percent difference between
the two are given in the table. Relative percent difference (RPD) is determined using the
following equation, where E is the expected concentration and A is the actual concentration as
determined by the enumeration.
For the Battelle-prepared, phenol-preserved spores, only one enumeration resulted in a concen-
xlOO%
tration that was more than 25% different from the expected concentration. The average
concentration of the Battelle stock solution was 6 x 109 spores/mL (ranging from 5.3 x 109 to
8.2 x io9 spores). Over the two-month period that the stocks were used and the enumeration
performed, the relative standard deviation of the eight results was 15%. The accuracy and
13
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Table 4-2. Anthrax Enumeration Data for PT Samples
Spore Solution
Description (units)
Battelle-prepared, phenol-
preserved stock solution
(108 spores/mL)
Battelle-prepared, phenol-
preserved serial dilution
validations
(104 spores/mL)
Vegetative anthrax
(104 cfu/mL)
Dugway-prepared
(106 spores/mL)
Date
January 28
January 28
January 30
February 2
February 10
February 26
March 1
March 23
January 28
January 30
March 2
March 23
March 23
March 24
March 22
March 23
March 24
Expected
Concentration
53
58
53
61
61
82
63
67
10
40
10
1,000
Unknown
260
Unknown
0.010
10
Actual
Concentration'3'
58
53
61
53
82
63
67
57
7.8
32
7.7
992
26
350
666
0.0081
8.0
RPD
9
9
15
14
55
23
5
14
22
20
24
1
NA
35
NA
19
20
(a) Each enumeration involved the development of three to five plates. The average, standard deviation, and relative
standard deviation for each set of Battelle-prepared, phenol-preserved enumeration data were determined, and the
average relative standard deviation of all enumerations was calculated to estimate the variability in the enumeration
process, which was 15%.
NA = not applicable.
precision of these enumerations indicate that the concentration of the spore stock solution was
consistent over several months and was usually close to the expected concentration. The serial
dilution validation data confirm that the PT samples containing the Battelle-prepared, phenol-
preserved spores were prepared accurately at various concentration levels. Also shown in Table 4-
2 are the enumerations performed to determine the concentration of the vegetative anthrax cells
and a stock solution of spores obtained from Dugway Proving Ground. For enumerations with
unknown expected concentrations, the concentration of that particular solution or the stock from
which it had been prepared had not previously been determined.
Table 4-3 gives the enumeration data for all of the interferent PT (shaded) and DW samples that
were spiked with anthrax spores. For possible interferent samples and samples prepared in DW,
the addition of spores was confirmed by enumeration for at least one sample representing each
matrix. The results of the DW samples enumerated in late January and early February indicated
that the relative difference between the expected concentration and the actual concentration
ranged from 17 to 96%. The larger percent differences for the DW samples as compared with the
PT samples were not a surprise, considering that DW is presumably an interferent-prone matrix.
These data suggest that spore health is dependent on whether the solution is in DI water or DW.
14
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Table 4-3. Anthrax Enumeration Results for Fortified Interferent and Drinking Water
Samples
Sample
Description
Expected Actual
Date Concentration Concentration'"'
(2004) (105 spores/mL) (105 spores/mL)
RPD
Cone. CA DW
Cone. CA DW
Unconc. CA DW
January 28
January 30
January 30
10
100
40
0.38
8.7
96
91
80
0.5mg/LOC
2.5mg/LOC
230 mg/L Ca
90 mg/L Mg
46 mg/L Ca
18 mg/L Mg
Cone. CA DW
Unconc. CA DW
Cone. OH DW
Unconc. OH DW
Cone. NY DW
Unconc. NY DW
Cone. FL DW
Unconc. FL DW
February 2
February 3
February 3
February
February 3
February 3
February 3
February 3
February 3
February 3
February 3
February 3
15
15
15
15
15
15
15
15
6.9
6.5
5.7
6.9
13
12
9.1
7.5
54
57
62
54
17
21
39
50
Cone. NY DW
Cone. CA DW
2.5 mg/L OC
230 mg/L Ca
90 mg/L Mg
March 3
March 3
March 3
March 3
1,000
1,000
1,000
1,000
2.5 mg/L OC
Cone. CA DW
March 23
March 23
1,000
1,000
962
448
4
55
230 mg/L Ca
90 mg/L Mg
Cone. NY DW
March 24
March 24
1,000
1,000
788
486
OC = Organic carbon (humic and fulvic acids)
Shading on table distinguishes the interferent and cross-reactivity PT samples from the DW samples.
(^ The uncertainty of the enumeration technique is approximately 15%.
15
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However, the effect of DW on spore health seemed to be less significant when the concentration
of spores was higher. For example, in March, when the DW and interferent samples were spiked
with higher concentrations of anthrax spores, the difference between the expected concentration
and the actual concentration for the interferent samples was between 0 and 21% and for the DW
samples between 7 and 55%. Enumerations were performed to characterize the concentration of
spores in each sample matrix. For each test matrix, spores were enumerated within a day of
testing. In the Chapter 6 tables, the actual concentrations of the test samples have been corrected
for the result of the appropriate enumeration for that sample. Because not every test sample was
enumerated and some of the test samples were the result of dilutions of enumerated samples, not
every actual concentration will be represented directly in Table 4-2 or Table 4-3.
The concentrations of the possible cross-reactive interferents of soybean lectin (analogue of ricin)
and lipopolysaccharide (analogue of botulinum toxin) were not confirmed independent of the
COA received from the supplier because of the lack of available analytical methodologies for
these analytes. However, samples containing Bacillus thuringiensis (analogue of anthrax) were
enumerated and were determined to be approximately an order of magnitude less than expected
because some spores were lost during washing with water. Because the lowest detectable concen-
tration of anthrax was much more concentrated than Tetracore, Inc., had claimed, additional PT
samples containing higher concentration levels of anthrax were prepared and analyzed. Additional
resources were not expended to determine the cross-reactivity of Bacillus thuringiensis at
comparable concentration levels.
4.4 Technical Systems Audit
The Battelle Quality Manager conducted a technical systems audit (TSA) to ensure that the
verification test was performed in accordance with the test/QA plan(4) and the AMS Center
QMP.(11) As part of the audit, the Battelle Quality Manager reviewed the standards and methods
used, compared actual test procedures with those specified in the test/QA plan,(4) and reviewed
data acquisition and handling procedures. Observations and findings from this audit were docu-
mented and submitted to the Battelle Verification Test Coordinator for response. No findings
were documented that required any significant action. The records concerning the TSA are
permanently stored with the Battelle Quality Manager.
4.5 Audit of Data Quality
At least 10% of the data acquired during the verification test was audited. Battelle's Quality
Manager or designee traced the data from the initial acquisition, through reduction and statistical
analysis, to final reporting, to ensure the integrity of the reported results. All calculations
performed on the data undergoing the audit were checked.
16
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4.6 QA/QC Reporting
Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.:
of the QMP for the ETV AMS Center.(11) Once the assessment report was prepared, the Battelle
Verification Test Coordinator responded to each potential problem and implemented any
necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up
corrective action was taken. The results of the TSA were sent to the EPA.
4.7 Data Review
Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-4 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
Table 4-4. Summary of Data Recording Process
Data to Be Recorded
Dates and times of test
events
Responsible
Party
Battelle
How Often
Where Recorded Recorded
ETV data sheets Start/end of test,
and at each
change of a test
parameter
Disposition
of Data
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Sample collection and
preparation information,
including chain-of-
custody
Battelle
ETV data sheets
and chain-of-
custody forms
At time of sample
collection and
preparation
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Detection device
procedures and sample
results
Battelle
ETV data sheets
Throughout test
duration
Manually incorporated
in data spreadsheets
Anthrax enumeration
data
Battelle
Enumeration data
forms
With every
enumeration
Manually incorporated
in data spreadsheets
Reference method
procedures and sample
results
ATEL
Data acquisition
system, as
appropriate
Throughout
sample analysis
process
Transferred to
spreadsheets and
reported to Battelle
17
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Chapter 5
Statistical Methods and Reported Parameters
The methods presented in this chapter were used to verify the performance parameters listed in
Section 3.1. The Tetracore ELISAtest kits produce qualitative results; i.e., the color change
within a sample well on a Tetracore ELISA plate indicates only the presence or absence of a
contaminant, not a measure of the concentration present. Therefore, the data evaluation methods
were used in that context.
5.1 Qualitative Contaminant Presence/Absence
Accuracy was assessed by reporting the number of positive results out of the total number of
samples tested for the Tetracore ELISA at each concentration level of contaminant-only PT
sample tested for anthrax spores, botulinum toxin, and ricin.
5.2 False Positive/Negative Responses
A false positive response was defined as a positive response when the DI water or DW sample was
spiked with a potential interferent, a cross-reactive compound, or not spiked at all. A false
negative response was defined as a negative response when any sample was spiked with a
contaminant at a concentration greater than the lowest detectable concentration of the Tetracore
ELISA for each analyte in DI water. Interferent PT samples, cross-reactivity PT samples, and DW
samples were included in the analysis. The number of false positive and negative results is
reported.
5.3 Consistency
The reproducibility of the results was assessed by calculating the percentage of individual test
samples that produced positive or negative results without variation within replicates.
18
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5.4 Lowest Detectable Concentration
The lowest detectable concentration for each contaminant was determined to be the concentration
level at which at least two out of the three replicates generated positive responses. These concen-
tration levels are determined for each target contaminant in solutions of DI water.
5.5 Other Performance Factors
Aspects of the Tetracore ELISA performance such as ease of use, field portability, and sample
throughput are discussed in Section 6. Also addressed are qualitative observations of the
verification staff pertaining to the performance of the Tetracore ELISA.
19
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Chapter 6
Test Results
6.1 Qualitative Contaminant Presence/Absence
The responses for the Tetracore ELISA using the contaminant-only PT samples containing
anthrax, botulinum toxin, and ricin are discussed in the following sections. The Tetracore ELISA
provides indication of only a positive or negative response based on whether the color of a
solution in a sample well changes to a color different from that of the method blank sample.
6.1.1 Anthrax
The results obtained for the performance test samples containing anthrax spores are given in
Table 6-1 a. The first five concentration levels listed were initially analyzed, and the results
indicated that none of those samples (up to 50 times the vendor-stated LOD) produced detectable
results. The Battelle-prepared, phenol-preserved serial dilution validation enumeration on
February 2 confirmed the concentration of the stock solution used to prepare the solutions
analyzed by the Tetracore ELISA. In addition, the serial dilution validation enumeration on
January 30 (1 x 10s spores/mL expected) confirmed that anthrax solutions can be accurately
diluted using standard techniques. After discussions with Tetracore, Inc., the following
speculative explanations for these results were considered:
1. The target proteins on the spore's surface may have been stripped off or chemically altered by
phenol in the storage solution. (The absence or alteration of these proteins would probably
decrease the sensitivity of the Tetracore ELISA to the affected spores.)
2. The sensitivity of the Tetracore ELISA to anthrax spores is dependent on the method used to
prepare the spores; therefore, the spores prepared at Battelle may result in decreased
responsiveness compared with spores prepared elsewhere.
20
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Table 6-1 a. Anthrax Contaminant-Only PT Sample Results
Purpose of
Analysis
Actual Fortified
Concentration^'
200 spores/mL(b)
2 x 104spores/mL(c)
Original test/QA
plan PT samples
Expanded
sensitivity
determination
Alternate spore
preparation
Vegetative cell
sensitivity
8 x
2 x
8 x
8 x
8 x
8 x
8 x
8 x
8 x
8 x
O
O
O
3
104spores/mL
10s spores/mL
105spores/mL
108spores/mL
107spores/mL
106spores/mL
106 spores/mL
10s spores/mL
104 spores/mL
103 spores/mL
x 105cfu/mL
x 104cfu/mL
x 103cfu/mL
x 102cfu/mL
Anthrax
Description
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Vegetative
Vegetative
Vegetative
Vegetative
Prep
Location
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Dugway
Dugway
Dugway
Dugway
Battelle
Battelle
Battelle
Battelle
Phenol-
Preserved
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
NA
NA
NA
NA
Positive
Results Out
of Total
Replicates
0/3
0/3
0/3
0/3
0/3
3/3
3/3
3/3
0/3
0/3
0/3
0/3
3/3
3/3
0/3
0/3
(a) Actual concentrations were corrected for the enumeration of the stock solution from which each sample was
prepared. The uncertainly of the enumeration technique is approximately 15%.
^ ' Lethal dose concentration.
(c) Vendor-stated LOD.
NA = not applicable. Vegetative cells were not prepared from any stock solution; they were grown and placed in
solution.
Additional testing beyond that described in the test/QA plan was performed to explore these
possible explanations and to gain more information about the performance of the Tetracore
ELISA. It included evaluating whether Battelle's storage of the stock solution of anthrax spores in
a 1% solution of phenol had any impact on the performance of the Tetracore ELISA, increasing
the concentration of spores beyond what was required by the test/QA plan, and subjecting the
plates to Ames strain anthrax spores prepared by Dugway Proving Ground using a preparation
method that is different from the one Battelle uses.
21
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To address the possibility that storing spores in phenol affected the sensitivity of the Tetracore
ELISA, as well as the possibility that the Tetracore ELISA could not detect the spores prepared by
Battelle as readily as the spores prepared elsewhere, a series of samples was prepared using one
anthrax spore stock solution that had been stored in a phenol solution and one that had not. Using
the Battelle preparation, samples containing approximately 109, 108, and 107 spores/mL were
prepared and analyzed. All three concentration levels were detectable, whereas 106 spores/mL had
been shown to be not detectable during prior testing. Therefore, the lowest detectable concentra-
tion for Battelle-prepared spores was approximately 107 spores/mL. Spores that were prepared at
Dugway Proving Ground and received at Battelle in 2001 were then analyzed. Since 2001, the
Dugway stock solution had been refrigerated as a solution of spores in spent media. The solution
was washed in DI water as described for the phenol storage solution above and diluted by tenfold
factors several times to prepare solutions with various concentration levels (107, 106, 10s, and
104 spores/mL). Both the stock solution concentration and the dilution methodology were
confirmed by plate enumeration as shown in Table 4-2. These samples were analyzed to determine
the approximate sensitivity to these spores. None of these concentration levels were detectable.
Tetracore informed Battelle that its ELISA is more sensitive to vegetative anthrax than spores. To
evaluate this premise, a solution of vegetative cells was prepared by collecting a single vegetative
anthrax colony from an enumeration plate, placing it into DI water, and mixing it well. This
solution was diluted by a factor of 10 four times, and then the stock solution and two diluted
samples were enumerated to determine the concentration of vegetative cells in each sample. These
samples were analyzed to determine the approximate sensitivity for these vegetative cells. The
lowest detectable concentration of vegetative cells was 3 x 104 colony-forming units (cfu)/mL,
approximately the concentration of the vendor-stated LOD for anthrax spores.
6.1.2 Botulinum Toxin
The results obtained for the PT samples containing botulinum toxin Types A and B are given in
Table 6-lb. The results showed that the Tetracore ELISA was reproducibly sensitive to botulinum
toxin Type A at concentrations as low as 0.02 mg/L. However, for botulinum toxin Type B, the
results were very inconsistent. The lowest concentrated sample (0.004 mg/L) generated 2 out of 3
positive results, and the next two higher concentration levels (0.02 and 0.04 mg/L) resulted in
only 1 positive result out of 6 total replicates. The 0.2-mg/L samples generated 3 out of 3
detectable results, and the 0.3-mg/L sample generated 1 out of 3 detectable results. These results
are especially puzzling because the Tetracore ELISA was able to reproducibly detect 0.04 mg/L of
botulinum toxin Type B when it was spiked into possible interfering matrices, as well as DW.
There is no certain explanation for the inconsistent botulinum toxin Type B PT sample results.
22
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Table 6-1 b. Botulinum Toxin Contaminant-Only PT Sample Results
Purpose
of Analysis
Botulinum toxin
PT samples
Concentration
(mg/L)
0.004(a)
0.02
0.04
0.2
0.3(b)
Positive Results Out of
Total Replicates (Type A)
0/3
3/3
3/3
3/3
NA(c)
Positive Results Out of Total
Replicates (Type B)
2/3
0/3
1/3
3/3
1/3
(a) Vendor-stated LOD.
(b) Lethal dose concentration.
(c) This concentration level was not analyzed using Type A botulinum toxin.
6.1.3 Ricin
The results obtained for the PT samples containing ricin are given in Table 6-lc. With the
exception of the 0.0015 mg/L sample (which generated all negative results), all replicate samples
analyzed generated positive results.
Table 6-lc. Ricin Contaminant-Only PT Sample Results
Purpose
of Analysis
Ricin PT samples
Concentration
(mg/L)
0.001 5(a)
0.0075
0.015
0.075
15<">
Positive Results Out of
Total Replicates
0/3
3/3
3/3
O /O
3/3
o /o
3/3
^Vendor-stated LOD.
(b:i Lethal dose concentration.
6.2 False Positive/Negative Responses
Three types of samples were analyzed to evaluate the susceptibility of Tetracore ELISA to false
positive and negative results. These included interferent PT samples, made up of DI water
fortified with Ca and Mg and samples fortified with humic and fulvic acids with and without the
addition of target contaminants; cross-reactivity PT samples, made up of DI water fortified with a
contaminant similar biologically or chemically with each specific target contaminant; and DW
samples both concentrated and unconcentrated and both with and without the addition of target
contaminants. A false positive result was defined as a positive result in the absence of the target
contaminant, and a false negative result was defined as a negative result from a sample containing
detectable levels of each target contaminant.
23
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6.2.1 Interferent PT Samples
The results from the interferent PT samples are given in Table 6-2. For Tetracore ELISA plates
coated with antibodies specific to each contaminant, the number of positive results out of the
number of replicates is given for PT samples containing only the possible interferents and those
possible interferents in the presence of the listed concentration of target contaminant. For anthrax,
expanded testing included additional interferent PT samples with a higher concentration of
anthrax. Results for botulinum toxin Types A and B and ricin are presented.
For anthrax, no false positive results were generated from the interferent samples that did not
contain anthrax spores (blanks). The two spiked Ca and Mg interferent samples contained 6 and
8 x 10s spores/mL and were falsely negative with respect to the vendor-stated LOD. However,
because these concentrations were very similar to the concentration of the PT samples that were
also determined to be not detectable, it is not clear if the negative results were due to the
interferents. For both spiked humic and fulvic acid interferent samples containing 2 x 106
spores/mL, a concentration that had previously been shown not to be detectable in the PT
samples, the results were positive. There is not a clear explanation for these positive results.
Expanded testing was performed to evaluate the performance of the Tetracore ELISA when
analyzing higher concentrations of anthrax spores. During that testing, 1 x io8 spores/mL, a
detectable concentration in DI water, were spiked into two interferent solutions. In Ca and Mg, as
well as in humic and fulvic acids, none of the three replicates had false negative results.
Table 6-2. Interferent PT Sample Results
Interferent
Sample
46 mg/L Ca
18mg/LMg
230 mg/L Ca
90 mg/L Mg
0.5 mg/L
humic and
fulvic acid
2.5 mg/L
humic and
fulvic acid
Positive Results Out of Total Replicates
Anthrax (spores/mL)
Blank 2xl06(a)
0/3 0/3
8 x 105(b)
0/3 0/3
6 x 105(b)
0/3 3/3
2 x 106(b)
0/3 3/3
2 x 106(b)
IxlO8
NA
3/3
8 x 107(b)
NA
3/3
1 x 108(b)
Botulinum Toxin (mg/L)
Type B Type A
Blank 0.04 0.04
0/3 3/3 NA
0/3 3/3 3/3
0/3 0/3 NA
0/3 3/3 1/3
Ricin (mg/L)
Blank 0.015
0/3 3/3
0/3 3/3
0/3 3/3
0/3 3/3
NA = not applicable. Sample not analyzed during expanded testing of anthrax or testing of botulinum toxin Type A.
(^ Expected concentration.
(b:i Actual concentration.
24
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For botulinum toxin, no false positive results were generated from the interferent samples that did
not contain botulinum toxin. When botulinum toxin Type B was added to the interferent samples
at 0.04 mg/L, all the results were positive except for the 0.5 mg/L humic and fulvic acid sample.
This apparent interferent was unexpected because the higher concentration of humic and fulvic
acid did not cause false negative results. Two interferent samples were fortified with botulinum
toxin Type A. The Ca and Mg sample generated three out of three positive results (no false
negatives), but the humic and fulvic acid sample generated only one out of three positive results
(two false negatives). For ricin, there were no false positive or false negative results with respect
to the interferent samples.
6.2.2 DW Samples
The results from the DW samples are given in Table 6-3. For Tetracore ELISA plates coated with
antibodies specific to each contaminant, the number of positive results out of the number of
replicates is given for the DW samples containing no target contaminants and also the DW
samples in the presence of the listed concentration of each target contaminant. For anthrax,
expanded testing included additional DW samples containing a higher concentration of anthrax.
Results for botulinum toxin Types A and B and ricin are also given.
Table 6-3. DW Sample Results
DW Sample
Unconcentrated
CADW
Concentrated
CADW
Unconcentrated
FLOW
Concentrated
FLOW
Unconcentrated
NYDW
Concentrated
NYDW
Unconcentrated
OHDW
Concentrated
OHDW
Positive Results Out
Anthrax (spores/mL)
Blank 2xl06(a) IxlO8
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
7 x 105®
0/3
7 x 105®
0/3
8 x 105(b)
0/3
9 x 105(b)
0/3
1 x 106(b)
0/3
1 x 106(b)
0/3
7 x 105®
0/3
6 x 105®
NA
3/3
5 x 107(b)
NA
NA
NA
3/3
5 x 107(b)
NA
NA
of Total
Replicates
Botulinum Toxin (mg/L)
Type B Type A
Blank 0.04 0.04
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
NA
3/3
NA
NA
NA
3/3
NA
NA
Ricin
Blank
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
(mg/L)
0.015
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
NA = not applicable. Sample not analyzed during expanded testing.
(a) Expected concentration.
(b) Actual concentration.
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For anthrax, there were no false positive results for the unspiked DW samples. Also, the DW
samples initially spiked with an expected concentration of 2 x 106 spores, but with actual
concentrations of between 7 x io5 and 2 x io6 spores/mL, generated false negative results with
respect to the vendor-stated LOD. As had been observed for the humic and fulvic acid interferent
samples, the negative results cannot necessarily be attributed to the DW matrix. The DW samples
spiked with approximately 1 x IO8 spores/mL generated no false negative results.
For botulinum toxin, there were no false positive results for the unspiked DW samples or false
negative results for either the Type B or Type A botulinum toxin. In 24 out of 24 Type B replicate
samples spiked with 0.04 mg/L botulinum toxin Type B, all the results were positive, again
contrasting with the PT sample results, where 0.04 mg/L was only detected in 1 out of 3
replicates. Similar results were generated for ricin. No false positives were observed for the
unspiked DW samples, and no false negative results were observed for the 0.015 mg/L spiked DW
samples.
6.2.3 Cross-Reactivity PT Samples
The results from the cross-reactivity PT samples are given in Table 6-4. For Tetracore ELISA
plates coated with antibodies specific to each target contaminant, a PT sample fortified with a
spore or chemical similar to each target contaminant was analyzed in the absence of any of the
target contaminant. The number of positive results out of the number of replicates is given for
each sample. All of the results were correctly reported as negative.
Table 6-4. Potentially Cross-Reactive PT Sample Results
Positive Results Out of Total Replicates
Botulinum
Anthrax Toxin Ricin
Bacillus thuringiensis (1 x io4 spores/mL)(a)
Lipopolysaccharide (0.04 mg/L)
Lectin from soybean (0.015 mg/L)
(-^ Concentration was determined after the fact to be below the lowest detectable concentration. Therefore, the non-
detectable results may not indicate a lack of cross-reactivity.
6.3 Consistency
For the Tetracore ELISA analysis of anthrax spores and ricin, the results were 100% consistent.
The Tetracore ELISA plates coated with antibodies specific for ricin generated results for 30
sample sets of three replicates, and all of them produced results that were either all positive or all
negative. Similarly, for anthrax, all 47 sets were entirely positive or negative. The botulinum toxin
results were not as consistent as the anthrax and ricin results. The PT samples containing
botulinum toxin Type B generated 3 out of 5 sample sets in which the results were not consistent.
26
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All four botulinum toxin Type A PT sample sets, however, generated consistent results. The
botulinum toxin Type B interferent and DW samples were 100% consistent over 16 sample sets.
Overall, 98% of the Tetracore ELISA samples analyzed generated results in sets of three in which
all the individual replicates had the same result, whether positive or negative.
6.4 Lowest Detectable Concentration
The lowest detectable concentration of each target contaminant was defined as the lowest
concentration of contaminant-only PT sample to have at least two out of three positive results. For
anthrax spores, that concentration was 8 x 106 spores/mL (for Battelle-prepared spores) and 3 x
104 cfu/mL for the vegetative anthrax cells. The spores from the Dugway preparation were not
detectable at levels up to 8 x io6 spores/mL. For botulinum toxin Type A, the lowest detectable
concentration was 0.02 mg/L; but, for Type B, it was less clear because of the inconsistent results
for the PT samples. The PT sample results indicated that the lowest detectable concentration was
0.004 mg/L, but some concentrations above that were not detectable. However, 0.04 mg/L was
consistently detectable in the interferent and DW samples, so the LOD is at least 0.04 mg/L. For
ricin, the lowest detectable concentration was 0.0075 mg/L.
6.5 Other Performance Factors
Battelle technicians, who had been trained by Tetracore to perform testing using the Tetracore
ELISA, performed all of the required laboratory testing. The technicians had no problem
performing the tests as they were trained. In addition to the antibody-coated Tetracore ELISA
plates and other reagents, a multichannel pipette, disposable tips, and plastic troughs for filling
the multichannel pipette with solution were required for testing. Because the Tetracore ELISA
plates were made from transparent plastic, a color change in a sample well could be observed
visually against a white background, such as a piece of paper. At times it was difficult to
determine whether a sample was different from the negative control sample. A plate reader would
eliminate that judgment call by the operator.
The performance of the Tetracore ELISA was not demonstrated outside the laboratory because the
functioning of the tools/equipment required for the Tetracore ELISA is independent of location.
Also, the procedure for laboratory analysis would not change if testing was moved from the
laboratory. No carrying case is provided, but a small box could easily be used for transporting the
supplies needed. The main requirement for a non-laboratory setting would be a well-lighted, flat
work space. Depending on the conditions, it may be beneficial to cover the ELISA plates during
incubation to avoid contamination.
Similarly, testing using an untrained operator was not performed because of the background
knowledge necessary to understand the detailed procedures for performing the testing and to
correctly use a multichannel pipettor. The operators with a technical background could analyze 48
samples on a single plate in approximately five hours.
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Chapter 7
Performance Summary
Table 7-1. Anthrax Summary Table
Parameter
Contaminant-
only PT
samples
Qualitative
contaminant
results
Interferent
PT samples
DW samples
Cross-
reactivity
False positives
Actual Fortified
Anthrax
Sample Information Concentration'3'
8 x 108 spores/mL
Battelle-prepared, phenol- 8 x 107 spores/mL
preserved spores 8 x 1 06 spores/mL
8 x 105 spores/mL
3 x 105 cfu/mL
3xl04cfu/mL
Vegetative cells _ , ~3 ,, , T
5 3 x 103 cfu/mL
3 x 103 cfu/mL
8 x 106spores/mL
^ , 8 x 105spores/mL
Dugway-prepared spores g x 104spores/mL
8 x 103spores/mL
230 mg/L Ca 01^7 / T CM
„„ „ ,, 8 x 107spores/mL(b)
90 mg/L Mg F
2.5 mg/L humic acid , In8 , T m
„ ,. „ „ , . . , 1 x 10s spores/mL1 >
2.5 mg/L fulvic acid
Humic acid and fulvic acid 2 x 106 spores/mL(b)
Ca and Mg 2 x 106 spores/mL
Concentrated CA 5 x 107 spores/mL(b)
Concentrated NY 5 x 107 spores/mL(b)
Unconcentrated DW 2xl06 spores/mL
1 x 104 spores/mL ., ,
„ , unspiked
Bacillus thuringiensis
Positive Results Out of
Total Replicates
3/3
3/3
3/3
0/3
3/3
3/3
0/3
0/3
0/3
0/3
0/3
0/3
3/3
3/3
6/6
0/6
3/3
3/3
0/24
0/3
No false positives resulted from the analysis of the interferent, DW, or cross-
reactivity samples. However, two humic and fulvic acid samples, spiked at
concentrations below what was detectable in DI water, generated positive results.
Bacillus thuringiensis was prepared at concentrations much lower than the lowest
detectable concentration of Bacillus anthracis. Therefore, negative results with
these samples do not necessarily indicate a lack of cross-reactivity.
28
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Parameter
False negatives
Consistency
Lowest detectable
concentration
Other performance factors
Sample Information
No false negative results were generated for the analysis of interferent or DW
samples spiked with detectable levels of anthrax. Tetracore ELISA was not able to
detect anthrax at the vendor-stated LOD, but was able to at much higher
concentrations. All of the unconcentrated DW and six Ca and Mg samples were
spiked at concentrations less than detectable by the test strips and, therefore, were,
as expected, negative.
100% (47 out of 47) of the results were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or negative.
8 x 106 spores/mL - Battelle prep (vendor-stated LOD: 2 x 104 spores/mL); 3 x
104 cfu/mL - vegetative anthrax (no vendor-stated LOD); the Dugway preparation
of spores was not detectable at concentrations up to 8 x 1 06 spores/mL
A technically trained operator easily performed the Tetracore ELISA analysis.
Untrained, non-technical, first-time users would not likely be able to perform the
testing because of the need to use a multichannel pipettor, prepare solutions, and
read a technical operating procedure. The Tetracore ELISA could be used outside
the laboratory without a problem. At times it was difficult to determine whether
the color of the sample had changed; no reader was used. Sample throughput was
48 samples in 5 hours.
^ The uncertainly of the enumeration technique was approximately 15%.
*• ' Battelle-prepared, phenol-preserved spores.
29
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Table 7-2. Botulinum Toxin Summary Table
Parameter
Qualitative
contaminant
positive
results
Contaminant-
only PT
samples
Interferent
PT samples
DW samples
Cross-
reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Other performance factors
Botulinum Toxin Positive Results Out
Sample Information Concentration (mg/L) of Total Replicates
0.004 0/3
Type A °-02 3/3
0.04 3/3
0.2 3/3
0.004 2/3
0.02 0/3
TypeB 0.04 1/3
0.2 3/3
0.3 1/3
_. . „ . „ „ . 3/3 Type A
CaandMg 0.04 J^
6 6/6 Type B
Humic acid and fulvic acid 0.04
3/6 Type B
Concentrated DW 0.04 iSype^
Unconcentrated DW 0.04 ^n^^
12/12 TypeB
0.04 mg/L
, . f . . , unspiked 0/3
Lipopolysaccharide
There were no false positive results for the interferent, DW, or cross-reactivity
samples.
Two out of three results were false negative when 0.04 mg/L botulinum toxin
Type A was spiked into 2.5 mg/L humic and fulvic acids, and three out of three
were false negatives when botulinum toxin Type B was spiked into 0.5 mg/L humic
and fulvic acids. There were no false negatives for the spiked DW samples.
With the exception of 2.5 mg/L humic and fulvic acids spiked with 0.04 mg/L
botulinum toxin Type A (1 out of 3 positive), results generated for botulinum toxin
Type A were 1 00% consistent. The D W and interferent samples spiked with
botulinum toxin Type B were equally consistent, but the contaminant PT samples
containing botulinum toxin Type B generated consistent results in just 2 out of 5
sample sets. Overall, 98% of the results were from sample sets that were either all
positive or all negative.
0.02 mg/L (Type A); not clear for Type B because of sporadic results, (vendor-
stated LOD for botulinum toxin [non-specific]: 0.004 mg/L)
A technically trained operator easily performed the Tetracore ELISA analysis.
Untrained, non-technical, first-time users would not likely be able to perform the
testing because of the need to use a multichannel pipettor, prepare solutions, and
read a technical operating procedure. The Tetracore ELISA could be used outside
the laboratory without a problem. At times it was difficult to determine whether the
color of the sample had changed; no reader was used. Sample throughput was 48
samples in 5 hours.
30
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Table 7-3. Ricin Summary Table
Parameter
Qualitative
contaminant
positive
results
Contaminant-
only PT
samples
Interferent PT
samples
DW samples
Cross-
reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Other performance factors
Ricin
Concentration Positive Results Out of Total
Sample Information (mg/L) Replicates
0.0015 0/3
0.0075 3/3
Ricin PT samples Q Q15 ^
0.075 3/3
15 3/3
CaandMg 0.015 6/6
Humic acid and fulvic _ _ , - , . ,
., 0.015 6/6
acid
Concentrated CW 0.015 12/12
Unconcentrated DW 0.015 12/12
0.015 mg/L .. , „.,
. ,,b . unspiked 0/3
Lectm from soybean
No false positive results were generated for ricin in DW or interferent samples.
There were no false negative results for interferent or DW samples spiked with
detectable concentrations of ricin.
1 00% of the results for ricin were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or negative.
0.0075 mg/L (vendor-stated LOD: 0.0015 mg/L)
A technically trained operator easily performed the Tetracore ELISA analysis.
Untrained, non-technical, first-time users would not likely be able to perform
the testing because of the need to use a multichannel pipettor, prepare solutions,
and read a technical operating procedure. The Tetracore ELISA could be used
outside the laboratory without a problem. At times it was difficult to determine
whether the color of the sample had changed; no reader was used. Sample
throughput was 48 samples in 5 hours.
31
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Chapter 8
References
1. Personal communication with Dick Burrows, U.S. Army Center for Health Promotion and
Preventive Medicine.
2. U.S. EPA threat prioritization study provided by Steve Allgeier, U.S. EPA Office of Water.
3. Center for Defense Information Fact Sheet: Ricin, www.cdi.org/terrorism;ricin-pr.cfm.
4. Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio,
January 2004.
5. U.S. EPA Method 180.1, "Turbidity (Nephelometric)," Methods for the Determination of
Inorganic Substances in Environmental Samples, EPA/600/R-93/100, August 1993.
6. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, Washington, D.C., 1997.
7. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020,
March 1983.
8. U.S. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
Inductively-Coupled Plasma Mass Spectrometry," in Methods for the Determination of
Organic Compounds in Drinking Water, Supplement I, EPA/600/R-94/111, October 1994.
9. U.S. EPA Method 524.2, "Permeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking
Water—Supplement III, EPA/600/R-95/131, August 1995.
10. U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector," Methods for the Determination of
Organic Compounds in Drinking Water—Supplement III., EPA/600/R-95/131, August 1995.
11. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle, Columbus,
Ohio, March 2004.
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