s-PA-600/1-77-01
March 1977
A SOLID SUBSTRATE IMMUNOLOGICAL
ASSAY FOR MONITORING ORGANIC
ENVIRONMENTAL CONTAMINANTS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animals—but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-77-018
March 1977
A SOLID SUBSTRATE IMMUNOLOGICAL ASSAY FOR
MONITORING ORGANIC ENVIRONMENTAL CONTAMINANTS
By
Herbert R. Lukens and Colin B. Williams
IRT Corporation
San Diego, California 92138
Contract No. 68-02-2202
Project Officer
M. F. Copeland
Experimental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
-------
DISCLAIMER
This report has been reviewed.by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
-------
FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
This project assesses the use of an immunoassay technique (antibody-
antigen responses) as a possible warning system for exposure to a
toxicant. If successful, it could ultimately develop into a system for
monitoring human exposure to toxicants as film badges now monitor exposure
to radiation which would be a novel breakthrough in an old problem that
would have application in agriculture as well as occupational health.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
111
-------
ABSTRACT
A solid substrate "film-badge" type monitor has been developed that is
capable of detecting 2-aminobenzimidazole (2-ABZI) at less than one part per
million in water in less than 10 minutes. The monitor makes use of the reac-
tion which takes place between 2-ABZI in the sample and a monolayer of its
antibody that has been deposited on a thin film of indium on a glass substrate.
A second approach in which the antibody is mounted on polystyrene and
reaction of its antigen-binding sites with a fluorescein-labeled antigen are
subject to competition with nonlabeled antigen in the sample, has been demon-
strated in principle. Improvements in this alternate approach are proposed.
IV
-------
CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
1. Introduction 1
2. Conclusions 4
3. Recommendations . 5
4. Methods and Materials . 7
5. Results 13
6. Discussion of Results 29
7. References 35
Appendices
A. Physical Basis for the Visual Observation of the Antibody-
Antigen Reaction 36
B. The Antibody Molecule 39
-------
FIGURES
Number
1 Agglomeration of indium particles on glass substrate
(Reichart Metalograph Polaroid Photograph) 10
2 Tn/Tno of fresh AB-film versus BSA solution as a function
of time and BSA concentration 31
3 Influence of aging on the reaction between AB-film and
BSA solutions , ... 32
B-l Schematic of IgG structure 40
VI
-------
TABLES
Number
1 Indium Slides 9
2 Change in Transmittance with Antibody and Antigen-Antibody
Layers on 1000 A Indium Films 14
3 Reactions of Protein Solutions with Indium Film 16
4 Initial Attempt to React BSA with Indium Coated with Anti-BSA ... 16
o
5 Effect of KOH on the Transmittance of Antibody Mounted on 200 A
Indium Films 18
6 Reaction of BSA Solutions with Freshly Prepared AB-Film 19
7 Reaction of BSA Solution with Week Old AB-Film 19
8 Reaction of BSA Solution with AB-Film Freshly Prepared from Old,
Alkaline Anti-BSA Solutions 20
VII
-------
SECTION 1
INTRODUCTION
BACKGROUND
Increasing concern for monitoring the natural environment and workplace
can be better understood when it is noted that over one-hundred-billion pounds
of organic chemicals, and in particular over one-billion pounds of pesticides,
are manufactured annually in the United States alone (Ref. 1). Many sophisti-
cated and sensitive assays have been developed for the detection and monitoring
of environmental contaminants, but all suffer from one or more limitations when
considered from the viewpoint of sensitivity, speed, low cost, simplicity, and
specificity. In addition, movement of an individual throughout a particular
industrial facility or agricultural area could well expose him to differing
concentrations of a contaminant for differing periods of time, thus requiring
that all possible sites of exposure be monitored.
This requirement logically leads to the consideration of workers carrying
on their person exposure monitors, in the same manner that film badges are
worn to monitor exposure to ionizing radiation. The present work has explored
the feasibility of such badges based upon the immunochemical specificity and
sensitivity of antibody for its causative agent, which in this case would be
the contaminant of environmental concern.
Two approaches have been investigated. In one approach the antibody was
bound to a very thin metal film deposited on a glass substrate. Subsequent
reaction of the antibody with the contaminant was detected by changes in
optical transmittance of the glass, film, protein complex. In the second
approach, antibody was affixed to a polymeric surface, and reaction with the
contaminant was observed by noting the inhibition of the reaction with
fluorescein-labeled contaminant.
-------
It has long been known that immunological techniques provide a highly
specific and sensitive assay for many compounds of high molecular weights
such as, for example, proteins. Recent work in which low molecular weight
compounds have been coupled to carriers with resulting immunospecificity has
extended the range of compounds which can be detected using such techniques.
A specific example of such an assay has been recently demonstrated by IRT
Corporation under the sponsorship of the Environmental Protection Agency. An
assay was developed which allowed the detection of 2-aminobenzimidazole
(2-ABZI, molecular weight 133), using a fluorescence polarization technique
with a detection limit in the picograms/mA range (Ref. 2). Such an assay
requires relatively sophisticated equipment, and is limited to the detection
of the contaminant in aqueous solutions. It does, however, demonstrate that
antibody, which is an essential part of the assay, can be produced to low
molecular weight compounds with a high degree of sensitivity and specificity.
In a recent, extension of conventional immunological techniques, Giaever
(Re*f. 3) has developed a visual method ,of detecting an antibody-antigen reac-
tion. His method demonstrates that the protein, bovine serum albumin (BSA),
can be adsorbed onto a metal film, and that the antibody to BSA will subse-
quently react with the BSA. In the initial step, BSA is bound to the metal
film, and then reacted with anti-BSA, after which a change in the optical
density of the film occurs and visual evidence of the reaction is obtained.
In Giaever1s experiments he was unable to carry out the procedure in the
reverse order (Giaever, private communication), i.e., the anti-BSA being
bound to the metal film with subsequent binding of BSA to the anti-BSA. This
defined a major area in our research task since use of metal film as a sub-
strate for a film badge system would not be suitable for detecting an antigen
unless the antibody could be deposited on the metal film without destroying
its specificity and sensitivity.
Inasmuch as investigation of the metal-film substrate system was a major
objective of this work, the antibody deposition technique received consider-
able attention. The use of a polymeric substrate was analogous to the use
of the metal film. However, the properties of film on polymer were not suit-
able for detection by optical transmission. Therefore, the use of a
fluorescein-labeled compound was required.
-------
2-ABZI was chosen as the contaminant of interest since an assay for this
compound had been developed under a previous EPA contract (Ref. 2), and conse-
quently results exist which will allow a comparative assessment of the sensi-
tivity of the presently proposed assay to be made. However, limited quantities
of the antibody to 2-ABZI exist, and, therefore, it was determined that in the
initial stages of the program, where techniques were being developed and refined,
it would be appropriate to use a more readily available antigen-antibody system,
and consequently BSA and anti-BSA were used for this purpose.
OBJECTIVES
The objective of this program was to develop a solid substrate immuno-
logical assay for the detection of organic contaminants of environmental
concern in a manner similar to the well-known radiological "film badge" type
of monitor. Specifically, the following eight tasks were undertaken and
successfully completed.
1. Metal-coated glass slides were prepared to serve as a solid
support for the antibody matrix.
2. Antibody specific to BSA was deposited as a monolayer on the
metal glass slide, and tested for reactivity to BSA.
3. Antibody specific to 2-ABZI was deposited as a monolayer on
the metal-coated glass slide.
4. Antibody product was deposited on an organic polymer sub-
strate as indicator slides.
5. Organic polymer-antibody preparation was examined for sensi-
tivity, reproducibility, and specificity to BSA.
6. The glass indicator slides were evaluated for sensitivity and
selectivity for 2-ABZI as a function of contaminant concentra-
tion.
7. The selectivity and sensitivity of the glass indicator slides
were compared, to results obtained by a fluorescence polariza-
tion immunoassay technique.
8. Applicability of the slides prepared in Task 3 to monitor the
concentration of 2-ABZI in water was evaluated, and recommended
configurations and monitoring techniques are herein proposed.
-------
SECTION 2
CONCLUSIONS
A solid substrate "film-badge" type monitor has been developed that is
capable of qualitatively detecting 2-aminobenzimidazole (2-ABZI) at less
than one part per million (ppm) in water in less than 10 minutes. The badge
o
is prepared by vacuum vapor deposition of a thin (^100 A) film of indium (In)
on a glass substrate, and attaching a monolayer of 2-ABZI antibody to the
indium. The latter step is carried out under conditions that leave free the
immunospecific positions of a significant number of antibody molecules. After
deposition of the antibody on the indium, glass-substrate, the badge is air
dried, and its optical transmittance measured on a densitometer. It may then
be used by either immersion in an aqueous sample or by placing an aliquot of
the sample on the monitor. After a short incubation period in the presence
of the unknown, the badge is rinsed, air dried, and its transmittance once
again measured. A decrease in the transmittance from its preexposure value
indicates the presence of 2-ABZI. If only a part of the film is exposed to
the sample, a visible difference between pre- and postexposure optical proper-
ties may be noted with ease when the sample contains detectable amounts of
2-ABZI.
The use of an alternate approach using somewhat more simplified measuring
equipment has been demonstrated in principle. It consists of a polystyrene
substrate to which antibody has been attached by a chemical reaction, and
which becomes fluorescent upon exposure to fluorescein-labeled antigen. This
effect is inhibited by prior exposure to a sample containing a nonfluorescent
form of the antigen and is generally referred to as a "competitive binding
assay." The major disadvantage of this second approach in its present stage
of development is the high level of background fluorescence from the polystyrene,
which limits its sensitivity; however, this is correctable. The potential advan-
tage of "the polymer system is for quantitative measurements.
-------
SECTION 3
RECOMMENDATIONS
Both the metal film and polymer substrates deserve further development.
The indium film technique has been developed to the point where it is very
close to fieldable and is able to give qualitative answers. The polymer system
has been proven in principle, and its development, which would be straightfor-
ward, could lead to a system that gives quantitative results. Development of
the metal film system should now be limited to work with indium films, and
should focus on optimum conditions for antibody deposition, packaging, and
stability. The problem of the nonspecific binding of protein must also be
addressed. For example, the study of substrates to which antibody molecules
might be chemically coupled without destruction of the antigen binding func-
tion would be a possible approach to this question of nonspecificity. The
goal would be to maximize the number of free binding sites of antibody and
the stability of the antibody surface, so that the monitor has optimum sensi-
tivity and a reproducible performance both in absolute terms and in rate of
reaction. Study of packaging and storage of the monitor should be undertaken
with a view to developing long shelf life, and at the same time be compatible
with ease of use.
Further development of the polymer substrate system would serve as a
backup to the indium film system. In contrast to the indium film system,
the polymer system is not suitable for visual observation; rather, it would
require a simple fieldable electronic instrument to detect the inhibition
of the fluorescence of the labeled contaminant in question by an unknown
sample. It is recommended that one focus of further development be the
acquisition of a suitable nonfluorescent polymer, i.e., a polymer that does
not fluoresce at the same wavelength as the labeled reagent. Alternatively,
the use of radiolabeled compounds in conjunction with a photographic plate
-------
as a simplified counting system might be considered which may also solve the
nonspecific fluorescence problem. In addition, the reagents relevant to the
measurement of a contaminant of environmental concern should now be used, the
sensitivity defined, and a calibration curve obtained.
-------
SECTION 4
METHODS AND MATERIALS
PREPARATION OF METAL-COATED SUBSTRATE
The substrate chosen for these experiments was 22 x 22 mm microscope
slide-cover glasses of No. 1 thickness. Metal was deposited on these cover
glasses by means of a commercial vacuum coater system, which was equipped
with a standard 18-inch-diameter bell jar, multiple electrical feedthroughs,
pressure monitoring gauges, film thickness monitoring, auxiliary power supplies
for substrate heating, and linear motion feedthroughs. The system was operated
in the 10 torr range, and is capable of handling a wide range of resistance
heated boats, coils, and crucibles for tungsten filament evaporation. The
unit provides evaporation filament power from a 2 kVA multitap power trans-
former. It also includes a 6 kW electron-beam gun for electron-beam evaporation.
Indium films were prepared by tungsten filament evaporation out of a
tantalum boat. Nickel films were prepared by electron-beam evaporation. The
majority of films prepared were of indium, since system success was encountered
early with indium, and its further development was the most efficient use of
the available experimental time.
The procedure for preparing the metal-coated glass slide film, was first
to clean the glass thoroughly by sequential treatment with mild detergent,
distilled water, reagent grade acetone, and absolute alcohol, followed by
drying in a warm airstream. The slide was then placed in the vacuum chamber
and cleaned with argon plasma for 15 minutes. .
Metal was placed in a quartz or tantalum boat, the pressure reduced to
less than 10 mm Hg, and then heated to melting with a shutter in position
above the boat to allow removal of possible contaminants which would result in
-------
deposition of contaminants on the slide. When the metal surface on the
shutter appeared clean it was removed without breaking the vacuum, and metal
was deposited on the glass. The thickness of the deposit was measured with a
Sloan thickness monitor.
o
Initially, metal films of approximately 1000 A were prepared under
various conditions of substrate temperature, rate of deposition, and purity
of metal. Table 1 lists typical combinations of parameters for the indium.
0
Subsequently, indium films approximately 100 A thick were prepared. It is
important that the metal is not deposited as a continuous film, but rather
agglomerates into small droplets as described in Appendix A. These are
illustrated in Figure 1, and optimally they measure 1000 A across and are
in the foam of an oblate spheroid.
COATING THE METAL FILM
The deposition, of protein, such as BSA or immunoglobulin, was carried
out by contacting a defined area of the metal film with a saline solution of
the protein (0.15M NaC&), either by partial immersion of the film or by placing
drops of solution on the film. After the deposition of protein, the films were
thoroughly rinsed with distilled water and allowed to dry in air.
Testing the initial protein film for reactivity with another compound was
carried out by contacting the film with an aqueous solution of the compound by
either of the two methods previously outlined, followed by rinsing and drying.
The principal technique by which the initial depos-ition of a protein (or
other compound) and/or subsequent reaction with another compound was detected,
was by measurement of the optical transmittance of the films with a McBeth-
Ansco Optical Transmission Densitometer. Operation of the densitometer requires
that the sensor system be placed on the film so that there is minimal loss of
light and maximum reproducibility of measurement. In order to .obviate dis-
turbance of the films, for example, by scratching of the surface by the
densitometer, they were covered with a clean microscope slide-cover slip prior
to the transmission measurements. The transmittance of the added cover slip
was slightly less than 100%, so care was taken to use the same cover slip
throughout a given experiment to take into account this additional absorption.
-------
TABLE 1. INDIUM SLIDES
Slide
No.
1
2
3
4
5
6
7
Indium
Purity
0.9999
0.9999
0.9999
0.9999
0.99999
0.99999
0.99999
Substrate
Temperature
(°C)
21
100
21
21
21
21
21
Thickness
o
(A)
1006
1007
1000
1000
1000
1000
1300
Rate of
Deposition
(A/sec)
7
7
50
7
7
50
7
, 9
-------
Figure 1. Agglomeration of indium particles on glass
substrate (Reichart metalograph polaroid
photograph)
10
-------
Also,, the densitometer was checked with respect to its "zero" and
"calibration" readings prior to each measurement to guard against transient
perturbations, and adjustments were made where necessary.
Changes in reflectance and in transmittance were also examined by the
naked eye,, since they were qualitatively observed with ease. This was aided
by the fact that experimental treatments were addressed to less than a whole
film, and a change in film thickness over the treated area resulted in an
optical contrast with the untreated portion.
CULLULOSE SUBSTRATE SYSTEMS
Both Whatman No. 42 filter paper and cellulose thin-layer chromatography
(TLC) sheet were cut into strips, and the strips were then immersed in a saline
*
solution containing 12.5 mg of carbodiimide for two hours, after which the
strips were rinsed with water and immersed in a saline solution of anti-BSA
reagent for three hours. The strips were then rinsed with saline and used as
described in the Experimental and Results Section. Also, several cellulose
TLC strips were reacted with normal rabbit immunoglobulin via the carbodiimide
procedure.
POLYSTYRENE
Polystyrene strips were coated with antibody protein by direct immersion
in a saline solution of the protein, and, after rinsing, reacted with protein
solutions, including fluorescinated BSA (BSA-F).
Polystyrene discs were treated by the method of Filippusson and Hornby
(Ref. 5) to form surface polyaminostyrene. The method involves initial nitra-
tion by immersion in an equimolar mixture of nitric and sulfuric acids for
20 minutes at 0°C, followed by rinsing with distilled water. The surface NCL
o
groups are then reduced to amino groups by immersion in 6% (w/v) Na-SO. in
2M KOH at 70°C for two hours, with stirring, followed by rinsing with dilute
*
l-cyclohexyll-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate,
from Aldrich Chemical Company.
11
-------
HC&, and then water. One disc was set aside at this point as a blank. The
amino groups of two discs were diazotized by reacting with fresh 1.5% (w/v)
NaN02 in 0.6M HC£ in an ice bath for 20 minutes with stirring. The discs were
rinsed with 016M HC& and then with 0.001M HC£, and.immediately contacted with
a saline phosphate solution of anti-BSA reagent for two hours while cooling in
an ice bath.
Anti-BSA and BSA solutions were dialyzed against saline solution to remove
low molecular weight impurities, and then prepared at 300 ppm in saline. Also,
normal rabbit IgG (which contained no antibody to BSA) was prepared at 300 ppm
in saline by dialysis.
Fluorescein-labeled BSA was prepared by reacting 68.7 mg of BSA and 2 mg
of fluorescein isothiocyanate (mole ratio of 5 FNCS to 1 BSA) in saline of
pH 9 for two days at 4°C. The product, BSA-F, was purified by passing through
a Sephadex G-25 column using saline as the eluent.
A viewing box with an ultraviolet lamp was used for visual observation
of fluorescein-labeled antigen, and a fluorescence photon counter was used
for quantitative measurement of the labeled antigen. The latter was equipped
o
with a 5000 A cutoff filter so that only photons with wavelengths greater than
o
5000 A were counted, thereby reducing interfering fluorescence.
12
-------
SECTION 5
RESULTS
METAL FILM SYSTEMS
Indium Films, BSA Experiments
It was found that the changes in transmittance of indium films in the
o
1000 A range were relatively small subsequent to exposure to protein. For
example, the films of Table 1 were subjected to the following sequence of
experiments.
• The transmittance, TQ, of each quadrant of the indium-coated
slide was measured prior to treatment.
• About 0.1 m£ of anti-BSA solution (containing 290 yg of rabbit
immunoglobulin directed against BSA per m£ of saline solution)
was placed in each of two quadrants, and allowed to incubate at
room temperature for a measured length of time, t^. The film
was then gently and repeatedly rinsed with distilled water and
allowed to air dry.
• The transmittance, Tj, of the once-treated quadrants was
measured.
• About 0.1 m!i of BSA solution (containing 290 yg of BSA per mJl
of saline solution) was placed on one of the quadrants previously
treated with anti-BSA, and allowed to incubate for a measured
length of time, t2- The film was then gently and repeatedly
rinsed with distilled water and allowed to air dry.
• The transmittance, T2, of the twice-treated quadrant was
measured.
The results are given in Table 2. It can be seen that while there is
considerable change in transmittance effected by the first treatment, the
second treatment produces a relatively small change, and in both cases the
variation is quite large. Accordingly, subsequent experiments were directed
toward tMnner indium films, with the objective of improving the sensitivity.
13
-------
TABLE 2. CHANGE IN TRANSMITTANCE WITH ANTIBODY AND ANTIGEN-ANTIBODY
LAYERS ON 1000 X INDIUM FILMS
Slide
No.
To
(minutes)
Tl
C2
(minutes)
T2
1 0.164 31 0.161 22 0.161
2 0.242 30 0.205 21 0.202
3 PHYSICAL DAMAGE TO FILM (SCORING)
4 0.173 34 0.153 26 0.141
5 0.092 30 0.084 20 0.080
6 0.154 32 0.144 15 0.144
7 0.161 30 0.090 19 0.090
( 14
-------
Indium films of 50, 100, and 200 A thickness were separately treated
for 30 minutes with saline solutions containing 2.8, 28, and 280 ppm of BSA,
and with a saline solution containing 294 ppm of anti-BSA. The results of
these experiments are given in Table 3. It can be seen that the deposition
of protein on indium film, whether BSA or its antibody (IgG), is easily
accomplished and measured, and that relatively greater sensitivities are
o
obtained with thicknesses in the range 100 to 200 A.
The indium films that were coated with anti-BSA were then exposed to 294
ppm BSA solution. In no case was the transmittance significantly changed by
exposure to the BSA solution, as shown in Table 4.
In order to check the possibility that the negative results of Table 4
might have been due to a characteristic of the experimental procedures, a
o
100 A film was reacted first with 294 ppm BSA solution, and (after rinsing,
drying, and measuring transmittance) then reacted with 294 ppm anti-BSA solu-
tion. The results: were as follows.
Condition of Film Transmittance
Before the experiment 0.385
After contact with BSA 0.31
After contact with anti-BSA 0.27
Thus, it was clear that we were able to duplicate the effect reported by
Giaever (Ref. 3), but as he found, the reverse order of deposition resulted
in greatly reduced sensitivity (Giaever, private communication).
The most likely explanation for these results is that the immunospecific
end of the antibody (IgG) reacts with indium with much greater frequency than
the opposite end, the so-called Fc end, of the molecule (see Appendix B).
Under such a circumstance there would be relatively few immunospecific ends
available for binding to the BSA; hence, there could not be a significant
change in the film thickness, and consequently no significant change in the
transmittance.
15
-------
TABLE 3. REACTIONS OF PROTEIN SOLUTIONS WITH INDIUM FILM*
Transmittance for Various Thicknesses
Before and After Exposure
Solution
BSA,
BSA,
BSA,
Ant i- BSA,
2.8
28
294
294
ppm
ppm
ppm
ppm
50
Before
0.
0.
0.
0.
69
725
74
67
o
A
After
0.685
0.70
0.70
0.66
100 A
Before
0.365
0.36
0.385
0.385
After
0
0
0
0
.325
.305
.315
.285
of Indium
200 A
Before .
0.
0.
0.
0.
083
093
106
098
After
0.071
0.078
0.086
0.068
No exposures to more than one solution or more than one time were used
in these experiments.
TABLE 4. INITIAL ATTEMPT TO REACT BSA WITH INDIUM COATED WITH ANTI-BSA
Thickness of
Indium Film
0
(A)
:oo
100
50
Transmittance
Before Exposure to BSA
0.068
0.285
0.66
Transmittance
After Exposure to BSA
(294 ppm)
0.069 •
0.280
0.66
16
-------
The primary reactive groups of protein are free amino groups and free
carboxyl groups. In order for there to be a marked difference between the
two ends of the IgG molecule with respect to reactivity with indium, there
must necessarily be a significant difference in the relative numbers of
these reactive groups at each end of the molecule. Therefore, it appeared
that manipulation of chemical factors could result in modification of the
reaction such that the Fc end of IgG would predominantly bind to the indium,
leaving specific reactive groups free.
Inasmuch as the two groups are opposite with respect to acid-base proper-
ties, it was anticipated that the desired effect might be obtained by simple
adjustment: of pH. In particular, one could possibly increase the reactivity
of the carboxyl groups (which are more prevalent at the Fc end) by increasing
the pH of the antibody solution.
Consequently, an experiment was carried out in which an aliquot of 294
0
ppm anti-BSA solution, approximately 0.03M in KOH, was contacted with a 100 A
indium film, and then (after rinsing, drying, and reading transmittance) the
coated film was exposed to a 294 ppm BSA solution. The results were as follows,
Condition of Film
Before the experiment
After contact with anti-BSA
After contact with BSA
Transmittance
0.43
0.36
0.31
Significant changes, of approximately 15% in transmittance were apparent
under these conditions, and suggested that the optimum concentration of KOH
be determined. Subsequent experiments conducted with 200 A films are summar-
ized in Table 5.
As can be seen, the desired sequence of protein deposition was achieved
with a small improvement in the sensitivity over a wide range of alkalinities.
17
-------
TABLE 5. EFFECT OF KOH ON THE TRANSMITTANCE OF ANTIBODY MOUNTED ON 200 A
INDIUM FILMS
Anti-BSA Solutions,
Concentration of KOH
Condition of Film
Before the experiment
After contact with anti-BSA
.After contact with BSA
0.03M
0.079
0.063
0.055
0.004M
0.076
0.071
0.060
O.OOOSM
0.07S
0.072
0.060
Since the speed with which an unknown sample can be analyzed is of
considerable importance in a practical situation, it is of interest to deter-
mine the rate of reaction of protein with its antibody. Consequently, trans-
mittance was used to follow the BSA, anti-BSA reaction as a function of time
o
with 100 A films only, since, as previously demonstrated, this thickness gave
a near-optimum sensitivity.
The abbreviations below are used in the presentation of the results.
AB-film: An anti-BSA monolayer on an indium substrate film.
AG-AB film: The triple-layer BSA attached to the AB-film.
T : Transmittance of the AG-AB-film after lengthy
00
reaction of BSA solution with AB-film.
T : (T - TJ x 1000.
T : (T of the AB-film less T } x 1000.
no v a,-1
In the first series of experiments, an anti-BSA solution was made alka-
line and used within one hour to prepare AB-film; the AB-film was then reacted
with 10'm£ of BSA solution within 24 hours. The extent of the resulting reac-
tion as a function of time for BSA solutions of 0.4, 1.1, and 7 ppm concentra-
tions is given in Table 6.
18
-------
TABLE 6. REACTION OF BSA SOLUTIONS WITH FRESHLY PREPARED AB-FILM
0.40 ppm
Time
(minutes)
0
2
5
15
30
BSAa
T
n
30
20
12
3
0
1.1 ppm BSA
Time
(minutes)
0
2
5
9.5
15
21
T
n
73
30
27
8
2
0
7 ppm BSA°
Time
(minutes)
0
1.1
2.2
3.3
5.3
6.5
8.5
16
1
n
86
63
40
28
10
25
10
0
The regression line is given by log T = -0.065t + 1.443.
h ^
The regression-line is given by log T = -0.093t + 1.806.
The regression line is given by log T = -0.106t + 1.862.
A similar experiment, but with AB-film, prepared and stored one week in
the open at room temperature, gave the results shown in Table 7.
TABLE 7. REACTION OF BSA SOLUTION WITH WEEK OLD AB-FILM
2.8 ppm BSA*
Time
(minutes)
0
1
3
8
18
1
n
30
27
23
15
0
*The regression line is given by
log T = -0.037t + 1.473.
19
-------
Identical experiments^ but with AB-film freshly prepared from anti-BSA
solutions that had been alkaline for approximately one week, gave the results
shown.in Table 8.
TABLE 8. REACTION OF BSA SOLUTION WITH AB-FILM FRESHLY PREPARED FROM OLD,
ALKALINE ANTI-BSA SOLUTIONS
a
0.4 ppm BSA
Time
(minutes)
0
1
4
8
16
24
T
n
17
15
10
9
4
0
h
2.8 ppm BSA
Time
(minutes)
0
1
2.5
5
8
13
19
T
n
45
36
25
18
13
6
0
r*
28 ppm BSA
Time
(minutes)
0
1
2
3.5
5.5
8.5
21.0
T
n
42
30
22
18
13
7
0
Degression line: log T = -0.037t + 1.210.
U ^
Regression line: log T = -0.065t + 1.610.
Degression line: log T = -0.086t + 1.572.
The anti-BSA solution had been alkaline for five days prior to prepara-
tion of the slide used versus the 2.8 ppm BSA, and it was eight days old prior
to preparation of the slide used versus the 0.4 and 28 ppm BSA solutions.
The behavior of anti-2-aminobenzimidazole (anti-2-ABZI) deposited on indium
was next investigated. As in the case of anti-BSA, solutions of the antibody
in saline were made slightly alkaline prior to deposition on the indium.
Several attempts to deposit 2-ABZI on the anti-2-ABZI films failed. .At
least such effects were not evidenced by any changes in transmittance of the
film. Furthermore, after such attempts, films were exposed again to anti-2-
ABZI (which would have adhered to 2-ABZI, but not to anti-2-ABZI) without
affecting the transmittance.
20
-------
One hypothesis for the failure of 2-ABZI to react with its antibody was
that the alkaline treatment that inhibited a reaction of the "antibody-end"
of the IgG molecule with the indium film also inhibited its reaction with its
hapten. This would be true if one of the amino groups of the hapten (2-ABZI
has one each primary, secondary, and tertiary amino groups) was necessary for
the reaction with its antibody. The alkaline pH suppresses the reactivity of
the amino groups. Therefore, a solution of 2-ABZI, which was on the alkaline
side of neutrality, was neutralized with HC&, and a fresh attempt to react
the hapten with the antibody films was carried out. This attempt was success-
ful, as shown by the fact that the value of transmittance dropped from 0.355
to 0.330 upon contact with the 2-ABZI solution for 30 minutes.
Sensitivity
In order to test the sensitivity of the system, an anti-2-ABZI film on
o
126 A indium was contacted with a 2.5 ppm solution of 2-ABZI in three points
on a single slide with the following results.
Condition
Before contact with 2-ABZI
After contact with 2.5 ppm
2-ABZI for 30 minutes
Point A
0.345
0.320
Point B
0.335
0.315
Point C
0.330
0.320
Average T
0.337
0.318
The average difference of 0.019 in transmittance between the pretreatment
and posttreatment (with 2-ABZI) films is significant at better than the 5%
limit of confidence by Student's t-test of differences between means.
A similar test against 0.25 ppm 2-ABZI gave the following results.
21
-------
Condition
Before contact with 2-ABZI
After contact with 0.25 ppm
2-ABZI for 30 minutes
T
0.
0.
, Individual Measurements
335
325
0.325
0.325
0.345
0.310 .
Average T
0.333
0.320
The average difference of 0.013 in T between the pre- and posttreatment
films is significant at better than the 20% limit of confidence by the t-test.
An important aspect of this experiment was that the exposure to 0.25 ppm 2-ABZI
resulted in a difference in optical density that was readily noticeable to the
unaided eye.
In a similar test, 0.025 ppm 2-ABZI failed to produce any change in trans-
mittance within 30 minutes which was detectable visually or by available
instrumentation. Increasing the exposure to 24 hours resulted in the metal
substrate being physically degraded by the experimental 0.025 ppm 2-ABZI
solution.
Specificity
A number of experiments were carried out to test the specificity of the
system for measuring 2-ABZI, which are described below.
• o
An indium film (126 A) was coated with a layer of BSA and then dipped in
a 2.5 ppm solution of 2-ABZI for 30 minutes, and gave the following measured
values of transmittance.
Condition
Average Transmittance
With BSA layer
After contact with 2-ABZI
solution for 30 minutes
0.402 ± 0.009
0.398 ± 0.003
22
-------
It can be seen that the transmittance was not significantly altered by
placing the BSA-coated indium film in contact with a 2.5 ppm solution of
2-ABZI.
o
A second 126 A indium film was coated with .anti-2-ABZI, and then con-
tacted for 30 minutes at different points with solutions of 2-ABZI, benzimida-
zole, and 2-amino 5-chlorobenzimidazole. These imidazole compounds effected
the following changes in transmittance.
Condition
Prior to contact
After contact
Point
Contacted
With benzimidazole
0.315
0.310
Point Contacted
With 2-amino
5-chlorobenzimidazole
0.295
0.290
Point
Contacted
With 2-ABZI
0.345
0.320
These experiments indicate that 2-ABZI does not react with a random pro-
tein (BSA), but does react with its antibody (anti-2-ABZI), as shown in column
3, and that it has greater affinity for its antibody than do analogous imida-
zoles (benzimidazole and 2-amino 5-chlorobenzimidazole), as shown in columns
1 and 2.
Similar experiments, in which an attempt was made to react diethylstil-
besterol (DES) with anti-2-ABZI, proved negative, and confirmed the high
degree of specificity of the antibody for its hapten, as shown in the follow-
ing results.
Condition
Average Transmittance
Prior to contact with DES solution
After contact with DES solution
0.354 ± 0.005
0.352 ± 0.006
23
-------
In the course of conducting these experiments, it was determined that
2-ABZI would also coat the bare indium-coated glass slides (slides that did
not have an anti-2-ABZI coating), and that it was possible to subsequently
react both anti-2-ABZI and BSA with the 2-ABZI, as shown by the following
results.
Condition
Slide No. 1
Average T
Slide No. 2
Average T
Bare indium film
After 30-minute contact with
0.25 ppm 2-ABZI
Subsequent to 30-minute contact with
anti-2-ABZI
Subsequent to 30-minute contact with
BSA
0.408
0.370
0.318
0.465
0.422
0.380
Thus, although the reaction of 2-ABZI with a protein film is specific
to its antibody, the reaction of proteins to a film of 2-ABZI on indium film
appears not to be specific.
It was found, in addition, that the 2-ABZI analogs, benzimidazole and
2-amino 5-chlorobenzimidazole, also coat the indium film. However, DES did
not coat the indium film, so it appears that it is not coatable with all
organic compounds.
Nickel Films
Experiments were conducted to investigate the possible advantages of
using nickel films instead of indium. Two thicknesses of nickel were used,
O O
namely 100 A and 160 A. The nickel films were immersed in both neutral and
alkaline saline solutions containing 294 ppm of anti-BSA for 30 minutes.
The films were then rinsed and dried. The transmittance of the films were
24
-------
then measured and immersed in neutral saline solutions containing 7 ppm of
BSA for 30 minutes, and again rinsed and dried. Measurements of transmittance
showed no change after the addition of both anti-BSA and BSA. Additional
experiments, in which an attempt was made to reverse the process, i.e., first
lay down BSA on the nickel and then anti-BSA, also showed no change in
transmittance.
POLYMERIC SUBSTRATES
Experiments were conducted to investigate the feasibility of affixing
antibody to a strip of polymer in such a manner that the strip could be used
to detect antigen. Unlike the indium-on-glass technique, the reaction of
antigen with the antibody was not expected to give a visible effect. However,
the reaction would use up binding sites in proportion to antigen concentration,
so that a subsequent reaction with fluorescein-labeled antigen would be pro-
portionally inhibited. Thus, the method would work by first exposing the
strip to the sample suspected of containing the antigen, and then determining
the extent of suppression of the reaction between labeled antigen and the
strip.
Whatman No. 42 Filter Paper
One of the strips that had been reacted with anti-BSA via the carbodiimide
reaction was immersed in the fluorescein-labeled BSA (BSA-F) for 40 minutes,
and then rinsed with saline.
A second strip was immersed in the BSA reagent for 40 minutes, rinsed, and
then reacted with BSA-F reagent and rinsed again. Both strips were examined
under ultraviolet light, and it was found, as was expected, that the strip
that had been reacted with BSA prior to BSA-F was less fluorescent than the
strip that had not been exposed to BSA. However, the fluorescence of both
strips was irregular, which indicated nonuniformity of the substrate or the
treatment.
25
-------
Cellulose TLC Strips
Two cellulose TLC strips, reacted with anti-BSA via the carbodiimide
reaction, and two strips that were reacted with normal rabbit IgG reagent
via carbodiimide, were exposed to BSA-F and rinsed. It was found by observing
ultraviolet fluorescence that the BSA-F had not reacted with the strips con-
taining normal IgG to the same degree that it had reacted with the other strips,
which indicated a specificity of reaction. However, again the fluorescence was
unevenly distributed, and it was concluded that another support medium might
be more appropriate.
Polystyrene Culture Tubes
To each of three polystyrene culture tubes (a, b, and c) was added 0.8 mfc
of anti-BSA reagent, and to a fourth tube (d) was added 0.8 m£ of normal rabbit
IgG reagent. Each tube was incubated three hours at room temperature, and then
rinsed three times with saline solution.
To tubes a and b were added 1 m£ of BSA reagent, and they were then incu-
bated for 18 minutes, followed by a triple rinse with saline. Then, 0.5 m&
of BSA-F reagent was added to each of the four tubes, and they were then incu-
bated for 18 minutes, followed by a triple rinse with saline. A fifth tube
(e, untreated) was used as a blank. All five tubes were examined for equal
periods of time with a fluorescence photon counter. The results obtained were
as follows.
Tube
e
d
a
b
c
Gross Counts
(millions)
0.95
1.20
1.73
1.47
2.48
Treatment
None
Normal IgG, then BSA-F
Anti-BSA, BSA, then BSA-F
Anti-BSA, BSA, then BSA-F
Anti-BSA, then BSA-F
26
-------
These results indicate the following: (1) IgG attached to the polysty-
rene; (2) the reaction of BSA-F with anti-BSA (shown in tube c) was specific,
since it did not react with the normal IgG in tube d; and (3) the reaction of
BSA-F with anti-BSA was inhibited by prior exposure of the anti-BSA to BSA in
tubes a and b.
High-Impact Polystyrene
White, high-impact polystyrene strips were found to have an excessively
high blank fluorescence level, which made them unsuitable for further
experiments.
Black Polystyrene
Black, buna-loaded polystyrene sheet was examined as a candidate sub-
strate material. One tab was taken as a blank, and another was simply exposed
to the anti-BSA reagent for 30 minutes, rinsed, exposed to the BSA-F reagent
for 10 minutes, and rinsed. The strips were then counted with the fluorescence
photon counter. The blank gave 360,000 counts per minute, and the treated
strip gave 820,000 counts per minute. Thus, the ratio of sample fluorescence
to background fluorescence was greater for this material than for culture tubes,
and the material is a candidate substrate for the purpose at hand.
Polystyrene Discs
One of the discs with an anti-BSA coating prepared as described in Sec-
tion 4 was reacted with BSA-F reagent. Its fluorescence intensity, in counts
per minute, were observed with the fluorescence photon counter and compared
with those of the blank disc and a disc having an unreacted anti-BSA coating.
The results were as follows.
Disc
Blank
Treated with anti-BSA only
Treated with anti-BSA, then BSA-F
Count Rate
.430,000
230,000
1,590,000
27
-------
It can be seen that these samples gave the best signal-to-noise ratio
of those examined to this point. The anti-BSA apparently diminishes the
incident and/or fluorescent light.
28
-------
SECTION 6
DISCUSSION OF RESULTS
In comparing the two metal films, indium and nickel, the quality of indium
for the purpose at hand was quickly demonstrated, while it soon became apparent
that a relatively greater amount of effort would be required to define the use-
fulness of nickel films. Since, in these studies we are only concerned with
demonstrating the feasibility of the technique, indium was used exclusively in
rate and sensitivity experiments.
The hypothetical basis for experiments that led to the deposition of anti-
body in such a manner that its specific reactivity was partially retained was
useful in that pragmatic context. However, the experimental success cannot be
taken as proof of the validity of the hypothesis, nor can the follow-up hypo-
thesis regarding unblocking of sites specific for 2-ABZI be considered as
proven by experimental success. It is recognized that the model depicting
IgG as consisting of linear and parallel chains is idealized, and that the
molecule is highly convoluted (Ref. 6). It would be as reasonable to suppose
that the changes in pH effected the molecule's chemical configuration with
respect to exposed groups. Obviously, however, the initial hypotheses were
useful in that they served to guide the experimental efforts in a direction
which resulted in a practical solution to the problem of interest.
As expected, BSA and anti-BSA system served as an adequate model in the
developmental work on the indium film system, in the sense that the procedures
developed needed only slight modification for application to 2-ABZI and its
antibody. It is interesting that the sensitivities of the indium substrate
system in its present form are comparable for both BSA and 2-ABZI.
For example, 2-ABZI was easily detected at 0.25 ppm, but not detected at
0.025 ppm. By comparison, the BSA data in Table 6 vary about the regression
lines as follows:
29
-------
0.4 ppm BSA: ±65%
1.1 ppm BSA: ±23%
7 ppm BSA: ±33%
Clearly, the 0.4 ppm BSA left an observable change in the optical density
of the film (as did 0.25 ppm of 2-ABZI); the large variance associated with
the measurements, however, indicates that 0.4 ppm BSA was near the limit of
detection. Also, it may be noted from the values of T at time zero in
Table 6 that the net change in optical transmittance was much less for 0.4 ppm
than for 1.1 ppm or 7 ppm BSA.
The regression lines of Table 6 are plotted in Figure 2, and the regres-
sion lines of Tables 7 and 8 are plotted in Figure 3.
Although the curves of Figures 2 and 3 have a progression of slopes that
suggest a relation between rate of transmittance change and BSA concentration,
it is premature to conclude that such is the case. For one thing, the standard
deviations of two of the curves (1.1 and 7 ppm) of Figure 2 are such that there
is no significant difference between their slopes. Also, the conditions rele-
vant to Figure 3 were strictly comparable in only two of the four curves. More
importantly, it is clear that the change in transmittance follows an apparent
first-order kinetics over the range of concentrations used. This suggests a
primacy of the following relationship, since attachment of BSA to a binding
site on the film will decrease the transmittance in a linearly proportional
manner:
-dS/dt = kS (1)
where
2
S = number of binding sites/cm to.which BSA may attach
t = time
*
k = rate constant
Equation 1 integrates to the expression: £n(S/So) = -kT, which gives straight
lines when plotted on semilogarithm paper, as in Figures 2 and 3.
30
-------
1.0
o
c
0.1
0.01
RT-13653
a = STANDARD DEVIATION
OF DATA POINTS
ABOUT THE J.INE
I
0.4 ppm (a = ±65%) -
1.1 ppm (o = ±23%)
7 ppm (a = ±33%)
10
TIME (MINUTES)
20
Figure 2. Tn/Tno of fresh AB-film versus BSA solution as a function of
time and BSA concentration
31
-------
1.0
o
c
0.1
0.02
TWO CURVES
(1) OLD SLIDE (TABLE 2)
vs 2.8 ppm BSA
(2) ALSO, OLD ALKALINE
ANTI-BSA vs 0.4 ppm
BSA (TABLE 3)
OLD ALKALINE ANTI-
BSA vs 2.8 ppm BSA
(TABLE 3)
OLD ALKALINE ANTI-BSA vs
28 ppm BSA (TABLE 3)
10
20
RT-13652
TIME (MINUTES)
Figure 3. Influence of aging on the reaction between AB-film and
BSA solutions
' 32
-------
For the present purposes, a detailed understanding of the kinetics is
not necessary. It is relatively more important that we now know a technique
which is capable of detecting a substance at the sub-ppm level, and that the
time required for the. detection is short (under ten minutes). Also, the data
indicate that it is best to prepare AB-films with fresh solution, and that
such films tend to deteriorate if left exposed to air, light, and dust.
Although equivalent effort resulted in more progress with the polymeric
system than the nickel system, it was less productive than was the case with
the indium system. Nevertheless, the attachment of antibody to a diazotized
polyaminostyrene surface has been demonstrated, and it appears to be a suitable
method for preparation of a surface for subsequent reaction with antigen and
labeled antigen. Unfortunately, the substrates tested to date are inherently
fluorescent, and consequently a meaningful estimate of the true sensitivity
of the system could not be made. The hope that pigmented polystyrene would
be superior by virtue of elimination of all but surface fluorescence proved
disappointing, apparently, by virtue of pigment fluorescence.
Inasmuch as styrene has a pale blue fluorescence, the fluorescence of
pure polystyrene should have been blocked by the 5000 A cutoff filter in the
photon counter. The observed fluorescent background of the polystyrene discs,
therefore, was probably due to impurities, which indicates that a pure poly-
styrene would be more suitable for the desired application.
Alternatively, since it was observed that a coating of anti-BSA reduced
the measured fluorescence of a polystyrene disc (probably due to a decrease
in transmittance) it is possible that polystyrene might serve as a substrate
where a change in transmittance is measured as an indicator of hapten presence,
as in the indium film experiments. The disadvantage of this system is that
the change in transmittance is not easily discerned with the naked eye. The
advantage would be that the polymer substrate is less fragile than the metal-
on-glass substrate.
A major advantage of the polymer system operating in the fluorescence
mode, and with a field instrument for measuring fluorescence, is that it is
easily made quantitative via a calibration curve. The fluorescence intensity
33
-------
is inversely proportional to the hapten in the sample, since the latter uses
up antibody sites that would otherwise be available to the labeled hapten.
Based on the foregoing discussion, it appears that continued work on both
systems would be profitable. The indium film system is almost fieldable in its
present form, and can be implemented relatively quickly to give qualitative
results. It will be desirable to develop a packaging system and define a stor-
age system that will optimize stability and utility in the field. Once a
suitable polymer substrate is located (or, if necessary, specially synthesized),
the polymer system can be brought quickly to a point of providing quantitative
analyses with a portable fluorometer.
With respect to the special synthesis of polymer, it is recommended that
radiation polymerization be used, since it will obviate the need to use cata-
lysts, which can enhance the fluorescence of the polymer.
It would also be of interest to attempt to increase the change in trans-
mittance of the indium monitor on exposure to hapten. At the present time,
hapten only effects a change of about 10%. Such an increase could be accom-
plished by optimizing deposition of antibody, whereby a greater fraction of
molecules attach to the indium by their Fc ends rather than their Fab ends.
It is recognized that the failure to detect 25 ppb of 2-ABZI with the
indium badge was probably due to the use of an unstirred solution where dif-
fusion was rate limiting at such a low concentration. However, the indium
badge approach was conceived of as being a simple, straightforward, inexpensive
test that requires no special stirrers, instruments, or other field equipment;
thus, the unstirred solution was more realistic in that context.
34
-------
SECTION 7
REFERENCES
1. Statistical Abstract of the United States, U. S. Department of Commerce.
Bureau of the Census.
2. Lukens, H. R., Williams, C. B., Levison, S. A., Dandliker, W. B.,
Muryama, D: A Fluorescence Immunoassay Technique for Detecting Organic
Environmental Contaminants. United States Environmental Protection
Agency, Washington, DC, EPA-650/1-75-004, May 1975.
3. Giaever, I: The Antibody-Antigen Reaction — A Visual Observation.
•J. Immunology 110, No. 5 (1973) p. 144.
4. Filippusson, H., Hornby, W. E.: The Preparation and Properties of Yeast
B-Fructofuranosidase Chemically Attached to Polystyrene. Biochem. J.
120 (1970) p. 215.
5. Putnam, F. W.: The Primary Structure of Human Immunoglobulins: The Key
to Antibody Structure. Proceedings of the Robert A. Welch Foundation
Conferences on Chemical Research XVIII.. Immunochemistry, W. 0. Milligan
(Ed.), (1975) pp. 7-63.
35
-------
APPENDIX A
PHYSICAL BASIS FOR THE VISUAL OBSERVATION
OF THE ANTIBODY-ANTIGEN REACTION
The ability of an antibody to bind selectively to its antigen with a high
degree of specificity is well known. The ability of proteins, and in particu-
lar antibodies, to bind to metal surfaces is less well known, and of much more
recent origin. Giaever (Ref. 3) has demonstrated that bovine serum albumin
(BSA) can be made to bind to indium-plated glass slides, and that in turn
anti-BSA can be made to bind to the BSA-coated slide.
The unique aspect of this discovery is that as the BSA is bound to the
indium-plated glass slide it changes the light-scattering properties of the
indium, and the indium looks darker. As the anti-BSA is bound to the BSA,
the scattering properties change once more and the slide appears darker still.
The sensitivity of this technique has been demonstrated to be comparable
to existing radioimmunoassay techniques (i.e., nanogram to picogram/mft range)
it is simple to perform, and should be relatively inexpensive.
The observation by Giaever that protein absorption on an indium-coated
slide causes the slide to darken can be understood in terms of the theory of
light scattering by small particles, and as he points out, this theory is
based on Maxwell's equations.
As Giaever recognized, the unique nature of a thin coating of indium on
glass is that instead of forming a continuous coating, it deposits in the form
o
of small droplets, typically 1000 A across. (Other materials also deposit in
this'form, but usually the individual spheres coalesce to form a continuous
film at much smaller thicknesses.) These particles are smaller than a wave-
length of visible light, and consequently, the appearance of these films will
be described by the theory of scattering from small particles.
36
-------
Scattering from the uncoated indium is determined by noting that the
skin depth of the indium at optical frequencies is negligibly small compared
to the particle size. Hence, the particle may be approximated as having
infinite conductivity. In this approximation, the spatial dependence of the
scattered light intensity at point (r,6) from a small particle is given by:
i(r,0) = - rr- (i + cose) - cose
r A L
where In is the incident light intensity, A is the wavelength, and a is the
particle radius. In this formula, 6 is measured from the direction of the
incident light. Here, we see that light is scattered preferentially in the
o
backward direction since [5/8(l+cos 0)-cos0] is a maximum when 6 = IT. Although
interaction of the light scattered from an array of particles will change the
quantitative form of this equation, the qualitative conclusion remains: the
array of particles will be highly reflective, i.e., the film will look
"metallic".
Next, we consider the effect of coating these particles with a nonconduc-
tor. Scattering from such a structure, a nonconducting layer on a conducting
sphere, has apparently not been treated in the literature. However, for our
present purposes, we can consider the limiting case of scattering from a non-
conducting particle and recognize that the observed scattering will approach
this value as the coating becomes thicker. Under the same conditions as above,
the scattered intensity from a dielectric particle is:
2(27T)4 a6 I In - l|2 _
U s-t ^ /\ \ /-• I *t " a.
r—: (1 + cos 0) G I^T— sin
9 r A
where
. 1 \
in2ej •
_
2u ^
37
-------
J,/2 is the Bessel function of the first kind, of order 3/2. From this
expression, we can determine that the scattering is approximately isotropic
for a/A « 1, with the backscattered intensity slightly less than the forward
intensity. For increased a/A, the forward intensity grows at the expense of
the backscattered intensity.
Consequently, it can be concluded that a dielectric coating on the indium
spheres will reduce the intensity of the reflected light, i.e., the surface
will darken. A second layer will further reduce the backscattered light,
yielding additional darkening. In addition to these effects, due entirely to
the angular dependence of the scattering, additional darkening would be pro-
duced if the dielectric layer was absorbing at optical frequencies. This
would be manifest in the above formula through a reduction in I(r,6) produced
by a nonzero imaginary part of IT.
38
-------
APPENDIX B
THE ANTIBODY MOLECULE
Although there are several kinds of antibody molecules, the most prolific
one is immunoglobulin G (IgG), which has a molecular weight of ^140,000. It
is commonly depicted as comprised of four parallel chains of protein, two of
which are heavy chains (molecular weight of ^50,000 each) joined together by
a disulfide bond, and two of which are light chains (molecular weight of
^20,000 each) joined one each to a heavy chain by disulfide bonds, as shown
in Figure B-l. Also shown in Figure B-l are the dominant end groups, COOH
and NH7, and the plane of cleavage with papain digestion that splits IgG into
a crystalline (Fc) portion and an antigen-binding (Fab) portion.
39
-------
OH OH
0=C C=0
Fc
PORTION
!-^
Fab PORTION
LIGHT
CHAIN
r
J 1
..
Y
—
-^"
\i
^^^ DISULFIDE BONDS
-^/^
/ . PLANE OF
/ ^^ CLEAVAGE
"^~~ BY PAPAIN
<^
^1 N
_^^- HEAVY CHAINS
-^_— LIGHT
-CHAIN
t-J HUM
n2 M2 2 n2
RT-14137
Figure B-l. Schematic of IgG structure
i 40
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-018
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
A SOLID SUBSTRATE IMMUNOLOGICAL ASSAY FOR MONITORING
ORGANIC ENVIRONMENTAL CONTAMINANTS
5. REPORT DATE
March 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Herbert R. Lukens and Colin B. Williams
8. PERFORMING ORGANIZATION REPORT NO,
9. PERFORMING ORGANIZATION NAME AND ADDRESS
IRT Corporation
Box 80817
San Diego, California 92138
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
68-02-2202
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
HERL-RTP
14. SPONSORING AGENCY CODE
EPA/600/11
15. SUPPLEMENTARY NOTES
1f>. ABSTRACT
A solid substrate "film-badge" type monitor has been developed that is
capable of detecting 2-aminobenzimidazole (2-ABZI) at less than one part per
million in water in less than 10 minutes. The monitor makes use of the reac-
tion which takes place between 2-ABZI in the sample and a monolayer of its
antibody that has been deposited on a thin film of indium on a glass substrate.
A second approach in which the antibody is mounted on polystyrene and
reaction of its antigen-binding sites with a fluorescein-labeled antigen are
subject to competition with nonlabeled antigen in the sample, has been demon-
strated in principle. Improvements in this alternate approach are proposed.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Monitors
Chemical Analysis
Antigen antibody reactions
Assaying
06 T
07 E
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
lINfT.ASSTFTF.n
21. NO. OF PAGES
48
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
41
-------
INSTRUCTIONS
1. REPORT NUMBER
Insert the EPA report number as it appears on the cover of the publication.
2. LEAVE BLANK
3. RECIPIENTS ACCESSION NUMBER
Reserved for use by each report recipient.
4. TITLE AND SUBTITLE
Title should indicate clearly and briefly the subject coverage of the report, and be displayed prominently. Set subtitle, if used, in smaller
type or otherwise subordinate it to main title. When a report is prepared in more than one volume, repeat the primary title, add volume
number and include subtitle for the specific title.
5. REPORT DATE
Each report shall carry a date indicating at least month and year. Indicate the basis on which it was selected (e.g., date of issue, date of
approval, date of preparation, etc.).
6. PERFORMING ORGANIZATION CODE
Leave blank.
7. AUTHOR(S)
. Give name(s) in conventional order (John R. Doe, J. Robert Doe, etc.). List author's affiliation if it differs from the performing organi-
zation.
8. PERFORMING pRGANIZATION REPORT NUMBER
Insert if performing organization wishes to assign this number.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Give name, street, city, state, and ZIP code. List no more than two levels of an organizational hirearchy.
10. PROGRAM ELEMENT NUMBER
Use the program element number under which the report was prepared. Subordinate numbers may be included in parentheses.
11. CONTRACT/GRANT NUMBER
Insert contract or grant number under which report was prepared.
12. SPONSORING AGENCY NAME AND ADDRESS
Include ZIP code.
13. TYPE OF REPORT AND PERIOD COVERED
Indicate interim final, etc., and if applicable, dates covered.
14. SPONSORING AGENCY CODE
Leave blank.
15. SUPPLEMENTARY NOTES
Enter information not included elsewhere but useful, such as: Prepared in cooperation with, Translation of, Presented at conference of,
To be published in, Supersedes, Supplements, etc.
16. ABSTRACT
Include a brief (200 words or less) factual summary of the most significant information contained in the report. If the report contains a
'significant bibliography or literature survey, mention it here.
17. KEY WORDS AND DOCUMENT ANALYSIS
(a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major
concept of the research and are sufficiently specific and precise to be used as index entries for cataloging.
(b) IDENTIFIERS AND OPEN-ENDED TERMS - Use identifiers for project names, code names, equipment designators, etc. Use open-
ended terms written in descriptor form for those subjects for which no descriptor exists.
(c) COSATI FIELD GROUP - Field and group assignments are to be taken from the 1965 COSATI Subject Category List. Since the ma-
jority of documents are multidisciplinary in nature, the Primary Field/Group assignment(s) will be specific discipline, area of human
endeavor, or type pf physical object. The application(s) will be cross-referenced with secondary Field/Group assignments that will follow
the primary posting(s).
18. DISTRIBUTION STATEMENT
Denote releasability to the public or limitation for reasons other than security for example "Release Unlimited." Cite any availability to
the publk. with address and price.
19.&20. SECURITY CLASSIFICATION
DO NOT submit classified reports to the National Technical Information service.
21. NUMBER OF PAGES
Insert the total number of pages, including this one and unnumbered pages, but exclude distribution list, if any.
22: PRICE
Insert the price set by the National Technical Information Service or the Government Printing Office, if known.
EPA Form Z2JO-1 (»-73) (Reverse)
------- |