Table Of ContentEXECUTIVE SUMMARY
Immunochemistry sah broad applications for a wide variety of environmental
contaminants. However, the potential for applying immunochemical methods to environmental
measurements is just beginning to be realized. Immunochemical methods are based on
specific antibodies combining with their target analyte(s). Many specific antibodies have been
produced for targets fo environmental and human health concern. Such antibodies can be
configured into various analytical methods. The most popular immunochemical technique ni
environmental analyses today is immunoassay. Immunoassays have been shown to detect
and quantify many compounds of environmental interest such as pesticides, industrial
chemicals, and products of xenobiotic metabolism. Among the most important advantages of
immunoassays are their speed, sensitivity, selectivity and cost-effectiveness.
Immunoassays nac eb designed as rapid field-portable, semi-quantitative methods or
as standard quantitative laboratory procedures. They are well suited for the analysis of large
numbers of samples and often obviate lengthy sample preparations. Immunoassays can eb
used as screening methods to identify samples needing further analysis by classical analytical
methods. Immunoassays era especially applicable ni situations where analysis by
conventional methods si either not possible or is prohibitively expensive.
Environmental immunoassays have broad applications for monitoring studies. The
EPA has used immunoassay methods for monitoring groundwater and cleanup activities at
hazardous waste sites. Immunoassays nac also eb used as field screening tools ot confirm
the absence and or presence of particular contaminants or classes of contaminants for special
surveys. Other federal and state agencies era employing immunoassay technology where
appropriate such as for extensive monitoring studies that generate a large sample load.
nI addition to detection methods, other immunochemical procedures can be used for
environmental analysis. Immunoaffinity techniques now used extensively ni pharmaceutical
and biotechnology applications can be adapted to extract, and cleanup environmental
samples. Selective and sensitive sample collection systems such as air and personal
exposure monitors nac be designed based no the principal of immunoaffinity. Although
immunoaffinity procedures era not addressed ni this tutorial, they are mentioned here to
illustrate to the reader that immunochemical methods nac be adapted to a wide variety of
monitoring situations.
The U.S. EPA Environmental Monitoring Systems Laboratory at Las Vegas, Nevada
(EMSL-LV) has a program to develop and evaluate immunochemical methods for
environmental analysis. The EMSL-LV immunochemistry program consists of the following
major components" identification of need for na immunochemical method, identification of
existing technologies, development of new technologies, adaptations of existing technologies,
evaluations of existing technologies, field demonstration of portable technologies, and finally
technology transfer. Overall program goals, as well as prioritization of compounds for
methods development, era based upon input from client EPA Program Offices as well as the
EPA Regions. Analytical needs are defined as ot target analytes, matrices, detection limits
and application of the method.
Methods and immunologic reagents have been developed for the polychlorinated
biphenyls (PCBs), XTB (benzene, toluene, xylene) and various pesticides and nitroaromatic
vi Executive Summary
compounds through the EMSL-LV immunochemistry program. Additional methods are under
development for pyrethroid and organophosphorus pesticides.
The EMSL-LV immunochemistry program conducts laboratory and field evaluations to
assess method performance. The evaluation, characterization and testing of a particular
analytical method si necessary to ensure the intended esu of the method si met. Evaluations
are conducted according to EPA guidelines requiring the determination fo precision, within and
among laboratories bias, method detection limit, matrix effects, interferences, limit of reliable
measurement and ruggedness of the method. Demonstrations under the Supeffund
Innovative Technology Evaluation (SITE) program have been used to document method
performance under real-world environmental conditions. SITE demonstrations of
immunoassay methods for the PCBs, pentachlorophenol, and BTX have been completed,
other demonstrations era being planned. After a SITE demonstration the methods can be
submitted to the Superfund Field Screening Methods compendium for inclusion and
distribution.
Technology transfer activities include providing guidance and training to EPA regional,
EPA headquarters, and state personnel on the esu of immunoassays. A computer animated
graphics program has been developed to provide instruction no the theory and applications of
immunoassays. This graphics program may be a useful training aid to the tutorials contained
ni this document. Other instructional activities planned include the development of training
videos for performing immunoassays. A "hands on" workshop at the EMSL-LV si also being
considered. Individual training for EPA pemonnel sah been conducted and will remain na
option for interested individuals.
Another vehicle to facilitate the implementation of immunochemical methods are
annual meetings of researchers, developers and end-users of immunochemical methods. The
EMSL-LV has sponsored two meetings to discuss the direction of immunochemical methods
research, development, application, and acceptance within the regulating and regulated
communities. The last Immunochemistry Summit Meeting was held ni September 1993 and
included representatives from EPA and other federal and state agencies, large chemical
companies, biotechnology companies, and research institutes. tI si anticipated that this type
of meeting will continue to eb na annual forum for concerns and issues regarding
environmental immunochemical methods.
Considering the advantages and versatility of immunochemical methods, it si surprising
that the technology has not been more widely accepted by environmental analytical chemists.
Although many immunoassay methods have been reported ni the literature, their potential has
not been practically realized. Part of the problem si misunderstanding and perhaps skepticism
on the part of analytical chemists. A thorough understanding of the advantages and
limitations of immunoassay methods si essential to applying the technology ni situations where
they offer the most promise. tI si the intent of this document to dispel the mystery ni
understanding and performing na environmental immunoassay.
This document presents six specific immunoassay methods. The methods are based
no ht :: same working principle but illustrate different applications of the technology for various
analyt'cal situations. Two methods are presented to describe immunoassays for lipophilic
analytes using the triazine herbicides as examples. Although either method can be used for
environmental samples, both are presented to illustrate different formats for the same analyte.
The third method si for the insecticide carbaryl which si applicable for both environmental and
Executive Summary vii
biological samples. Methods for p-nitrophenol, paraquat and for various triazine mer-
capturates are examples of water soluble analytes. The triazine mercapturates method
illustrates the application of immunoassay for urine samples and hence exposure assessment
studies. Accompanying solid phase extraction procedures to extract triazines from water and
atrazine mercapturate from urine are also provided. llA of the methods described are
intended to serve as examples of the utility of immunoassay technology.
nI addition to the six specific immunoassay methods, this document also describes
analytical laboratory techniques necessary ot perform immunoassays. Suggestions for
general laboratory considerations such as protocol design, sample preparation, data handling
and analysis, and safety precautions are also given. Examples of troubleshooting and quality
control practices era included which can be applied to assays not contained ni this tutorial.
Protocols for preparing buffers, determining reagent integrity and for optimizing assay
conditions are also useful for immunoassays ni general. An appendix of commonly used
terms ni immunoassay should facilitate understanding of the technology.
Although immunoassays are now being employed for environmental analysis, there
may still be a need for training non-analysts ni the esu of immunoassay or updating the
experienced analytical chemist on na unfamiliar analytical format. tI si hoped that the
methods and procedures found ni this users guide will be beneficial and help to standardize
the immunochemical analysis of small molecules. Comments and written requests for
additional information yam be directed to Jeanette .M naV Emon, U.S. Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, .P .O xoB 93478,
Las Vegas, Nevada .8743-39198
ACKNOWLEDGMENTS
The authors thank noD Gurka dna Llewellyn Williams of the
Environmental Protection Agency, Environmental Monitoring
Systems Laboratory, saL Vegas, Nevada, Rosie Wong American of
Cyanamid, Princeton, New Jersey, dna George Fong, Florida
Department of Agriculture, Tallahassee, Florida for helpful review
and comments. The authors also thank IA Reed dna Virginia
Kelliher, Senior Environmental Employment (SEE) Program
enrollees assisting the Environmental Protection Agency under a
Cooperative Agreement with the Association National for Hispanic
Elderly for their helpful review dna comments. The authors
especially thank IA Reed for sih expert editorial assistance.
viii
PREFACE
Immunochemical methods for the analysis of environmental contaminants are relatively
new no the analytical chemistry scene. These methods are based no the use of a specific
antibody sa a detector for the analyte of interest. Immunoassays are rapid, sensitive, and
selective, and are generally cost effective for large sample loads. They have been applied to
diverse chemical structures (i.e. triazines, sulfonylureas, organophosphates, polychlorinated
biphenyls, cyclodienes) and are adaptable ot field use. These characteristics make
immunochemical analysis a valuable tool for esu by the environmental analytical chemist.
This document si designed to facilitate the transfer of this valuable technology to the
environmental analytical chemistry laboratory. Field personnel who may need to employ a
measurement technology at a monitoring site may also find this manual helpful.
This document si a tutorial designed ot instruct the reader ni the use and application of
immunochemical methods of analysis for environmental contaminants. A brief introduction
describes basic principles and the advantages and disadvantages of the technology, and
gives a listing of references which supply more detail. Preparation of the laboratory for use of
this technology and the general scientific considerations prior to using the technology are
discussed. Detailed step-wise procedures are given for analysis of selected analytes, triazine
herbicides, carbaryl, paraquat, and p-nitrophenols ni environmental samples as well as triazine
mercapturates ni urine samples. nI addition to the specific immunoassay methods, a series of
support techniques necessary to perform immunochemical methods are described. These
support techniques include pipetting, sample preparations, testing for matrix effects, optimizing
reagent concentrations, data analysis, recordkeeping, and equipment maintenance. A general
troubleshooting guide si provided to aid both the novice and experienced analyst.
This document provides specific instruction for certain analytes, but also serves to
familiadze the novice reader with many generic concepts needed to successfully utilize
immunochemistry technology including: applications, sampling, sample preparation,
extraction, cleanup, quality assurance, methods development and optimization, data handling
and troubleshooting. tI si not necessary for the reader to actually perform the immunoassays
given ni this User's Guide to obtain familiarity with these concepts. The Guide is written so
that the information presented can be applied to other immunoassays not given here. Thus,
the strength of the Guide si sti universal applicability to immunoassay methods.
ix
SECTION 1
INTRODUCTION
This manual is designed to introduce the analytical chemist to the general concepts
and use of immunoassays for the analysis of pesticides and other small molecules. By writing
this manual we hope to encourage the analytical chemist to consider this technology among
the repertoire of methods available for solving analytical problems. As with any other
analytical technique, it will be just as important for the analyst to be able to identify when
immunochemical technology is appropriate, as it si to learn how to conduct na
immunochemical analysis. Field personnel who may need to employ a measurement
technology ni the field may also find this manual helpful.
The manual is organized first to provide some general information on the technology,
second to provide tutorials consisting of some specific examples of immunoassays and thirdly
to provide guidelines and information on those procedures specific to immunochemical tech-
niques which may not currently be ni use ni the typical analytical chemistry laboratory. All of
these procedures were developed ni an academic laboratory and may need to be adjusted to
meet regulatory requirements of the various agencies within the government as to method
performance and their procedural guidelines. This caveat also can apply to a Contract Laboratory.
1.1 Brief history
The development of chromatographic instrumentation by pesticide analytical chemists
was closely paralleled by the development of immunoassay techniques by clinical analytical
chemists. Immunoassays are routinely used ni clinical situations for the analysis of proteins,
hormones and drugs. The success that these immunochemical procedures has achieved ni
the clinical area is being transferred to the area of pesticide analysis.
The first antibodies developed against pesticides were reported ni the 1970's. Recently
this technology has been refined for use ni the pesticide analytical chemist's laboratory to the
point that commercial test kits are now available. With the availability of kits, it is imperative
that the analyst understand the underlying principles of the methodology in order to evaluate
the strengths and limitations of any one "kit" for their specific application. Since many of these
easy-to-use test kits are designed for the non-analyst, it is important that these users also
have the same fundamental understanding.
1.2 Advantages/Disadvantages
Immunoassays are a~ complement to the analytical chemist's repertoire of
methods for the detection of pesticides and other environmental chemicals. Immunoassays
are rapid, sensitive and selective for the analyte of interest and generally cost effective for
large sample loads. Immunoassays have been applied to diverse chemical structures and are
adaptable to field use. As with any technology there are disadvantages. Antibodies may bind
to structural analogs of the analyte of interest (termed cross-reactivity). This technology si not
easily adapted to a multianalyte method, since each antibody binds primarily to a single
analyte or class of analytes. Reagent stability si often cited as a problem, but can be
overcome based on knowledge gained from the clinical field. This technology also requires a
large sample load to justify development of a new assay for an analyte of interest, due to the
expense of producing antibodies and establishing the procedure. For intermittent analysis, it
might be more cost effective to use existing commercially prepared test kits.
2 Environmental Immunochemical Analysis
3.1 Principles
Immunoassays rely no the reaction fo na analyte or antigen (Ag) with a selective
antibody (Ab) ot give a product (Ab )gA that nac be measured. This reaction si characterized
by eht Law of Mass Action (shown below), thus immunoassays era physical assays.
a k
Ab + Ag -" "" Ab. Ag
~k
nI sti most generic form, immunoassay si na analytical method dependent on the
specific binding of na antibody with sti target analyte. This specific interaction can provide
quantitation of the target analyte. Many types of labels have been used for quantitating
immunoassays including radioactivity, enzymes, fluorescence, phosphorescence,
chemiluminescence, and bioluminescence. Each fo these labels sah sti own particular
advantage. However, the esu of enzymes dna colorimetric substrates si probably the most
common for environmental analysis. Several different types of enzyme immunoassays (EIAs)
have been developed, the two broadest categories being heterogeneous and homogeneous
enzyme immunoassays. Heterogeneous immunoassays require a separation of bound and
free reagents throughout the assay. This si easily accomplished by simply washing the solid
phase (e.g. test tube, microtiter plate wells, cuvettes, etc) with buffer and surfactant.
Homogeneous immunoassays do not require separation or washing steps. However, ni these
immunoassays the enzyme label si required ot function ni the sample matrix which often
poses difficulties. euD to this restriction, homogeneous immunoassays era popular ni the
clinical field, while heterogeneous assays era used predominately for environmental matrices.
A common heterogeneous immunoassay si the enzyme-linked immunosorbent assay
(ELISA). The methods ni this guide are based no the ELISA format schematically shown ni
Figure 1 (adapted from eiW & Hammock, 1982). The following si a generic description for
preparing the microtiter plates and reagents for an ELISA. Preparation of microtiter plates: A
constant amount of the coating antigen si bound to the surface of polystyrene microtiter plate
wells by passive adsorption. After a pre-determined incubation period the wells are washed to
remove unbound coating antigen.
Preparation of ELISA reactants: )1 A constant amount of anti-analyte antibody (first
antibody) si incubated with increasing amounts of analyte ni separate test tubes (tubes ,B ,C
and ,D Figure .)1 This incubation period enables the formation of analyte-antibody
complexes. The number of analyte-antibody complexes formed and eht remaining amount fo
free reactants si dependent upon the amount fo analyte present ni eht samples or standards.
2) The incubation mixture si added to the prepared microtiter plate wells. The coating
antigen competes with remaining free analyte for available antibody. A washing step removes
all materials not bound to the microtiter well. )3 A second antibody covalently coupled to na
enzyme (the enzyme label) si next added which binds ot the first antibody now bound ot eht
coating antigen. fI eht first antibody was developed ni a rabbit, eht appropriate second
antibody would be goat anti-rabbit GgI covalently coupled ot alkaline phosphatase (or another
enzyme label). Excess second antibody si then washed out. )4 Finally substrate si added ot
produce a color change. This ELISA si typically called na inhibition assay since a high
concentration fo analyte ni eht samples or standards inhibits eht first antibody from binding ot
the coating antigen no eht microtiter plate well. This si due ot eht number fo analyte-antibody
complexes formed ni the initial incubation (tubes A-D Figure .)1 ehT amount of enzyme
Introduction 3
product formed is directly
proportional to the amount of first
antibody bound to the plate and
is inversely proportional to the
amount of analyte in the samples
and standards (tubes A-D, Figure A B C D
.)1 nI this example, the
A B
maximum color intensity is
observed in the wells containing ,p
the contents from tube A (Figure
)1 where all the available first goL noitartnecnoC
antibody si bound to the coating
antigen. As increasing amounts
enerytsyloP
of analyte are added, the color
gnitaoC negitn~.
intensity decreases leading to a
E Enzyme delpuoc ot
sigmoidal analyte close response dnoceS ydobitnA
curve similar to that shown ni A Firlt ydobitna
V'v'-j VV V
Figure .1 1 etylanA/netpaH
~t" etarlsbuS ot
tcudorP
There are several
variations in this "format'. For
example, the first antibody and
analyte may be added directly to
the coated microtiter plate well. Figure .1 Schematic of the procedure for conducting an immobilized
A second, commonly used, antigen ELISA.
format is the direct competition
assay. nI this immunoasssay format, the antibody is immobilized on the solid phase. Analyte
ni the sample competes with a known amount of enzyme-labelled hapten for binding sites on
the immoblized antibody (Figure 2). nI step ,1 the anti-analyte antibody si adsorbed to the
microtiter plate well. nI the competition step, the analyte and a hapten-labelled enzyme are
added to the microtiter plate well. All unbound reagents are washed out. The final step si the
addition of substrate. As ni the first format described above, the production of color si
inversely related to the concentration of analyte. This particular format si commonly employed
ni the commercial immunoassay test kits.
Formats will vary and it would be useful to know the format being tested as it may be
important to the performance of the assay. For example ni the second ELISA schematic, the
sample si ni contact with the enzyme labelled hapten. fI the enzyme si sensitive to a matrix
effect, then you will get inhibition of the enzyme activity which may then lead to a false
positive result. fI this is the case, the format of choice, would be the first format ni which, the
sample does not come ni contact with the enzyme.
Another important thing to remember with regard to formatting, si that the same
immunoassay reagents can be formatted for a highly quantitative laboratory test, for a semi-
quantitative test, or for rapid yes/no field tests. nI general, assays that are simple and very
rapid, tend to be less sensitive. Assays designed for laboratory use may perform less repro-
ducibly ni the field. As with any analytical method, immunoassays are designed to perform to
certain specifications under the conditions given. An assay designed to measure na analyte
ni a groundwater sample ni the laboratory may not perform the same when analyzing ground-
water at a field workstation, or even analyzinq surface, instead of groundwater, ni the
4 Environmental Immtmochemical Analysis
laboratory. That is not to say the
assay si not performing correctly.
tI si only to say that it will perform
differently, although the difference
should be consistent under given
En~m
conditions.
3). 0 (cid:12)9
As a final note to for-
matting and optimization; the
tutorial methods given ni this
user's manual use the reading of
2).
absorbances after a fixed period
of time or until the zero control
sample attains a given absorb-
ance value as an endpoint. An
alternative to the endpoint mode
is to use a kinetic read mode. nI Anti.
1).
(cid:12)9 y Y Y
this case, the absorbances ni the
wells of the microtiter plate are
i,i i
read at fixed intervals (several
seconds) immediately after addi-
tion of the substrate. nI this way,
the rate of the enzyme reaction is
Figure .2 Schematic of an immobilized antibody ELISA.
monitored. A major benefit of
kinetics reading is the minimi-
zation of well-to-well variation for replicate analyses due to the time difference in the addition
of substrate. Other advantages to this method include a decrease ni analysis time and a
decrease in the amount of reagent needed to obtain a useful signal. nI addition, one can
check the linearity of the enzyme reaction, thus further verifying integrity of the assay.
1.4 Applications
An easy way to introduce immunoassay into the analytical laboratory si to use specific
immunoassays as screening methods to determine dilution levels for routine instrumental
analysis. Used ni this manner, immunoassays can minimize instrument clown time by
protecting sensitive components such as electron capture detectors. The U.S. Environmental
Protection Agency has used immunoassay methods for monitoring cleanup activities at
hazardous waste sites. Many EPA Regions have expressed satisfaction ni utilizing
immunoassay methods for these types of field monitoring activities. nI some Regions
immunoassay methods are used for groundwater monitoring as a screening tool. For these
monitoring situations, a sample yielding a positive result, is confirmed by an alternative
method. The EPA, ni conjunction with the state of Idaho, si currently evaluating the use of
immunoassay to monitor water ni the vadose zone for pesticides, as a Yaw of determining the
efficiency of irrigation management practices to prevent leaching. Additional studies are being
conducted by the EPA such sa those through the Superfund Innovative Technology Evaluation
program (Gerlach et al., 1993). Other state and federal agencies (e.g. the U.S. Army Corps of
Engineers, the U.S. Geological Survey, the U.S. Department of Agriculture, the National
Institute of Occupational Safety and Health, and the U.S. Food and Drug Administration) are
implementing or evaluating the use of immunochemical methods for their respective
monitoring programs.
Introduction 5
The California Department of Food and Agriculture Chemistry Laboratory si an
example of a State regulatory laboratory introducing immunoassay for part of their normal
operation. Their goal si to replace, ni a cost effective manner, instrumental analysis for
specific compounds ni their routine compliance monitoring program with immunoassay. This
includes considerations of protocol design, dealing with outliers, curve fitting techniques,
consideration for generating "defensible" data ni terms of processing and analysis and having
rapid, real time access to quality assurance data.
