Environmental monitoring by immunoassay n e s e assays are useful for toxicology research and hwnan health monitoring Martin Vanderlaan Bruce E. Watkins Lany Stanker Lawrence Livermore Nm‘oml Laboratory Livermore. Cnlif: 94550
The EPA, the U S . Department of Agriculture (USDA), and the Food and Drug Administration are often criticized by Congress and citizen groups for inadequate monitoring of toxic chemicals (I). Critics charge that too few chemicals are monitored, that the number of samples is inadequate to ensure detection of contamination, and that there is too long a delay between sample collection and communication of results back to the site. AU of these problems stem from the cost, sophistication, and time involved in multiresidue analytical chemistry. Even with the best analytical methods, problems arise when research laboratory procedures are scaled up to handle thousands of samples under massscreening conditions. Furthermore, detection of all potential contaminants cannot be incorporated into a single multiresidue procedure, so one must either create multiple work-ups on the same sample or ignore some compounds. These problems are liiely to become more urgent as demands increase for adequate determination of the fate of pesticides and toxic chemicals in food and in the environment. The problems involved in analyzing large numbers of samples, therefore, motivate the search for low-cost, rapid, automated residue detection methods. Immunoassayssatisfy these criteria and have become common analytical methods in the clinical laboratory. The most frequent use for immunoassays in the c h i c is to identify proteins, bacteria, and viruses-targets that are much larger than the small organic molecules that need to be identified in environmental samples. Nonetheless, immu-
FIGURE 1
Antigen-antibody Interactions Haptens have been conjugated to carrier proteins and may be used to immunize animals. Part of the immune response consists of antibody molecules that bind only to the hapten portion of the antigen. These antibodies have combining sites that recognize the cube or yramid haptens and ara influenced by such factors as ionic bonds, hydrophobic nteractions, and geometric fit. Once generated. the antibodies will bind to the haptens whether or not they are conjuyted to proteins. The remainder of the antibody molecule can be used to provide a site or conjugation of other functional groups, such as dyes (green or white spheres shown above),enzymes, or radionuclides. This other functionali IS measured in immunoassays (e.g., by measuring enzymatic activity or radioactivity) an from this measurement the quantity of the hapten can be inferred.
P
This anids not subject to US. copyright. Published 1988 American Chemical Society
2
Enuron. Sci. Technol.. Vol. 22. NO.3,1988 247
nologists demonstrated more than three decades ago that antibodies could be generated to selectively bind low-molecular-weight molecules, such as dinitrophenol (2). The immune response to this molecule has been extensively used as a research tool to study the regulation of the immune system. Within the past 15 years there has been a heightened interest in generating antibodies to environmentally significant chemicals for trace residue analysis. Dinitrophenol is, after all, not only a convenient chemical for studying immunology but also an EPA priority pollutant (3). The extent to which immunoassay techniques are finding greater accepance in the environmentallaboratory is revealed by a growing number of publications, recent meetings called by regulatory agencies, and a proliferation of companies marketing immunoassays for environmental and food residue analysis.
Antibodies and inunmoasays Figure 1 illustrates the antigen-antibody interactions on which immunoassays are based. Antibodies are symmetrical serum proteins, with two sites that enable binding to target molecules (antigens). In the illustration, cube-shaped and pyramid-shaped combining-site indentations in the antibodies provide a basis for recognizing counterpat target molecules. This geometric depiction of antigen-antibody binding is obviously a simplification. In reality, the antibody combining site is a complex, folded surface, and binding is influenced by hydrophobic and ionic forces as well as geometric fit. The bulk of the antibody protein is not involved in the combining site and can provide a site for covalent attachment to marker molecules, such as enzymes, radionuclides, or the white and green fluorophores shown in Figure 1 (4). “Hapten” is the term applied to a small molecule (less than about loo0 daltons) that does not induce an immune response by itself but is recognized by some antibodies; immune response is induced by injecting a complex of the hapten and a protein into an animal (5).In Figure 1 the haptens shown are linked to carrier proteins. A wide range of antibodies is produced in response to immunization with a hapten-protein conjugate. Figure 2 shows this diversity in antibodies. The response to a dioxin-protein conjugate is shown to illustrate many of the featuresof the immune resvnse to hapten-protein conjugates in general. Three antibodies and the lymphocytes that secrete these antibodies are shown. Each lymphocyte produces identical antibodies with a unique com248 Environ. Sci Technot.. Vol. 22.NO 3. 1988
I
II haoten I
’
II
Ntl
Linker
bining site; therefore, the multiplicity of antibodies in the serum of the immunized animal reflects the multiplicity of lymphocytes. Most of the antibodies produced will be like t h m represented by antibodies A and B, which react with the protein alone or with some complex of the protein, hapten, and linkage groups. Only a few of the antibodies will be l i e antibody c, which binds with high affinity to the hapten alone with no contribution of the protein and l i r to the binding site. There will be many members in the general class of hapten-binding antibodies (e.g., c, c‘,c”,etc.) that are produced by different lymphocytes. These lymphocytes differ from each other in the fine details of their respective combining sites. The binding specificity of serumcontaining polyclonal antibodies reflects the aggregate binding specificity of the individual monoclonal antibodies. The exact mixture of antibodies present in the serum is dynamic in time, reflecting the expansion and contraction of the various subpopulations of lymphocytes clones. The mixture also differs from one immunized animal to the
Lwrnohocyte B
next. Accordingly, the specificity of polyclonal antisera will be variable over time and between animals. Each antibody bas a componding lymphocyte from which it has been secreted. Hence, serumderived antibodies are polyclonal, which is to say they are produced by many clones of lymphocytes in the animal. Some of the antibodies react with the protein; others react with complex combining sites that contain the hapten, l i g e groups, and the protein; and still others react with the hapten alone. This last subset of antibodies is the one most desirable for immunoassays aimed at detecting hap ten-like chemicals. In 1975 George Kohler and Ceasar Milstein revolutionized the production of antibodies by developing a method for culturing the particular lymphocytes that secrete antibodies (Figure 3) (6,7).After several months of immunization, a mouse is sacrificed and its spleen cells are used as the source of antibody-secreting lymphocytes for making monoclonal antibodies. Even though these cells normally do not grow in culture, we can create antibody secreting cell lies by fusing them with
cells that do grow in culture-in this case, myeloma tumor cells. Spleen cells and tumor cells are mixed in the presence of polyethylene glycol (PEG), where they fuse into a new cell type, called a hybridoma, which grows in culture l i i the tumor cell and produces antibodies like the spleen cells. On a perell basis the fusion efficiency is very low (only about one cell in 1W will fuse) but on a per-spleen basis the fusion efficiency is high, producing about l W hybridoma colonies per fusion. Hybridomas are seeded at roughly one colony per well in multi-well plates for cloning. The ability to screen hybridomas and select the particular rare clones that produce the most useful antibody reagents does not exist if one is producing polyclod antisera. The obligation of having to screen the clones carries with it an increase in cost and effort. The greatest task in making monoclonal antibodies is the rapid screening of hybridomas to find the 1-10 per spleen that produce the best antibodies. Each hybridoma secretes its own monoclonal antibody into the cell culture medium of its well, and medium samples are screened by immunoassay. Ideally the format of the screening assay will closely resemble the format of the eventual assay in which the antibodies will be applied. Once selected, the clones are expanded into the larger wells of a %-well plate and then to bulk spinner flasks. There is at least a threemonth period from the time of fusion until large quantities of antibodies are available. During this time there are continuous rounds of subculturing the cells, assaying the media from wells, selecting the desired clones, and expanding the culture to ensure that, in the end, we are dealing with stable, uniform cells and that each cell is secreting the best antibody for the particular application. At this point cells may be frozen for long-term storage and used to produce large quantities of antibodies in ascites fluid (6, 7). These antibodies are called monoclonal antibodies (Mabs) because they are produced by a single strain of cloned cells in culture. Before Kohler and Milstein, the only means of obtaining hapten-specific antibodies was to purify them from the polyclonal serum of immunized animals. Now, instead of having to purify rare antibodies from many similar proteins in the serum, one can isolate the rare cells that produce these antibodies, indelinitely grow these cells in culture, and generate large quantities of identical antibody molecules. For environmental monitoring, Mabs have two important advantages over conventional, serumderived, polyclonal antibodies. First, comparison of
immunoassay results, both among laboratories and over t h e within the same laboratory, is greatly facilitated by Mabs. The Mab is a uniform, invariant reagent that may be widely distributed, easily standardized, and incorporated into regulated methods. The Mab can be produced in large enough quantity for literally bfflions of assays. In contrast, the polyclonal mixture of serumderived antibodies is a limited reagent because it varies over time in the animal, and it also varies from one animal to the next. Second, and more importantly, the desired antibodies in polyclonal antisera are always diluted in a mixture of irrelevant antibodies which often are a source of interference in the immunoassay. A typical surface available in an antibody combining si is estimated to be about 20 x 25 (8, whereas environmental chemicals are typically smaller (e.g., dioxin is a pl er mole cule with dimensions 3 x [q). Immunologists have observed that, for haptens with molecular weight less than 750, the overwhelmingbulk of antibodies produced will have combining sites that extend beyond the hapten to include determinants on the protein that is conjugated with the hapten tb make it immunogenic (Io). Most of these antibodies will read poorly with the free hapten alone, making them unsuitable for immunoassays.
f
A
With Mabs, on the other hand, one can design screening tests to select those rare hybridoma clones that have antibodies with a high affmity for the free hapten and to reject those antibodies tbat recognize. portions of the conjugated protein as well as the hapten. Once an antibody has been produced it must be incorporated into an immunoassay, and the assay must be validated. Since a comprehensiveintrcduction to immunoassays for analytical chemists has recently appeared ( I I ) , we will limit the present discussion to only the most important principles governing the use of antibodies in immunoassays. Competition Enzyme Linked Immunosorbent Assays (cELISAs) are one of the most common immunoassay formats (12. 13). Figure 4 shows that the competition for a l i i t e d amount of antibody by a plastic-immobilized hapten and a free hapten in solution follows the general laws of mass action and is influenced only by the relative amounts of the reactants and the a6inity of the antibody-hapten interaction (12). The basic principle of competition immunoassays is that a limited amount of antibody will partition between free antigen in the sample and immobilized antigen on a solid surface. The first step in the procedure is to immobilize one form of the target molecule on a plastic surface and wash away the unbound
Envimn. Sci. Techiwl., MI. 22. No. 3,1988 WS
material. S i l e adsorbtion is sutlicient for this immobilization. In the second step, fme antigen is added in solution either from a standard or as part of the sample b e i i a n a l y d , and then anti-
body is added. The antibody separates the target molecules in solution from those adsorbed to the surface of the plate. With no hapten in solution, a maximum amount of antibody binds to the plate (A). High concentrations of h a m in solution block binding of the antibody to the plate (C). At intermediate hapten concentrations some antibody will be bound to the plate and some will remain in solution (B).At the end ofthe incubation period (typically 1 h) the solution phase is washed away, leaving behind that fraction of the antibody that is bound to the plastic-immobilized hapten. The 6nal step in the assay is the detection of the antibody that remains bound to the solid phase. For cELISAS this is done by detecting enzymatic activity associated with the antibody. The enzyme can either have been directly conjugated to the hapten-specific antibody used in the second step or introduced via an enzyme-conjugated second antibody that binds to the antihapen antibody. A wide variety of enzymes are suitable-peroxidase, alkaline phosphatase, and urease, for example-& there is also a wide choice in substrates for these enzymes. Enzymatic activity is most often demoastrated by the release of a colored product, which is then despectraPh' y. Sensitive assays based on the detection of fluorescent, radiolabeled, luminescent or electrically charged enzyme products also have been reported (14). The exact shape of the standardcurve shown in Figure 4 i s not important, and available curve-fitling programs can approximate this curve using either log-logit transfornutions of the data or 4-pameter logistic equations (15). Because of the sigmoidal shape of the inhibition curve, the 50% inhibition (Iso) concentration is the position that can be determined with greatest precision (12). If the hapten concentration of an unknown sample is near the 150 value, the amount of hapten can be quantified by c o m p i y n with the standard curve. Conceneatlons that are on the upper or lower asymptote of the standard curve can at best be specified as Wing above or below the companding thresholds.
Immumasssys for pesticides R o important articlos review early work in the development of immunoassays for pesticides. A 1971 paper by Ercegovich-which discusses immunoassays for DDT, malathion, and aminotriazole-puts forth the first strong suggestion that environmental !2W Emiron. Sci. lochmi., MI 22. NO. 3. isan
chemists consider immunoassays as alternative analytical methods (16). In 1980 Hammock and Mumma reviewed theii exprience synthesizing haptenpmtein conjugates and producing polyclbnal antisera (17). Hammock, Mumma, and oolleagw have developed many immunoassays for pesticides. Theii work shows that immunoassays are particularly appropriatein cases where conventional analytical methods are time consuming (e.g., paraquat [la),where the tar@ is a complex, biologicallyderived molecule (e.g., Bacillus rhwingensis ismelensis tarin [19),or where only specific immen are of interest (e.& s-bidethrin [2a).Furthermore, they have shown that the target molecule for the assay need not be the active ingrrdient in a pesticide formulation, but may be one of the components present in larger quantity (e.g., triton X-100 [21]). Hemmock, Mumma, and colleagues have m y updated theii reviews in the area of monitoring agricultural chemicals (22,23). AU immun-y methods give wmparable sensitivities, Suggesting that it is the atfinity of the antibcdy rather
than the famat that influeaces the sensitivity of wmpetition assays (24). The assays with the highest sensitivity have Is0 values near ow part per billion @pb)in the final assay bu&r, whereas poorer assays have Is0values in the 50100 ppb range. w c a l l y , insensitive assays ria& the fact that the antisera contains many antibcdies to the linkage groups and p m i n and relatively few groups suitable for detecting free hap
ten.
Not surprisingly, for the experiments with polyclonal antisera there is some cross reactivity among srmcturally related co-. For example, the antibodies to 2,4,.5-trichlorophenoxyacetic acid (2,4,5-T) cross react with 2,4dichlomphenoxyaoetic acid (2,4D) (25); the antibodies to difluorbenzumn react with similar insecticides (26); . . the antibcdies to benomyl and tmdmefon react with metabolites and break down products of these fungicides (27.28). W l e 1 lists some of the immunoassays recently developed for pticides and includes refenmm for papers published since 1980. The Mab immunoassays show
greater specificity. For example, our Mabs to permethrin react 10 times less effectively to cypermethrin, which differs from permethrin only by the substitution of a cyano group for a hydrogen (36).
Cnrcinoge~,toxic chemicals When applied to human blood, urine, and tissue samples these assays can provide a quantitative assessment of dose. In epidemiologic studies they can help correlate specific exposures to diseases (39). Several of the immunoassays for industrial chemicals detect urinary metabolites of carcinogens as part of an industrial hygiene program. When applied to samples from animals exposed to carcinogens, antibodies provide quantitative data on the distribution of the carcinogen among tissues and between different cell types within the same tissue (40). These studies provide insight into the metabolism of the chemicals, the formation and repair of DNA damage, and the toxicology of carcinogens. Table 2 shows that the development of immunoassaysfor chemicals, carcinogens, mutagens, and carcinogen-DNA adducts has been an area of active research. For the most part, the DNA adduct immunoassays presented in Table 2 use mouse Mabs and are therefore far more specific than the pesticide assays listed in Table 1. For example, the antibodies to benzo[a]pyrene-DNA recognize the toxicologically relevant 7,8,9,10 antidiolepoxidebenzo[a]pyrene (BPDE) adduct, but do not recognize adducts formed by the synisomer of BPDE
Herbkidas
Chbrsulfuron 2,4,5.-T and 2.4-1 Diclofopmethyl Paraquat
ELSA RIA EIA, FIA
90 7 70
antisera antisera antisera
Terbunyn
Atrazine insecticides
Diflurbenzuron Parathion Permethrin Funglcidea
Triedimefon
nonitoring several human populations at high risk for cancer for their bodyburden of carcinogen-DNA adducts. studies are in progress on smokers and coke oven workers for the presence of bem[a]pyrene adducts (62),and there are also studies underway on high risk populations for aflatoxins and ethylated bases (39). Similar studies on the presence of DNA-adducts in wildlife could provide data on the biological availability of toxic chemicals. The measurement of hazardous materials in foods by immunoassay has attracted the attention of regulatory agencies, processed food manufacturers, and researchers interested in the con(41). Also, the DNA adduct assays are nection between diet and cancer (63). more sensitive than many of those re- Immunoassays have been used for deported for pesticides. For purposes of tecting pesticides that may become part comparison with Table 1, the current Iso of the human diet via the food chain. values have been converted to units of For example, our assays for the insectinglmL, although either units of h o l e s cide permethrin was funded by the of adduct per mg of DNA or frequency USDA for the purpose of detecting of modifiedbases were used in the orig- these residues in meats (36). inal references. In general these assays Immunoassays have also been develcan detect about one modified base in oped for the detection of veterinary 107-108 bases, quantities which corres- drug residues such as dimetronidazole pond to several hundred to one thou- and for anabolic hormones such as disand adducts per cell, or from 10 to 100 ethylstilbestml in animal tissues (61). tholes of adduct per assay. The low Mycotoxins may be present in nuts and levels of DNA adducts in human popu- grains, and several immunoassays for lations have necessitated highly sensi- the aflatoxins and ochratoxins have tive immunoassays, and several elabo- been developed (49, 50, 64). In addirate modificationsto cELISA have been tion, the cooking of meats forms potent made to maximize sensitivity using flu- mutagens known as aminoimiorescent or radioactive substrates for zoazaarenes (AIAs); we recently reported immunoassays for these chemithe enzymes (42, 43). At least three research groups have cals (sa). made Mabs to alkylated bases (e.g., 6The quantitation of AIAs illustrates a methyldeoxyguanosine (44). There are particularly appropriate application of many excellent reviews on the immu- the immunoassay technology because noassay of DNA adducts (41-47) AIAs are very difficult to measure by In addition, at least three groups are conventional analyrical methods. AJAs
are polar and are present at only the ppb level in the complex mixture of other compounds found in cooked meats. A I A s also are chemically similar to naturally occurring chemicals present in much greater quantities. AU of these properties contribute to difficulties in AIA purification and identification by high-pressure liquid chromatography (HPLC), which requires about a person-month per sample for complete analysis. These same pmperties pose no problem for the immunoassay, which quirea only a single cleanup column before the sample may be analyzed. The potential for immunoassays to greatly reduce assay costs is perhaps best illustrated in the assay of the toxic pollutants polychlorinated dibenzodioxin (F'CDD) and polychlorinated dibenzofuran (FCDF). The high toxicity of these compounds requiresthat assays be sensitive enough to detect contamination below one part per billion. In the environment, F'CDD- and PCDF-contaminated samples often contain large quantities of other chlorinated hydrocarbons such as 2,4,-D, 2,4,5-T, polychlorinated biphenyls (F'CBs) and chlorinated phenols. To permit detection of the dioxins and furans at the desired level of sensitivity, these related chemicals must be removed by extensive clean-up procedures (9). Quantitative dioxin analysis, a sophisticated procedure, requires multiple chromatographic steps to remove interfering substances and costs at least $500 per sample. Recognizing the need for improvements in the dioxin assay and the potential of immunoassay, researchers developed rabbit antisera that recognizes Enwmn. Scl Techml.. MI. 22. NO 3,lgsS 251
Some immunoassays for DNA adducts, carcinogens, and toxic chemlcals Immunoassay tormar
Clwmlcal 3e(.cltld ____
1~ve4ue (ng/mLp
DNA adducts
BenzMaIpyreneDNA Ethylated and methylated bases Aflatoxin Bl-DNA
ELIS 05 ELtS 02 RIA ELlSA 6.0 taminopyreneDNA ELlSA 0.05 Mutagens, carcinogens, and toxic chemicals Aflatoxins RIA 3.3 Ochratoxins ELSA Niltrofluoranthrenes RIA 120 RIA Dibenzofurans and dibenzodioxins ELSA PCBS RIA Benzidine metabolite RIA 0.25
bAminobiphenyl metabolite 3,3'dchlorobenzidine Aminoimidazoazaarenes Diethvlstilbestrol
-
RIA ELlSA ELlSA RIA
2.5 500 1.o
0.1
Mob or
antlrwra
Mab antiser Mab Mab Mab
152 Envimn. Sci. Techrol.. Vol. 22. No. 3, i900
-47 48 41
Mab antisera Mab antisera Mab antisera antisera
49
anlisera antisera Mab antisera
58
.ELSA = Enzyme Linked l m m m m m n l Assay. RIA = Faoloirnmunaassay FIA Immunoassay. EIA Enzyme ImmunOasSay ~Concemratlon~nthe 8nal m a y medurn (dsually 0 1-0 3 mL In nach asswl
PCDDs, PCDFs, and PCBs (52, 53, 56). The polyclonal antisera did not adequately distinguish among these closely related chemicals and there was considerable rabbit-twabbit variability in the specificity of the antisera. Furthermore, the radioimmunoassay (RJA) required frequent synthesis of radiolabeled dioxin and reportedly took several days to complete. Despite these limitations, researchers demonstrated that immunoassays for highly water-insoluble compounds were possible and that they could be applied to soil and tissue samples. Encouraged by their results, we followed their general scientificapproach, but rather than working with polyclonal antisera and RJAs, we generated mouse Mabs and developed a cBLISA (54). The Mabs, as expected, showed far greater specificity than did the rabbit polyclonal antibodies, reacting only with intermediate chlorinated dioxins and dibenzofurans. The antibodies do not react with PCBs, chlorinated phenols, or chlorinated pesticides and should be suitable for determining trace levels of dioxin contamination in the presence of these compounds. The cELISA has been successfully applied to the analysis of dioxins and furans in PCB transformer oil,motor oil, fly ash, several technical grade chemicals, distillation residues, and soils. Although ' some sample cleanup steps are still needed, the assay generally requires about half that needed for gas chromatography/mass spectrometry (GC/MS) analysis. The cELISA and GClMS results show good quantitative correlation, with the detection l i t for the
Refennm no.
50 57 52, 53
54
55, 58 57
II
59 60
-
61 FluarePcenC'
I
cELISA of about 1 ng of dioxin. We estimare the cost will be $10 per assay and analysis should require less than a day from time of sample collection. Coostrsintson immunoassays Despite the promise of immunoassays, they do have certain liitations. For example, because any one antibody will permit detection of a l i i t e d set of s u u a u d y similar cross-reacting residues, one must decide in advance what compound is to be measured and select appropriate antibodies. The metabolism of the compound, for example, may be important in selecting exactly which compounds are the real targets of interest for assay. The immunoassays for dimetiidamle, benomyl, benzidine, and aminobiphenyl all are aimed at detecting metabolites of these chemicals rather than parent compounds. Although a cocktail of antibodiescan be assembled to provide for multiresidue analysis, the set has to be built up gradnally, one antikdy at a time. To some extent, experience gained in developing antibodies to a single hapten aids in the development of future antibodies to other haptens. Nonetheless, each compound requires the synthesis of its own hapten-protein conjugate, the immunization of its own animals, the screening of hybridomas if Mabs are desired, and eventually the extensive characte.rization of each new set of antibodies. The process takes roughly a year, and must be repeated for each new compound. If one does not know what chemicals to expect in a sample, a conventional multiresidue analytical approach is still
mferable. On the other hand, if the problem is one of screening a lage number of samples for a limited number of compounds, then immunoassay may be the more appropriate technology. Even if good methods already exist for a compound, immunoassays may still be the method of choice if the sample load is high enough, because immunoassays are so amenable to parallel sample processing and automation. Having noted the single-residue orientation of immunoassavs, it is worth commenting on strate& by which broad specificity, or multiresidue antibodies might be produced. A pesticide or a toxic chemical is often mediated by interactions with specific receptors or binding poteins in cells. Antibodies are receptor proteins as well, and if a Mab can be selected that mimics the binding of a target protein for toxicity, then such an antibody would be suitable for detecting all chemicals acting through that common receptor. For example, the effectsof PCDDs, FCDFs, and PCBs are probably mediated by their interactions with a specific cellular protein, known as the Ah rec e w r (65).Our Mabs to dioxin mimic this receptor, although the Mab is only a model for the binding receptor, and its binding is not a functional measure of toxicity. Extending this argument further and applying it to pesticides, antibody development might precede and influence the development of new pticides. Antibodies could be selected to cross-react with pesticides classed according to their functional property of binding to a common receptor. It is easier to generate Mabs than it is to clone and express the gene for actual target proteins by recombinant DNA techniques. Indeed, the antibody could be selected based on its binding similarity to a postulated receptor, even if the exact receptor is not known. Not only would such antibodies react with currently available pesticides, but they may also recognize future pesticides that are mediated by bmding to the same receptor. mpeets Immunoassay has great potential to reduce the cost of screening large numbers of samples. Once the assays are developed, experiments that are now prohibitively expensive will become feasible. For example, the immunoassay for paraquat allowed for the study of occupational exposure among workers involved in the aerial spraying of this herbicide to cotton fields (18). Samples from workers' clothing, breathing zone air, and hand rinses were analyzed. Such a comprehensive study done by GC methods would have been vastly more difficult because of
the necessary sample preparation. Once the burden of costly and cumbersome assays for other compounds is removed, environmental studies that require extensive sampling become more feasible. One factor contributing to the low cost of immunoassays is that, once the initial research and development efforts have been made, running the immunoassays requires a minimum of training. Indeed, pregnancy tests now available in drug stores are based on immunoassays for low-molecularweight hormones, which shows that even the lay public can utilize immunoassays. Similarly packaged kits for screening pesticide residues or toxic wastes could give citizens the ability to monitor their own environment. This could allow health officials to focus their efforts only on those cases where clear evidence of contamination exists, rather than having to spend the majority of their time screening potential problem sites. Given the initial investment of time and needed resources, how can a large number of antibodies to different compounds be developed, standardized, and distributed? Where target molecules and commercial markets are clear, the antibodies will be produced by private industry. The commercial availability of immunoassays for aflatoxins is a case in point. Assay kits are available from such companies as Agritech (Portland, Me,), Noogen (Lansing, Mich.), and Naremco (Springfield, Md.), among others. At present, however, there are only a few compounds attracting private companies. This limited interest stems in part from the newness of the field and from the interdisciplinary nature of the staff required to bridge immunology, synthetic chemistry, and environmental science. Commercial interest in immunoassays is also partially limited by uncertainty as to how regulatory agencies will accept these new tests. Recent meetings held by the EPA, the USDA, the Association of Official Analytical Chemists, and the pesticide section of International Union of Pure and Applied Chemistry have dealt with this new technology. A recent EPA advisory panel concluded, “The EPA should make clear their needs and priorities with respect to analytical objectives. A list of chemicals for which no reliable analytical method exists . . . is very valuable, but prioritizing of the list, based on probability of occurrence and toxicological concern, is also required. For chemicals with less pressing need or lower commercial potential (‘orphan chemicals’) the EPA should provide research and development funds to develop ap-
propriate antibodies. . . . To facilitate the incorporation of available and future immunological methods into EPA operations, the EPA should develop a core of individuals with expertise in this area. . . . The EPA should play an active role in carrying new methods through an in-house testing and validation procedure.” (66) With minor modifications these recommendations apply equally well to other agencies, The federal agencies charged with monitoring our environment and food must provide funding and encouragement to cover initial investments if new immunoassays are to be developed. The benefits of such investments will be great. For example, nationwide monitoring programs such as the one recently undertaken for dioxins can be freed of many of the costs and problems associated with analyzing large numbers of samples ( 6 7 ) . Cleanup efforts at waste sites will benefit as well. As a nation, we are now undertaking the massive problems of decontaminating waste. Whatever approaches are used, four prime questions remain: Are the efforts effective? How do rival methods compare? Exactly where must the decontamination be done at a given site? Have chemical residues been reduced to low enough levels? To the existing analytical burden of determining the spatial extent of pollution is added the temporal dimension, for one now must follow the decay of decontamination over time. The end result will be a vastly increased sampfe load. Environmental science has always been a multidisciplinary field, but the terms and concepts of immunology are new to most analytical chemists. Similarly, the clinical development of immunoassays means that most experience has been gained on the analysis of blood, urine, and tissue samples. The immunochemical analysis of samples from soils, ground water, waste chemical drums, or produce poses new challenges in sample preparation that have yet to be extensively addressed. A dozen or so immunoassays for various chemicals must be validated so that environmental chemists can become familiar with the techniques involved and so that immunochemists can become appreciative of the particular problems associated with performing analyses on samples extracted from environmental matrices. In the future there may be immunoassay formats better suited for the particular problems associated with environmental monitoring. The currently available assays such as cELISA and RIA all require that samples be collected and taken back to a laboratory for processing. Future developments in
instrumentation may allow immunoassays to be conducted in situ (e.g., in wells or on smoke stacks). Already antibodies have been coupled to a variety of solid-state electrical sensors and optical fibers (68- 70). Although all of these new assays are still experimental, they hold the promise that passive monitoring probes could be engineered to continuously monitor water or air for the presence of specific chemicals. Coupling the specificity of immunological reactions with all the advantages of micro-electronics is one of the challenges for the future.
Acknowledgment Funding for this work was provided by the U.S. Environmental Protection Agency under interagency agreement number DW 89931433-01-0, the U.S. Department of Agriculture under interagency agreement 12-37-5-043,and was performed under the auspices of the US.Department of Energy at Lawrence Livermore National Laboratory under contract W-7405-ENG-48.
References (1) “Better Regulation of Pesticide Exports and Pesticide Residues in Imported Food is Essential”; Office of the Controller General, Government Accounting Office: Washington, D.C., 1979; CED 7943. (2) Farah, F. S.; Kern, M.; Eisen, H.N. J . Exp. Med. 1960,112, 1195-1210. (3) Sittig, M. Priority Toxic Pollutants; Noyes Data Corp.: Park Ridge, N.J., 1980; p. 6. (4) Paul, W. E. Fundamental Immunology; Raven: New York, 1984. (5) Erlanger, B. F. Methods Enzymol. 1980, 70, 85-104. (6) Gafre, G; Milstein, C. Methods Enzymol. 1981, 73, 3-45. (7) Oi, V. T.; Herzenber, L. A. In Selected Methods in Cellular Immunology; Michell, B; Singii, S. M., Eds.; Freeman: San Francisco. 1980: DD. 351-72. (8) Poljak, R.-j.etal. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 3305-10. (9) Rappe, C. Environ. Sei. Technol. 1984, I8,78A-90AS (10) Delaage, M. et al. In Monoclonal Antibodies and New Trends in Immunoassays; Bizollon, Ch. A . , Ed.; Elsevier: Amsterdam. 1984: DD. 53-58. (11) Monroe,-’D. Anal. Chem. 1984, 56, 920A-931A. (12) Pesce, A. J.; Ford, D. J.: Makler. M. T. In Enzyme Immunoassay; Ishikawa, E.; Kawai, T.;Miyai, K., Eds.; Igaku Shoin: Tokyo, 1981. (13) Engvall, E.; Perlman, P. Immunochemistry 1971,8, 871-74. (14) Voller, A.; Bartlett, A.; Bidwell, D. Immunoassays for the the 1980s; University Park Press: Baltimore, 1981. (15) Rogers, R.P.C. In Practical Immunoassay: The State of the Art; Butt, W. R . , Ed; Dekker: New York, 1984; pp. 253-308. ’ (16) Ercegovich, C. D. In Pesticide Identijcation at the Residue Level; Gould, R. E , Ed. ; Advances in Chemistry 104; American Chemical Society: Washington, D.C., 1971; pp. 162-78. (17) Hammock, B. D.; Mumma, R . 0 . In Recent Advances in Pesticides: Analtyical Methodology; Harvey, J . , Jr.; Zweig, G . , Eds.; ACS Symposium Series 136; American Chemical Society: Washington, D.C., 1980; pp. 321-52. (18) Van Emon, J . M.; Hammock, B . D.; Seiber, J . N. Anal. Chern. 1986, 58, 186673. Environ. Sci. Technol., Vol. 22, No. 3,1988 253
(19) W i , S. 1. et al. J. Appl. Bacteriol. 1984, 57,447-54. (20) Wing, K. D.;Hammock, B. D.;Wustner. D. A. 1. Agric. Food Chem. 1918.26, 132833. (21) W i , S. 1.; Hammock, B. D. AMI. Bioc k m . 1982,125, 168-76. (22) Hammock, 8. D. et al. In Pesticide Science and Biotechnology: Greenlaigh. R.; Roberts. T. R.. Eds.: Blackwell Scientific: London, 1987;pp. 309-16. (23) Mumma. R. 0.; Brady. 1. E In Pesticide Science and Biotechnoloiy; Omnlaigh. R.; Roberts. T. R., Eds.; Blackwell Scientific: London,
("07. ,701.
.
-_
pp..,.*,**_I " I
Envimn. AMI. Chem. 1981, IO. 295. (56) Luster. M. 1. et al. Toxicol. Appl. Pharm c o l . 1979.50, 147-55. (57) Johnson. H. 1. et SI.J. AMI. Exicol. 1980.4, 86-91. (58) Johnson, H. 1. et al. 1. AMI. E x i d . 1981,5. 157-62. (59) Spierlo. F. W. et al. 1. AMI. Exicol. 1987,II.31-35. (60)Vanderlaan, M. et PI. Carcinogenesis, in press.
(61) Warwick M. 1.; Bales. M. L.; Shearer. G. I n Immunoassays in Food AMIySis: MarTis, B. A,; Clifford, M. N., Eds.; Elsevier: London. 1985;pp. 169-86. (62) Harris, C. C. etal. Pmc. Notl. Acad. Sci. U.S.A. 1985,82.6672-76. (63) Morris, B. A,; Clifford. M. N. In Immunoassays in Food AMIysi3: Elsevier: London, 1985;pp. 159-68. (64)Chu. E S. J. Food Pmt. 1984.47, 56269. (65) Safe. S. H. Annu. Rev. Pharmcol. Exicol. 1986,26,371-99. (66)Summary Stalcmcnt from a Peer Review and Biomarkers Panel Meeting, San Jose, Calif., June 1986; Slatemen1 prepared by ICAIR. Life Systems Inc., for the US. EPA contract number 68424246. (67) "The National Dioxin Study"; U.S. EPA.
"0
(24) Ekins R.: Jackson. T. In MO~OCIOMI Antibodies and New Trends in Immunoassays; Bizollon. Ch. A., Ed.; Elsevier: New York, 1984;p. 149. (25) Rinder, D. E; Flseker. I. R. Bull. E d ron. Contam. Exicol. 1981.26.375-80. (26) Wei. S. 1.; Hammock, B. D. J. Agric. Food Chcm. 1984.32. 1294-1301. ~.... (27) Newsome, W. H.; Shields, 1.E.J. Agn'c. Food Chem. 1981.29, 220-22. (28) Newsome, W. H. Bull. Environ. Contam. ~
~
~~~
Toricol. 1986. 36. 9-14. I
' Chem. I&, 33,962-65. (30) Knopp. D.; Nuhn, F!; Dobberkau. H. Arch. Toxtcol. 1985, 58, 27-32. (31) Schwalbe, M.;Dorn. E.;Beyermann. K. 1. Agric. Food Chem. 1984,32.73441. (32) Niewola. 2.et 81. Clin. Chim. Acto 1985,
Offce of Water Regulations and Standards; Washington. D.C.. 1986. (68) Sutherland. R. M. el al. Clin. Chcm. 1984,30, 1533-38. (69) Andrade, J. D.el al. IEEE P a m . Electron Devices 1985, ED-32, 1175-79. (70) Ngeh-Ngwainbi, 1. el PI.1. Am. Chem. soc. 1986,108.544447.
IdR . .-, 14Q.56 ..~
(33) Huber, S. 1.; Hock. B. 1. Plant Dis. and Prof. 1985,92,147-56. (34) Huber, S . 1. Chemosphere 1985, 14. 1795-1803. (35) Ercegovich. C. D. et al. J. Agric. Food Chem. 1981,29.559-63. (36) Stanker, L. H. et al. J. Apric. - Food Chem., in press. (37) Newsome. W. H. 1. Agric. Food Chem. 1985,33,528-30. (38) Newsome. W. H. In Pesticide Science ond Biotechnology; Grssnlaigh, R.; Roberts, T R., Eds.; Blackwell Scientific: London, 1987;pp. 349-52. (39) Wild, C. F! etal. 1. CcllulorBiochemistry 1986.30, 171-79. (40)Van der Laken. C. 1. et al. Mutot. Res. 1984, 130.208. (41)Santella. R. M.;Hsieh, L. L.; Perera. F. &sic Life Sci. 1986.38. 509. (42) Van der laken. C. 1. et al. Corehogmesir 1982,3,569-72. (43)Harris, C. C. Prm. Natl. Acad, Sci. U.S.A. 1979, 76. 533639. (44)Kreik. E.; Welling. M; van der Laken, C. J. In Methods of Monitoring Erposure to Carcinogenic ond Mutogenic Agents: Hemminki, K.; Vainia, H., Eds.; IARC Press: Lyon. France, 1985;pp. 297-305. (45) Wild, C. F! et al. Carcinogenesis 1¶U,4,
Marlin b d e h obtained a Ph.D. from New York Universiry 's Institute f o r Environmental Medicine and joined the staff at Lawrence Livermore National Laboratory (LLNL) in 1976.
I
-
I
l f. d.l. - 9.. .
(46) Strickland, F! T.;Boylc. 1. M. Pmg. Nucleic Acid Res. Mol. Biol. 1984.31, 1-49. (47) Baan, R. A. et 81. In Mutogem in Our Environment; Sorsa, M.; Vainia, H.,Eds.; Liss: New York, 1982;p. I I I. (48) Haugen, A. el al. Proc. Notl. Acad. Sei. U.S.A. 1981, 78,4124-27. (49) Grwpman. 1. D. el SI.Pmc. Nul. Acad. Sci. U.S.A. 1984,81. 7728-31. (50) Morgan, M.R.A.; McNerney. R.: Chm, H. W.4. In Immunoassays in FoodAMlysis; Morris, B. A.; Clifford, M. N.. Eds.; Elsevier: London. 1985;pp. 159-68. (51) Haas. R. A.; Hanson. C. V.; Monteclara, F. Presented at the 79th Annual Meeting of the Air Pollution Control Association. lune IORh
(5ijAibro. P: W. et al. T a r i d Appl. Pharmncol.
1979.50. 137-46.
(53) Luster, M. 1. et 81. AMI. Chcm. 1980,52, 5 M .1407-1.__I.
(54)Stanker, L. H. et al. Taricology 1987.45, 22943. (55) Newsome, W. H.; Shields. B. I. la. J. 2%
Envimn. scl. Rchrol.. MI. 22,No. 3,1888
Bruce E. W h s (I) has his Ph.D. in organic chemistryfrom the University of California, Berkeley. He joined the LLNL staff in 1984. Lany Stanker (r) obtained a Ph.D. in botany from the University of IlIinois. He worked in the Department of 7Lmor Virology at the M. D. And rson Hospital and l b m r Institute in Houston and joined the LLNL staff in 1982. Together. they led the IJW~OCIOMI ontibody group at LLNL, which has as its main focus the development of immunoassaysfor toxicology research and human health monitoring.
