Environmental Immunochemistry: Responding to a ... - ACS Publications

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Environmental Immunochemistry: Responding to a Spectrum of Analytical Needs 1

Jeanette M . Van Emon and Clare L. Gerlach

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Downloaded by KAOHSIUNG MEDICAL UNIV on April 3, 2018 | https://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch001

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Characterization Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, P.O. Box 93478, Las Vegas, NV 89193-3478 Lockheed Martin Environmental Systems, 980 Kelly Johnson Drive, Las Vegas, NV 89119

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The field of environmental immunochemistry brings together several specialties, including analytical chemistry, biochemistry, molecular biology, and environmental engineering. This multidisciplinary nature is both a benefit and a confusion to practitioners, rewarding a mastery of several scientific skills with the excitement of innovative technology. Environmental immunochemistry can be as simple as a disposable immunoassay test kit or as complex as an integrated system that employs immunochemical techniques as a component of a multistep process. The growing regulatory and user acceptance of immunochemical methods for dozens of regulated compounds ensures the continued growth in this technology - at the lab bench, at the hazardous waste site, and beyond. Applications are widespread, including determination of agricultural runoff, assessment of human and ecological exposure, quantification of food and pharmaceutical purity, and groundwater monitoring.

Environmental immunochemistry has grown dramatically in the academic, commercial, and regulatory areas over the past fifteen years. Before the 1980s, immunochemical methods were widely used in clinical applications (7) and their success in these critical studies led environmental scientists to consider immunoassay use for screening of hazardous compounds in various media. Body fluids are complex media, but new challenges were presented by soils, sludges, food, and agricultural products. Methods were developed, test kits were designed and manufactured, and many comparison studies were initiated to evaluate the performance of these environmental analytical newcomers. It is appropriate at this juncture in the development and use of environmental immunoassays, to review the success of the methods, assess the status of regulatory

0097-6156/96/0646-0002$15.00/0 © 1996 American Chemical Society

Van Emon et al.; Environmental Immunochemical Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


1. VAN EMON & GERLACH

Responding to a Spectrum of Analytical Needs

acceptance, and welcome the next tier of practitioners to the arena of immunochemical and related technologies. This volume presents in-depth research reports, environmental applications studies, data interpretation subtleties, and commercial success stories. It is hoped that the scope of this monograph will make it interesting to the experts, give new applications ideas to researchers, and provide a strong technical basis for novice users. The multidisciplinary character of immunochemical technology is one of the strengths of the Immunochemistry Summit Meetings, upon which this volume is based. The popularity of the Summit Meetings is due to the recognized value of interagency and intercorporation exchange of ideas. A l l immunoassays are based on the interaction between an antibody and a target analyte. Antibodies are produced in response to an immunogen by a complex mechanism (2). Recently, as analytical chemists have become more interested in the technology, better quantitative methods have been developed, and the user community has benefitted (5). Commercial manufacturers of immunoassay test kits have contributed to the availability of more and better analytical tools. University researchers continue to push the technical envelope, extending immunochemical capabilities well beyond their status in 1990. The combined enthusiasm of these groups is apparent in this volume and is palpable at the Summit Meetings. Research in the area of human exposure monitoring is described in this edition. R. E. Biagini and fellow researchers at the National Institute for Occupational Safety and Health (NIOSH) present work that demonstrates the efficiency of immunoassay test kits for measuring alachlor metabolites in urine. Absorption, partitioning, and excretion of toxic compounds is reliant upon several factors and this multivariate character presents a challenge in data interpretation as well as in analytical procedures. Examples of the research at NIOSH are presented, including the use of circulating antibodies and antibody techniques to monitor exposure in the urine of exposed workers. Research at NIOSH demonstrated that circulating antibodies to morphine can be present in the absence of urinary analytes. In another NIOSH study, an immunoassay test kit, originally developed for alachlor analyses in groundwater, was found to be 4-5 times more sensitive in detecting the primary human metabolite of alachlor, alachlor mercapturate, than in detecting the parent molecule. For some compounds, immunoassay techniques are orders of magnitude more sensitive than traditional gas chromatographic/mass spectrometric techniques. Benefits such as increased sample throughput, reduced cost, simpler sample preparation and no derivitization steps make this type of analysis very attractive. V. Lopez-Avila and J. M . Van Emon discuss their work in the coupling of immunoaffinity chromatography (IAC) extraction with on-line liquid chromatography and mass spectrometry (MS). The IAC technique is based on the ability of antibodies to separate a target analyte from the complex matrices that often challenge environmental analytical chemists. On-line analysis is done by high-performance liquid chromatography (HPLC) and electrospray MS. The system is particularly useful for the analysis of compounds that are water-soluble, nonvolatile, thermally labile, or highly polar. Sample throughput of the MS is increased by integrating IAC with the analytical instrumentation, providing automated, streamlined sample preparation.


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ENVIRONMENTAL IMMUNOCHEMICAL METHODS

I. Wengatz and co-workers at the University of California-Davis are developing immunoassays to assess human exposure to xenobiotics, such as pesticides (4). Metabolites of certain xenobiotics may serve as biomarkers of exposure in toxicity studies. The UC-Davis group is developing immunoassays for trace levels of triazine herbicides and their metabolites, and is using cross-reactivity information to enable antibody use for screening classes of analytes. Human exposure research is increasingly important as regulatory agencies move from prescriptive methods based on absolute contaminant concentration to risk-driven guidelines based on the bioavailability of the contaminant and its threat to human and ecological health. Other immunochemical-based technologies, such as biosensors, are being developed at the Naval Research Laboratory. L . Shriver-Lake and fellow researchers describe a continuous flow immunosensor that can be used to measure small molecules in discrete samples or in monitoring process streams. A fiber optic biosensor, based on a competitive immunoassay being performed on the fiber core of a long optical fiber, is also being studied. Response is measured by the change in the fluorescent signal. Electrochemical immunoassays are based on modifications of enzyme immunoassays with the enzyme activités being determined potentiometrically or amperometrically. O. Sadik provdes a status report on electrochemical immunosensors based on conducting electroactive polymers. Immunosensors provide the analytical advantages of conventional immunoassay methods, as well as the option of obtaining real-time monitoring measurements. A n electrochemical immunosensor is also described for the analysis of polychlorinated biphenyls. The promise of these methods is in the eventual development of a sensor that can be used remotely, gathering information without an operator and sparing personnel the possible exposure associated with some environmental work. S. Coulter and associates discuss a solid state system that combines the advantages of optical sensing and competitive immunoassays. This sensing package comprises a light source which provides the output through the waveguide, the sensing chemistry, and the appropriate detector. This chip-based sensor is easily manufactured and has a sensing arm and a reference arm. By combining fiber optic chemical sensor technology with immunoassay, these systems enlarge the panorama of analytical tools available to environmental scientists. Research that reduces the number of steps in an analytical procedure will be appreciated as the environmental analysis emphasis moves from the laboratory to the hazardous waste site. Immunoassay test kits and other immunochemical procedures are now used almost routinely to monitor the purity of food and drugs, and drinking water. Immunoassay test kits are increasingly used at hazardous waste sites regulated under Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) and Resource Conservation and Recovery Act (RCRA). An important step was taken in 1993 when the EPA's Office of Solid Waste and Emergency Response (OSWER) accepted nine immunoassay methods for its compendium, SW-846 (5). This regulatory acceptance means that these methods can be used for certain R C R A applications. The EPA's Office of Water and Office of Drinking Water are utilizing field methods such as immunoassays to determine the safety of the nation's water supplies for drinking, agriculture, and recreational use (6). The U.S. Geological





Survey uses immunoassays to analyze water samples (7). The U.S. Food and Drug Administration (FDA) uses immunochemical methods to determine the purity of processed foods and manufactured drugs. The U.S. Department of Agriculture uses immunoassays to measure the levels of pesticides in crops and their byproducts and in meat and poultry inspection. M . Trucksess and associates describe work done at the F D A to determine the levels of fumonisins in corn. Fumonisins are mycotoxins that have demonstrated toxicity in horses and swine, and have been implicated in certain cancers in humans. Immunochemical methods are now preferred for mycotoxin monitoring in foods because results are obtained much faster than with traditional methods, such as thinlayer, liquid, and gas chromatography. Enzyme-linked immunosorbent assay (ELISA) methods are commercially available and the F D A study compares and evaluates these technologies. The need for inexpensive, easy-to-use methods has been well documented (8,9). With this low-cost, however, users may sometimes relinquish maximum analytical performance, such as extremely low detection limits, very high precision, and even analyte-specific identification. But not necessarily. Many quantitative immunochemical methods are now available that achieve extremely low detection levels and rival their traditional laboratory counterparts. Often there is a need for both screening methods (e.g., in characterizing hazardous waste sites) and higher cost analytical procedures. Special quality assurance (QA) considerations are needed in immunochemical methods. W. A . Coakley of the U.S. EPA's Environmental Response Team and a technical support team from Roy F. Weston, Inc., describe a QA system that focuses on generic and core indicators of confidence. Generic indicators assess the reliability of the total sampling and analysis scheme. Core indicators are specific to the mode of analysis, in this case, immunoassays. Several features are key to the interpretation of immunoassay results: temperature, analyte specificity, non-analyte interference, moisture content, and dilution factors. Understanding the entire process and the potential effects of these and other factors is essential to the quality of information obtained with these innovative methods. The first role of immunoassay test kits was in screening applications where they provided a welcome addition to thefield-portableinstruments commonly used in hazardous waste site characterization. Their results are comparable to those from gas chromatography/mass spectrometry (GC/MS). Though immunoassays are frequently more sensitive than GC/MS, high immunoassay results are still routinely confirmed by laboratory procedures. In this aspect, technical strength has outpaced regulatory and user acceptance. Early uses at hazardous waste sites were conducted by EPA's Superfund Innovative Technology Evaluation (SITE) program (10). In 1988, the first SITE demonstration of a measurement technology evaluated immunoassays for pentachlorophenol (11). Subsequent SITE demonstrations evaluated immunoassay test kits for benzene/toluene/xylene (12) and polychlorinated biphenyls (13). The use of immunoassays to obtain quantitative results has escalated in the past several years. With this increased reliance upon sensitivity and specificity come several challenges in the area of data interpretation. T. L. Fare and fellow researchers


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at Ohmicron Corporation detail the importance of correct calibration techniques. Three basic approaches are discussed: empirical, semi-empirical, and equations derived from the Law of Mass Action. Types of error are described and recommendations are made regarding the processing of immunoassay data. R. W. Gerlach and J. M . Van Emon discuss a variety of data analysis and interpretation issues identified as a result of their work in field evaluations of environmental immunoassays. Analysis of multiple estimates for parameters such as false positive and false negative rates suggest that interval estimates are often better than point estimates. Response factors which control false negative and false positive rates are identified. The effect of explicit and implicit experimental design factors on data interpretation and their impact on the use of advanced non-linear calibration analysis are also reviewed. These papers are critical in understanding the strengths and weaknesses of various statistical procedures for interpreting data from an immunochemical study in the laboratory or in the field. The success of early environmental immunoassay studies led to increased research, publication, and commercial development. More immunoassays were developed for a wider number of compounds of environmental concern. This research effort resulted in test kits that were capable of achieving more reliable results and lower detection limits, with less cross-reactivity. Immunoassays are available for individual compounds and for groups of related compounds. For example, one can use immunoassay test kits to monitor a specific triazine, such as atrazine, or a group of closely related triazines. In some cases, if the ratio of cross-reactivity for specific compounds is known, monitoring for the group and multiplying by the correct factor can be a time-saving and inexpensive method for characterizing a hazardous waste site. Novel innovations are also expanding the range of commercially available detection systems. K. Dill describes the Threshold Immunoassay System, a commercial sandwich immunoassay detection system, that is based on a silicon chip with eight identically etched sites. The system reduces the distortions due to solid-phase/liquidphase interactions by using solution-phase binding and is capable of detecting a wide range of molecular weights, from pesticides to DNA. The normal sandwich immunoassay format was modified to indirectly detect the herbicide atrazine. This paper presents an excellent example of industrial response to technical market requirements. The market drives the research into faster, less expensive, and versatile analytical methods. The primary use of environmental immunoassays is in field-screening procedures because of the relative low cost and the ease-of-use in hazardous waste site environments, but research based on the high sensitivity of immunochemical methods has elevated the technology to a strong competitor in the quantitative laboratory as well. This next step to acceptance as a quantitative analytical procedure is critical. Pragmatic acceptance dictates regulatory acceptance. Regulatory acceptance stimulates commercial interest. Commercial interest results in more candidate methods. These new methods may then gain pragmatic acceptance. Thus, the circle of research and development is perpetuated, resulting in better procedures for chemists, better data for end users, and a better environment for everyone.





By linking immunochemical procedures to other analytical and sample prepara tion steps, analysts are exploring new avenues for technical advancement of immuno logic analytical procedures. In this volume, work is presented that describes supercritical fluid extraction with ELISA, electroimmunochemical processes, and the use of metal chelates in certain environmental applications. There are opportunities for research in teaming GC/MS methods with immunoassays. Capillary electrophore sis with laser-induced fluorescence offers another linking option for immunochemical methods. The results of the research so far indicates considerable promise in these hyphenated techniques and research is ongoing at university, private, and government laboratories. The future of environmental immunochemical technologies is very promising. Ongoing research and continued regulatory interest set the stage for an expanding technological base - in field and laboratory applications, in human exposure moni toring, and in food and agricultural uses. The editors wish to thank the hundreds of participants in the Summit Meetings whose interest and input have made the meetings successful and made this volume possible. The editors gratefully acknowledge the contributions of all the authors and the reviewers for their valuable comments in preparing this book.

Acknowledgments The editors are indebted to Allan W. Reed for his tireless efforts in format, layout, and production matters. A l Reed is a Senior Environmental Employement Program enrollee, assisting the U.S. EPA under a cooperative agreement. The U.S. Environ mental Protection Agency (EPA), through its Office of Research and Development (ORD), funded and collaborated in the research described here. It has been subjected to the Agency's peer review system and has been approved as an EPA publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Literature Cited 1. 2.

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Berson, S.A.; Yalow, R.S.; Bauman, Α.; Rothschild, M . A . ; Newerly, K . J. Clin. Invest. 1956, 35, 170-190. Van Emon, J.M.; Seiber, J.N.; Hammock, B.D. In Advanced Analytical Techniques; Sherma, J., Ed.; Academic Press: San Diego, CA, 1989, Vol. XVII. Hammock, B.D.; Gee, S.J.; Cheung, P.Y.K.; Miyiamoto, T.; Goodrow, M . H . ; Van Emon, J.M.; Seiber, J.N. In Pesticide Science and Biotechnology; Greenhalgh, R. And Roberts, T.R., Eds.; Blackwell: Oxford, 1986; 309. Hammock, B.D.; Mumma, R.O. In Pesticide Analytical Methodology; Zweig, G., Ed.; A C S Symposium Series No. 136, American Chemical Society, Washington, DC, 1980; 321-352.


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Office of Solid Waste and Emergency Response. Test Methods for Evaluating Solid Waste. Physical/Chemical Methods. Vols. I and II. Third edition; EPA/SW-846; U.S. Environmental Protection Agency: Washington, D C , 1995. Environmental Monitoring and Management Council. Environmental Monitoring Methods Index, Version 1.0, Users Manual; EPA/821-B-92-001; U.S. Environmental Protection Agency: Washington, DC, 1992. Goolsby, D.A.; Battaglin, W.A.; Thurman, E . M . Occurrence and Transport of Agricultural Chemicals in Mississippi River Basin in July-August, 1993; Circular 1120-C; U.S. Geological Survey: Washington, DC, 1993. Van Emon, J.M.; Lopez-Avila, V . Anal. Chem. 1992, 64(2), 79A-87A. Van Emon, J.M.; Gerlach, C.L. Environ. Sci. Technol. 1995, 29(7), 312-317. Superfund Innovative Technology Evaluation Program, Technology Profiles, Sixth Edition; EPA/540/R-93/526; U.S. Environmental Protection Agency: Washington, DC, 1993. Van Emon, J.M.; Gerlach, R.W. Bull. Environ. Contam. Toxicol., 1992, 48, 635-642. White, R.J.; Gerlach, R.W.; Van Emon, J.M. An Immunoassay for Detecting Gasoline Components; EPA/600/X-92/116; U.S. Environmental Protection Agency: Washington, DC, 1992. Johnson, J.C.; Van Emon, J.M. Development and Evaluation of a Quantitative Enzyme-Linked Immunosorbent Assay (ELISA) for Polychlorinated Biphenyls; EPA/600/R-94/112; U.S. Environmental Protection Agency: Washington, DC, 1994.


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