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Immunochemistry Research
An Overview of the EDRB, NERL Immunochemistry Program
The U.S. EPA supports the development and utilization of technologies that assist environmental scientists in making correct assessments of the level and extent of contaminants at hazardous waste sites as well as assessing risks and impacts of such contaminants. In recent years, field-portable instrumentation has effectively brought the analytical capabilities of the environmental laboratory to the site of contamination. Some technologies previously confined to the research laboratory are now available at low cost to field crews whose investigations benefit from on-site, real-time results. Field portability eliminated much of the high costs and high levels of technical expertise required for laboratory analyses.
Immunochemical methods as reliable techniques used in medical and clinical applications for years have been considered more recently for environmental applications. Immunoassays are based on selective antibodies combining with their corresponding target analyte or group of related target analytes.
Since 1987, the U.S. EPA Exposure & Dose Research Branch-Las Vegas (EDRB-LV) has led the Agency's effort to correctly develop various immunochemical methods for screening and quantitative analysis of compounds of environmental concern. The EDRB-LV Immunochemistry Program is an active participant in the research of new immunochemical technologies, the demonstration of commercial immunoassay test kits on-site, and the interdisciplinary effort to apply good laboratory practices even when the laboratory is essentially in a backpack. Our ongoing research in this area includes:
- synthetic organic chemistry for the development of the haptens, antigens, and immunogens necessary for specific antibody production;
- antibody-based personal exposure monitors to detect vaporous contaminants in the air;
- immunochemistry methods development (including immunoassay, immunoaffinity chromatography and electroimmunochemical methods);
- demonstration of immunoassay test kits at Superfund sites;
- development of a variety of immunoassay formats based on the central reaction of antibody recognition;
- development of new immunochemical methods for the detection of residues in foods and dislodgeable residues from foliage; and
- integration of immunochemical methods with traditional analytical procedures (e.g., immunoaffinity chromatography with HPLC and MS, and ELISA with supercritical fluid extraction).
The U.S. EPA and other federal regulatory agencies have actively investigated using immunochemical methods for various screening applications. These applications range from meat inspection by the U.S. Department of Agriculture to the determination of pharmaceutical purity and levels of antibiotics in milk by the U.S. Food and Drug Administration. The EPA benefits from rapid laboratory and on-site analysis of analytes of environmental concern. In addition, the laboratory use of immunochemistry can be valuable in the screening of samples that, because of their contaminant concentration or nature, might be harmful to a sensitive detector.
Immunochemical methodology includes the entire process of synthesis of reagents, assay development, sample preparation, analyte detection, and data analysis. Immunoassay kits have been developed that are easily transportable to a hazardous waste site or other monitoring site. The advantage to using field-portable methods is quick turnaround, maintenance of sample integrity, and cost savings. Worker health can be protected by the early identification of hazardous components in a field sample. Immunoassay kits are available in a variety of formats. The kits essentially package-specific antibodies, reagents, standards, enzyme conjugates, and chromogenic substrates in ready-to-use units. For immunoassay kits to function well as measurement tools, the end user must ensure that proper sampling protocols and sample preparation are be employed. End users must also be informed and educated as to the uses, limitations, and sensitivities.
Initially, environmental immunochemistry methods were limited to qualitative tests that provided results in a yes/no format based on an established threshold. Currently, methods are being developed to provide semiquantitative and quantitative results. Comparisons between immunochemical methods and more traditional methods (such as gas chromatography-mass spectrometry) typically have shown good agreement, especially when the sample preparation steps have equivalent efficiency. Immunochemical methods can be as simple as the direct use of kits to get a yes/no response or as complex as any quantitative analytical procedure, with cleanup and sample preparation steps, and expert data interpretation.
Immunochemical methods can be coupled with traditional techniques to allow the researcher to exploit the advantages of both, often with a synergistic effect. Research is being performed in the area of hyphenated techniques (i.e., immunoassay and immunoaffinity columns coupled with on-line and off-line instruments). An example at the EDRB-LV is the coupling of enzyme-linked immunosorbent assay (ELISA), with supercritical fluid extraction (SFE) for analyte/contaminant identification and quantitation. Another example, especially useful when mass spectral data are required, is the coupling of high-performance immunoaffinity chromatography for sample preparation with mass spectrometry. Microbore high-performance liquid chromatography (HPLC) generates low volume extracts which can be analyzed directly by ELISA. Robotics use in the laboratory increases the speed and reliability of routine immunoassays and can easily support large scale epidemiologic or exposure assessment studies.
Scientists in the EDRB-LV Immunochemistry Program team with private
industry or university researchers. These partnerships, fostered by the
Federal Technology Transfer Act of 1986, allow
government scientists to work with their non-government counterparts to
advance the state-of-the-science in all aspects of research and commercialization.
Formal and informal collaboration with researchers at universities, such
as the University of California-Davis and Tulane University, help to keep
EDRB-LV immunochemistry researchers current on immunochemical and related
research being conducted in the academic community. In addition, the Immunochemistry
Program is active in planning and participating in various technical meetings
and symposia, including the Pittsburgh Conference, American Chemical Society
meetings, the Gordon Conferences, and conferences sponsored by the Association
of Official Analytical Chemists.
The EDRB-LV immunochemistry laboratories perform various immunoassays, including radioimmunoassays, fluoroimmunoassays, and enzyme immunoassays. This breadth of capability gives the EDRB-LV considerable strength in performing methods comparisons between various immunochemical methods and other technologies.
Inhouse research is conducted, in part, through the Senior Environmental Employees Program (SEEP). Periodically, vacancies become available in the SEEP to support immunochemistry research. For further information contact vanemon.jeanette@epa.gov.
The EDRB-LV Immunochemistry Program laboratories also have a suite of analytical instrumentation, which aids in methods development and comparison, including:
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The initial application of immunochemical research was in clinical chemistry, an area that has used various immunoassays for medical tests for many years. Since the early 1980s, the use of immunoassays for environmental studies has been growing. Environmental immunochemistry includes several areas of expertise: analytical chemistry, biotechnology, immunology, synthetic organic chemistry, statistics, and chemometrics.
The EDRB-LV Immunochemistry Program designs its products to meet the requirements of its clients. These clients include the EPA Regions, the Superfund Program, the Office of Water, the Office of Pesticides, and, ultimately, the American public. Client applications include the analysis of pesticides in food, dislodgeable residues on crops, screening prior to chromatographic analysis, and various exposure assessments. Several analytes are under investigation at the EDRB-LV.
Sample preparations using immunoaffinity are based on the attraction between a selective antibody and a specific analyte. Sample extraction and preparation are accomplished using immunoaffinity chromatography columns. This procedure is being investigated for preconcentration and cleanup of complex samples such as foods, soils, and sediments.
ELISA is a detection technique that is part of the multistep process that begins in the synthesis laboratory. The first step is the determination of the target analyte (e.g., is it the parent compound, an environmental breakdown product, or a biological marker of exposure?). The exact requirements for a particular application must be determined before any analytical procedure is employed. This is particularly true for immunochemical methods development since the first step of hapten synthesis influences the selectivity of the resulting antibody. Haptens are designed, synthesized, and characterized. Next, the specific antibody is produced, characterized and used for assay development. The assay is formatted to best suit the end use. Extraction steps and cleanup procedures are optimized for the matrix. Then the method is tested on real world samples and the results are compared with those from accepted methods. The immunoassay is then ready for delivery to the end user.
Research into the analytical devices known as biosensors is another ongoing research area at the EDRB-LV. One type of biosensor uses an antibody as the biological sensing element in contact with an electrochemical transducer to relate the concentration of an analyte to a measurable electrical signal.
Collaboration with researchers at universities and other government laboratories enables EDRB-LV investigators to explore the use of flow immunosensors and fiber-optic immunosensors for detecting environmental pollutants. Scientists in the Immunochemistry Program at the EDRB-LV are investigating the teaming of conductive electroactive polymers with antibodies to develop a reuseable immunosensor capable of giving a reproducible analytical signal. There are many advantages to using electrochemical immunosensors including: the reversibility of the antibody-antigen interaction, the ability to perform immunoassays by binding molecules, and electronic signal generation on the same substrate. Electroimmunochemical methods show promise for the continuous monitoring of waste streams to measure compliance with regulations. The development of on-line electroimmunoassays offers a powerful method for the detection of PCBs, heavy metals, and pesticide residues. Electroimmunoassay is particularly suited to groundwater monitoring.
As with all analytical procedures, the results of immunochemical tests rely upon thoughtful data interpretation. When innovative methods are being compared with traditional procedures, it is important that correlation, bias, and comparability be established and defined. Chemometrics is used to provide innovative statistical methods to solve such chemical data analysis problems.
The EDRB-LV Immunochemistry Program has been active in methods development to support EPA monitoring needs. Immunoassays and related chelate-based methods are currently being demonstrated and evaluated for the detection of mercury.
The EDRB-LV immunochemistry program has also investigated the use of immunoassay for the detection of pesticides in foods. Supercritical fluid extraction has been coupled successfully with ELISA to analyze this difficult, often heterogeneous matrix. Supercritical carbon dioxide, with and without modifiers, has been used to extract various pesticides from foods, including samples from the FDA's Total Diet Study. These methods are being refined for use in national exposure assessment studies.
The development of immunoaffinity-based personal exposure monitors (PEM) is the first application of immunochemical techniques to direct vapor sampling. The prototype PEM used a very small diameter gas-permeable dialysis tubing as an interface between the vapor and the solution containing the antibodies. These PEMs can be formatted as badges and worn by farm workers, factory workers, and other potentially at-risk individuals, to detect exposure to pesticides and other compounds and presenting a human health concern. Early work has demonstrated the possibility of using immunochemical methods in this application. Currently research is directed toward simplifying and ruggedizing PEMs for field use. An alternative approach for the detection of airborne contaminants also being investigated at EDRB-LV is the collection of samples by active or passive samplers followed through quantitation through immunoassay.
A 96-well, microplate-based ELISA for the quantitative determination of polychlorinated biphenyls (PCBs) in soil has been developed and evaluated on samples from hazardous waste sites. The data generated during the development, evaluation, and application of the quantitative PCB ELISA strongly suggest that this technique can function in a highly accurate and precise manner as a detection and quantitation device when coupled to an efficient extraction procedure. This work promises to bridge the performance gap which exists between immunoassay test kits and instrumental methods.
The EDRB-LV Immunochemistry Program has been active in several field demonstrations and evaluations of immunoassay test kits in response to EPA Regional and Superfund Offices' needs. The first was in 1989 and was performed under the EPA's Superfund Innovative Technology Evaluation (SITE) program. Commercial tube and 96-well plate format immunoassay kits for the determination of pentachlorophenol (PCP) were evaluated by comparison of kit results with conventional gas chromatography (GC) and GC-mass spectrometry results. The results showed both the tube and plate immunoassays to be sensitive screening methods. Though slightly less accurate, they were faster, less expensive, and had a higher sample throughput than GC analysis.
In another SITE demonstration performed in 1992, the EDRB-LV evaluated a semiquantitative ELISA for polychlorinated biphenyls. The EDRB-LV also performed a SITE field evaluation of a test kit for benzene/toluene/xylene (BTX) as a rapid field screening method.
In addition to developing and applying immunochemical methods, the EDRB-LV Immunochemistry Program realizes the importance of communicating and transferring immunoassay technologies. A tutorial was designed to familiarize readers with the basics of immunoassay techniques. This guidance document is useful for novice readers as well as analytical chemists just beginning to apply this new technology.
The EDRB-LV Immunochemistry Program has sponsored an innovative approach to technical networking and communication. The first Immunochemistry Summit Meeting was held in 1992. Researchers from various EPA offices, other government regulatory agencies, chemical manufacturers, instrument companies, and test kit manufacturers met and discussed issues related to the technical development, implementation, and application of immunochemical methods in environmental studies. The success of the first meeting in 1992 led to subsequent yearly meetings. By providing an environment for the free exchange of ideas related to the advancement of immunochemical research and application, the Immunochemistry Program at EDRB-LV is ensuring continued communication among scientists with different approaches and viewpoints, as well as assessing the different needs of many end users.
An industry workgroup was formed as a result of discussions at the first Immunochemistry Summit Meeting. Known as the Analytical Environmental Immunochemical Consortium (AEIC), it provides a forum for promoting and fostering the effective use of immunochemical methods for environmental analysis.
In 1994 the Summit meetings became more research oriented than the previous meetings, indicating that acceptance is no longer the major concern of participants. Basic and applied research is continuing to expand the role of immunochemistry in all areas of analytical environmental chemistry. Several categories of interest have been identified by participants in all the Summit meetings.
The EDRB-LV Immunochemistry Program plans to continue its research into the use of immunochemical methods for environmental applications by fostering research into the development of more sensitive, reliable methods for the detection of analytes of environmental interest. One area of research is robotics which may streamline the routine use of immunochemical procedures in the environmental laboratory. Robotics can also perform extensive parallelism studies for the validation of individual immunoassays.
The Immunochemistry Program will also participate in on-site demonstrations of immunochemical methods to establish their practical usability. Immunoassay methods can easily be integrated into monitoring studies to assess the extent of occupational and non-occupational exposure to environmental contaminants.
In addition, we will continue to investigative innovative ways of coupling immunochemical procedures with traditional technologies, enhancing the capabilities of current methods. An example is microbore HPLC coupled with enzyme immunoassay detection. The high sensitivity of immunoassay is ideally suited to measurement of the low volume fractions produced by microbore HPLC.
The Immunochemistry Program is currently investigating practical applications including immunoassay methods for various pesticides in soil, water, and foods, and the use of immunochemical methods in air analysis. Many immunoassay test kits are available but, due to the complexity of various environmental matrices, refinements must be made to ensure usability for samples such as soil, sediment, leaf washings, food and biological tissues. The demonstration and evaluation of a mercury immunoassay test kit at a Superfund site is also underway.
The EDRB-LV Immunochemistry Program will continue to provide information so that end users can judiciously incorporate immunochemical methods into their suite of analytical procedures. Beginning with the first Immunochemistry Summit Meeting and subsequent development of the Immunochemistry Forum, the Immunochemistry Program is dedicated to advancing the networking among immunochemistry experts and potential end users.
As more becomes known about the advantages of immunochemical methods, the EDRB-LV Immunochemistry Program will continue to lead the Agency research, development, and applications efforts in this challenging, innovative area of environmental analytical chemistry. To maintain this edge, EDRB-LV researchers will continue to work with multiple clients to supply methods that are targeted to particular end-user needs.
The director of the Immunochemistry Program at EDRB-LV is Dr. Jeanette M. Van Emon.
Immunochemistry Program Bibliography
The EDRB-LV has published numerous documents and articles related to the research and application of immunochemical methods in environmental science.