A STATUS REPORT ON
CIELDmPORTABLE IMMUNOASSAY Having revolutionized many field and laboratory analyses, environmental immunochemical technologies are gaining acceptance in the environmental community. JEANETTE M . V A N E M O N
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he development of environmental immunochemical technologies and their growing acceptance within the regulatory community have revolutionizedmany field and laboratoryanalyses. Beginning in the 1980s, environmental scientists adapted methods that had earlier resulted in a Nobel prize for radioimmunoassay ( I ) . Pesticide analytical chemists, recognizing the shortcomings of gas chromatography (GC)for some applications, championed the use of immunoassays. Since this pioneering work, immunoassays have been developed for scores of contaminants in a variety of matrices and for a multitude of monitoring applications. There are many current challenges in the research and application of immunoassay methods. A primary goal is to advance immunoassaysto the next stage of environmental analytical acceptance by involving chemists in the development of quantitative methods. Analytical chemists are beginning to integrate immunochemical methods such as immunoassay as a detector for microbore high-performance liquid chromatography;immunoallinitychromatography is being integrated with standard detection procedures such as mass spectrometry (MSI. These tandem techniques result in more versatile methods for samples of environmental importance. Another challenge is in the development of uniform evaluationguidelines,particularlywhen the detection level of the immunoassay is lower than the accepted comparison method. A related issue is attainment of the level of confirmation necessary to support the required data quality objectivesof a particular study. Initially, many analysts used the rule of thumb that all immunoassay positive values (i.e., above the regulatory action level for the analyte of concern1 should be confirmed by a traditional method. This costly step will be avoided by institut-
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ing proper quality assurance/quality control procedures, and hy continuing to demonstrate the reliability of immunoassays for environmental decision making. All immunoassays rely on the interaction between an antibody and a target analyte. Antibodies are produced in response to an immunogen by a complex mechanism (2).A common format for environmental field-portable immunoassays uses an analyte-selectiveantibody immobilizedon a solid SUIface such as a test tube. The analyte from the sample and a known amount of enzyme-tagged analyte compete for a limited number of antibody binding sites (Figure 1). Quantitation is achieved by comparing the signal generated by an unknown sample with a standard curve. Immunoassay test kits essentially package antibodies, reagents, standards, and substrates in field-transportableunits that are ready to use (3).A 96-well microplate-based immunoassay format is often used in the laboratory because of its high sample capacity. Selective antibodieshave been developed for many compounds of environmental concern, ranging from small pesticide molecules to protein products from recombinant DNA. Figure 2 provides a general development scheme for an immunoassay Environmental immunoassays have been developed and evaluated for analytes including, but not limited to, major classes of pesticides, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons, pentachlorophenol (PCP), and BTEX (henzene, toluene, ethylbenzene,and xylene) compounds (41. Immunoassay test kit manufacturers have responded to the environmental analytical community’s needs hy providing and continuallyrefining fieldportable immunoassay kits and standard operating procedures. The test kit manufacturers, environmental regulators, and major chemical industries are cooperating to continue immunoassaydevelopment for 0013-936Xr5510929-312AsO9.00100 1995 American Chemical Society
environmental applications.A work group called the Analytical EnvironmentalImmunochemical Consortium (AEIC)fosters this cooperation. The AEIC holds frequent meetings to discuss research and regulatory issues and writes guidelines for immunochemical method development. Immunoassays are used in the laboratory as well as in the field. Pre-analysis of environmental samples prior to GC analysis, for example, can identify the need for dilution, thereby saving an expensive electron capture detector from contamination and downtime.
and acceptance of new analytical methods hy the environmental community. It will serve a larger community than the SITE program and will respond to the needs of a wider user community. %o immunochemistry projects are helping the Consortium launch its program. One is a pilot electronic bulletin hoard system (BBS)highlighting immunochemical research and application; the other is a field evaluation of a mercury immunoassay at Nevada's only Superfund site. These efforts are heing conducted through the Consortium, and both projects are expected to he completed in 1995.
Vehicles for field evaluation EPA has participated in field evaluations of several immunoassays. The Superfund Innovative Technology Evaluation (SITE) program was developed toevaluate remediation and monitoringtechnologies for use at hazardous waste sites around the nation. Immunoassay demonstrations are discussed in an EPA Technology Profile of the SITE program (5). In 1988, the first SITE demonstration of a measurement technology evaluated two immunoassays for PCP an immunoassay test kit and a 96-well microplate immunoassay The plate immunoassay compared well (Spearman rank correlation coefficient of 0.92, n = 47, SS% confidence interval) with EPA Method 604, a GC method (6). Both the test kit and microplate immunoassay were compared with SW846 Method 8270, a GC-MSmethod, and showed a slight positive hias, suggesting a minimal tendency to generate false-negative results. This SITE demonstration gives an example of near real-time diagnostic capabilitietthe ability to monitor remediation events-that immunoassays can provide in the field. An immunoassay immediately identified a problem with an on-site bioreactor, which was confirmed two weeks later by MS results. Another SITE project evaluated an immunoassay used to detect BTX compounds (7).The project demonstrated the adaptability and portability of immunoassays by allowing analytical measurements to be performed in a van a few yards from the contaminated site. For BTX concentrations of 100 ppb or more, a false-negative rate of 5% or less can be achieved. A recent SITE project evaluated several fieldportable methods for PCBs, including a 96-well plate immunoassay. Sample splits were analyzed hy both field-portable and laboratory immunoassays using various extraction methods ( 8 ) .The SITE studies served to advance regulatory and technical accepm c e of immunoassay testing. And the resulting synergistic yet competitive relationship between researchers, regulators, and commercial entrepreneurs has benefited the entire environmental analytical community. A partnership formed in 1994 between EPA and the departments of Energy and Defense resulted in the Consortium for Site Characterization Technology Whereas the SITE program's major focus is on innovative treatment and remediation technologies, the Consortium will be dedicated exclusively to development, demonstration, and evaluation of monitoring and characterization technologies. The Consortium can serve as the next step in the evaluation
A technician samples for immunoassay analysis, which will yield near-realtime data on a potentially contaminated site. Photo by EP4 Characterization Research Division.
Interest in field-portable environmental immunochemical methods is great among the on-site analytical community. This interest has been expressed at the annual Immunochemistry Summit meetings, sponsored by the Immunochemistry Program at the EPA National Exposure Research Lahoratory, Characterization Research Division-Las Vegas (CRD-LV). The simultaneous emergence of electronic BBSs and their widespread use has led to many requests for the establishment of a BBS for environmental immunochemistry. This will be a prelude to an electronic BBS encompassing all field analytical technologies. CRD-LV is developing a pilot BBS for environmental immunochemistry methods, including key contacts, literature citations, and case studies. The VOL. 29. NO. 7.19951 ENVIRONMENTAL SCIENCE 8 TECHNOLOGY.
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A generalized procedure for the development of an immunoassay
Laboratory-coordinated a study at the Allied Paper/ Portage Creek/Kalamazoo River Superfund Site, Allegan County, MI. The size and complexity of this site-235 miles long and comprised of soils, sediments, and paper pulp contaminated with PCB+ made field screening methods necessary. Using two immunoassaysand one ion-specificelectrode technique, field scientists completed a study to determine the reliability of these innovative techniques. They conducted a comparison study of these methods and a GC method using performance evaluation materials with known concentrations. The immunoassay field methods used a simple methanol shake extraction and provided semiquantitative results (Table 1). The immunoassay is just one step (detection) in the analytical scheme. In the above study, the GC method used an overnight Soxhlet extraction compared with the abbreviated extraction requirements of the immunoassav These differencesin sample preparation should be considered when comparing methods.
Ongoing research .2velop Eelectlve antibody
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times, etc.. and develop standard curve
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pilot BBS is expected to be released this summer on an exisline EPA BBS...Dossiblv CLU-IN or the Office of Researcyh and Developmen; (ORD) system. To pmvide data for the BBS and for information ahout the expected date of release, contact JeanetteVan Emon, EPA, CRD-LV fax (702) 798-2243. Several EPA regional studies have used immunoassays as screening tools and as semiquantitative detection devices. In 1992, at the request of EPA Region 5, the CRD-LV Technology Support Centerthen called the Environmental Monitoring Systems 3 14 A
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Vendors are developing and evaluating immunoassays for an ever-expanding list of analytes of environmental concern (seep. 316). Several pesticide immunoassays, such as those for benomyl, alachlor, and atrazine, have moved from the research laboratory to field evaluation. Rapid field screening immunoassays have been developed for general compound groups such as PCBs and PAHs. Now they are being developed to accurately measure inorganic compounds, including such heavy metals as cadmium and mercury (9,1O).When immunoassays can quickly and reliably map contamination at a hazardous waste site, subsequent sampling design and better use of in-laboratory analytical instrumentation results in more useful, in-depth information. Often, the analytes are the least of the problem when developing immunoassays. The crossreactivity of the antibody between the analyte of concern and other similar compounds can confuse the issue. Cross-reactivityin immunoassays is similar to interference or lack of chromatographic resolution in classical analytical chemistry Matrix effects from difficult media such as soil, sediment, and sludge can also prove a challenge when developing a fieldrugged, easy-to-use method. The sensitivity of the antibody can be exploited by diluting the sample to minimize interferences and still achieve the desired detection limits (11).It is common practice in immunoassay to run several dilutions of the sample to determine sample matrix effects. If the resulting curve is parallel to the standard CUNP. sample ma& effectscan be considered minimal. This ouick oualirv control Drocedure can determine if sample cleanup steps are necessary. Though streamlined sample cleanup steps are highly desirahle in field analytical methods, little or no sample cleanup may reduce the reliability of the immunoassay. Quantitative standard curves should be shown in buffer as well as in the presence of sample matrix. If cleanup steps are used before the assay, recovery at each step must be known. It is important to have a quantitative appreciation of the
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cross-reactivity of metabolites and decomposition products, structural homologs, and other compounds Semiquantitative results from two field-portable immunoassays likely to he encountered in the samTwo field portable immunoassays were used at the Allied PaperlPoi3age CreeWKalamazoo ple matrix (2).Pre-analytical proceRiver SuoerfundSite on known concentrations of oerformance evaluation materials in soil. dures such as sample drying, extracsediment end paper pulp samples to prove their capabiliies. Semiquantitabve r - ~ ~ ~are * tion, and cleanup are important in shown below. preparing samples fa r immunoPetfwrnance chemical analysis (8). evalualion AWWO AWW. Supercritical fluid extraction couNmhr d number d l a b numb# d falw h k rd material pled with enzyme-linked immuconcsnbnlion, samples nsgalivss. pwism. immuDou.I*I nosorbent assay (SFE-ELISA) has achieved higher extraction efficien0.5 12 0 ) 12 cies with lower organic solvent USE. 1.5 12 0 9 This technology was used to success8.0 12 3 5 fully determine PCB concentrations !5 10 1 8 10 0 4 in soil samples from a S u p e r h d site 6 0 6 (8, 12). Current SFE-ELISA research focuses on multiresidue methods to 62 4 44 determine pesticide residues in f o o < a and other matrices. vironmental Monitoring Methods Index, available ORD began investigating the use of immunoasthrough the National Technical Information Sersays for environmental monitoring applications in vice (13).This recognition of field methods is the next 1987. CRD-L\I an ORD laboratory, collaborates with researchers in the Superfund Office, the EPA Office step in regulatory acceptance of innovative methods. Vendors and users alike welcome this advance of Pesticide Programs (OPP), and the EPA Office of as the first step in uniform method verification and Water (OW). The CRD-LV Immunochemistry Prothe enhancement of the image of field methods. gamk goals are based on input from these and other EPA is not the only regulatory agency participatclient EPA program offices. Superfund, OPP, and OW ing in the development of environmentalimmunowere the first program offices to recognize the advantages of rapid, portable, easy-to-use immunoas- assays. Other federal and state agencies rely on immunoassays to provide high-quality data for less than says. the cost of more traditional laboratory analyses. The The EPA OW is studying immunoassays in the diversity of applications among these agencies is course of permitting under the Clean Water Act. Recently, OW tested specific analytes in grab samples widespread, ranging from blood and urine testing for from commercial laundry effluent. In these difficult matrices, they have found good correlation hetween a commercial immunoassay kit and GC analysis (data unpublished). The OPP work focuses on analysis of pesticide residues in soil, crops, and other matrices to assess occupational and nonoccupational exposure. As more efficacious pesticides with extremelylow biologicaUy active levels enter the marmolecule that can bind M a selective a ket, analytical methods with commensuratelylower detection levels must evolve. . valently bound m a hapten ora label, such The EPA program offices typically rely on analytical methods developed for their specific applicaHapten. A small molecule that can induce antibody tions. The individual offices are evaluating integraproduction when covalently bound to a carrier tion of immunoassays into their monitoring programs. In 1992, the Office of Solid Waste (OSW) began including immunoassays in its guidance document, SW-846. This compendium of analytical and test methods approved by OSW is used in determining regulatory compliance under the Resource Conservation and Recovery Act; it is not an exhaustive list of Agency-wide required methods. The EPA Environmental Monitoring and Measurement Council was chartered to coordinate methMonoclonal antibody. A homogeneous antibo ods research, foster consistency in monitoring methpopulation derived from one specificantib ods, and explore the feasibility of a national laboratory accreditation program. To address acceptance issues on an interagency level, the Consortium on Site Chancterization Technologywas formed to provide a sound scientificbasis for verifyingtechnologies. Publication of a list of field-portable technologies is anticipated later this year in the EPA EnVOL. 29. NO. 7.1995 I ENVIRONMENTAL SCIENCE &TECHNOLOGY rn 3 IS A
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Biokbraake, lnc. 3820 NW 48th street
Lincoln. NE 68624
Millipore Corporation 50 Ashbury R M d Bedford. MA 0173 18w) 845-5478,
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EM Science
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arious pharmaceuticals and biomarkers of expoire to the analysis of raw and processed agricultural commodities for pesticide residues. The U.S. Geological Survey (USGS) and the U.S. Army Corps of Engineers have responsibilityfor monitoring the nation’s waterways. The USGS recently used immunoassays in a study of water quality in the Mississippi River. Immunoassays were also used by the USGS to analyze the floodwaters of the Mississippi during the Great Midwestern Flood of 1993 (14). Immunoassays are being used by state and federal agencies to ensure that the food supply is not contaminated with potentially hazardous chemicals. The US. Food and Drug Administration uses immunoassays to check for mycotoxins, antibiotics,and pesticides in food commodities. The US. Department of Agriculture, through their Food Safety and Inspection Services, collects and analyzes samples of meat and poultry.
Comparability and acceptability
I Analvtes for which immunoassavs have been
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The comparability of results within and between kits and manufacturers is critical for successful incorporation of immunoassays into a larger study. Frequently, immunoassaysare more sensitive than GC methods and, although this sensitivity is a benefit, it could lead to difficulties when comparing data. Methods are diflicult to compare directly because immunoassays can have greater sensitivity hut can he limited by variations attributable to matrix effects. Simple or abbreviated extraction procedures reuire caution. In one study, splits of soil samples conlining PCBs were extracted by two different proceures, simple methanol shake extraction and Dnventional Soxhlet earaction. Detection of both ntracts was accomplished by immunoassay. Soxhlet ntraction proved more efficient for more contamiated samples. A 96-well microplate immunoasiy provided quantitative PCB results (8. Kits should ~ ~ c l u information de on extraction efficiencies over several concentration ranges for a variety of matrices as pan of their quality control measures. Scientists who have good experiences with imiunoassays are quick to praise the technology and recommend it for an expanding list of analytes and matrices. Vendors have an interest in having their products accepted by a wide range of end users and regulators. The tendency to oyersell a promising technology is great. Responsible environmental scientists are encouraging cautious evaluation of innovative methods, warning against acceptance based solely on enthusiasm and potential. Acceptance can he accelerated without compromisingtechnical validity, such as through performance-based methods to fulfill specific data quality objectives. In many monitoring situations, performance-based methods can be used without compromising technical validity or legal defensibility. Immunoassay test kits often are regarded as simpler and easier to learn than more complex analytical procedures such as GC and MS. However, the ease-of-use issue is not without drawbacks. As immunoassays become more readily available, results might be interpreted erroneously. The regulatory community, chemical registrants, the vendor community, and organizations such as the AEIC want to
increase the judicious use of immunoassays by training test kit users, publishing guidelines for immunoassay use, and fostering inclusion of uniform information in immunoassay test kits. The increasing public awareness of the availability and use of immunoassays is an area of promise and concern: promise that the technologywill be applied to many more screening situations in the future and concern that data from these uses might be misinterpreted or misused. The EPA C D L V Immunochemistry Program has sponsored annual Immunochemistry Summit meetings since 1992. These meetings are held in Las Vegas and include formal papers, poster presentations, panel discussions, and a multitude of opportunities for informal networking. The Summit Meeting format was designed to include researchers and regulators from various government agencies, chemical manufacturing companies, universities, and test kit and instrument manufacturing companies. Summit I provided a unique environment for technical networking between these groups. One of the results was the formation of the AEIC. This organization is now chartered, has had several meetings, and continues to establish industry standards. Key discussionpoints at the Summit meetings include research trends, regulatory acceptance, communication issues, and data quality criteria. lmmunochemistry Summit IV will take place August 2-3, A monograph of the papers presented at Immunochemistry Summit 111and the upcoming Summit lV will be published together in early 1996.
Note EPA, through its Officeof Research and Development IORD), partially funded a n d collaborated in the research described here. It has been subjected to the Agency's peer review and has been approved as an EPA publication. Mention of trade names 01 commercial products does not constitute endorsement or recommend them for use,
References I11 Berson, S. A. et al. I. Ciin. Invest. 1956, 35. 170-90. (2) Van Emon, 1. M.;Seiber, 1. N.;Hammock, B. D. In Ad
mnced Analytical Techniques; Shema, I., Ed.; Academic Press: San Diego, CA. 1989 Vol. XVII. 131 Van Emon. I. M.;Gerlach, C. L. Envimnrnentni Lob. 1994. 6121.24-54. 141 Van Emon, I. M.; Lopez-Avila, V. Anal. Chem. 1992, 6412).
._
7QA37rl
I51 'Superfund Innovative lechnology Evaluation Program, 'leohnolngyProfiles.S i Edition'; U.S.Lnvlninmrntal Pro-
tection Agency: Washington, DC. IYYJ; EPAi540. H-93 526. I61 Van tmon.I. M.: (krlach R. W Bull Envirvn. Canmm. I m . trol. 1992. 48. 63542. (71 While. R. I.; tierlarh. R. W ; Van tmon.I. hl. An Immu.
noassay fur L)cwcring(;asolme Components". U S. Environmental hntPcrion Agency Washinyon. DC. 1992. F.PAi 60U X-Y21116. 181 lohnson. i. C.; Van Emon. I hl. "Development and Evalualion of a Quanriratwe Enzyme-Linked Immunusor-
bent Assay fELISAi fur Polychlorinated Ripheqls"; U.S. Environmental Protmion Agmiy: Washington. DC, 1994.
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The increasing popularity of field immunoassay analysis is. in large part due to the highly portable equipment and minimal seblp requirements, Photo by W4 Characterization Research Division.
EPA/600/R-94/112. 19) Blake, D. A. et al. Anal. Biochem. 1994,217,7&75. (IO) Wylie, D. E. etal.Anal. Biockem. 1991, 194, 381-87. (11) Gee, S. I.; Hammock, B. D.; Van Eman, I. M. 'X User's Guide to Environmenfal ImmunochemicalAnalpis"; US. Environmental Pmtectian Agency:Washington. DC, 1994; EPA/540/R-94/509. 112) Lopez-Avila,V: Charan, C.: Van Eman, 1. M. Enuiron. Test. Anal. 1994,3(3), 34-39. 113) EnvironmentalMonitoringand Management Cauncil. Environmental Monitoring Methods Index Version 1.O. Userr Manual; US.EnvironmentalProtenion Agency:Washington, DC, 1992; EPA1821-8-92-001. 114) Goolsby, D. A,, Battaglin, W. A,; Thurman, E. M. "Occurrence and Transport of Agricultural Chemicals in Mississippi River Basin in July-August. 1993"; U S . Geological Survey, Washington, DC, 1993; Circular 1120-C.
Jeanette M.Van Emon is director of the Immunochemistry Program at the EPA Exposure Research Laboratory, Charanerization Research Division-Lus Vega. Chre L. Gerlach is the task leader for technical transfer at Lockheed Environmental Systems & Technologies Co., under contract with EPA
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