FEATURE
A STATUS REPORT ON
FIELD-PORTABLE IMMUNOASSAY Having revolutionized many field and laboratory analyses, environmental immunochemical technologies are gaining acceptance in the environmental community. JEANETTE M. V A N Ε Μ Ο Ν
T
he development of environmental immu nochemical technologies and their grow ing acceptance within the regulatory com munity have revolutionized many field and laboratory analyses. Beginning in the 1980s, environmental scientists adapted meth ods that had earlier resulted in a Nobel prize for ra dioimmunoassay (i). Pesticide analytical chemists, recognizing the shortcomings of gas chromatogra phy (GC) for some applications, championed the use of immunoassays. Since this pioneering work, im munoassays have been developed for scores of con taminants in a variety of matrices and for a multi tude of monitoring applications. There are many current challenges in the re search and application of immunoassay methods. A primary goal is to advance immunoassays to the next stage of environmental analytical acceptance by in volving chemists in the development of quantita tive methods. Analytical chemists are beginning to integrate immunochemical methods such as immu noassay as a detector for microbore high-perfor mance liquid chromatography; immunoaffmity chromatography is being integrated with standard detection procedures such as mass spectrometry (MS). These tandem techniques result in more ver satile methods for samples of environmental impor tance. Another challenge is in the development of uni form evaluation guidelines, particularly when the de tection level of the immunoassay is lower than the accepted comparison method. A related issue is at tainment of the level of confirmation necessary to support the required data quality objectives of a par ticular 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 concern) should be confirmed by a traditional method. This costly step will be avoided by institut-
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CLARE L. G E R L A C H
ing proper quality assurance/quality control proce dures, and by continuing to demonstrate the reli ability of immunoassays for environmental decision making. All immunoassays rely on the interaction be tween an antibody and a target analyte. Antibodies are produced in response to an immunogen by a complex mechanism (2). A common format for en vironmental field-portable immunoassays uses an analyte-selective antibody immobilized on a solid sur face such as a test tube. The analyte from the sam ple and a known amount of enzyme-tagged analyte compete for a limited number of antibody binding sites (Figure 1). Quantitation is achieved by com paring the signal generated by an unknown sample with a standard curve. Immunoassay test kits essen tially package antibodies, reagents, standards, and substrates in field-transportable units that are ready to use (3). A 96-well microplate-based immunoas say format is often used in the laboratory because of its high sample capacity. Selective antibodies have been developed for many compounds of environmental concern, ranging from small pesticide molecules to protein products from recombinant DNA. Figure 2 provides a general de velopment scheme for an immunoassay. Environmental immunoassays have been devel oped and evaluated for analytes including, but not limited to, major classes of pesticides, polychlorinated biphenyls (PCBs), polyaromatic hydrocar bons, pentachlorophenol (PCP), and BTEX (ben zene, toluene, ethylbenzene, and xylene) compounds (4). Immunoassay test kit manufacturers have re sponded to the environmental analytical communi ty's needs by providing and continually refining fieldportable immunoassay kits and standard operating procedures. The test kit manufacturers, environmen tal regulators, and major chemical industries are co operating to continue immunoassay development for 0013-936X/95/0929-312A$09.00/0 © 1995 American Chemical Society
environmental applications. A work group called the Analytical Environmental Immunochemical Consor tium (AEIC) fosters this cooperation. The AEIC holds frequent meetings to discuss research and regula tory issues and writes guidelines for immunochem ical method development. Immunoassays are used in the laboratory as well as in the field. Pre-analysis of environmental sam ples 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 by the en vironmental community. It will serve a larger com munity than the SITE program and will respond to the needs of a wider user community. Two immunochemistry projects are helping the Consortium launch its program. One is a pilot elec tronic bulletin board system (BBS) highlighting im munochemical research and application; the other is a field evaluation of a mercury immunoassay at Nevada's only Superfund site. These efforts are be ing conducted through the Consortium, and both projects are expected to be completed in 1995.
Vehicles for field evaluation EPA has participated in field evaluations of several immunoassays. The Superfund Innovative Technol ogy Evaluation (SITE) program was developed to eval uate remediation and monitoring technologies for use at hazardous waste sites around the nation. Immu noassay demonstrations are discussed in an EPA Technology Profile of the SITE program (5). In 1988, the first SITE demonstration of a mea surement technology evaluated two immunoassays for PCP: an immunoassay test kit and a 96-well mi croplate immunoassay. The plate immunoassay com pared well (Spearman rank correlation coefficient of 0.92, η = 47, 95% 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-MS method, and showed a slight positive bias, suggesting a minimal tendency to generate false-negative results. This SITE demon stration gives an example of near real-time diagnos tic capabilities—the ability to monitor remediation events—that immunoassays can provide in the field. An immunoassay immediately identified a prob lem with an on-site bioreactor, which was con firmed two weeks later by MS results. Another SITE project evaluated an immuno assay used to detect BTX compounds (7). The project demonstrated the adaptability and portability of im munoassays by allowing analytical measurements to be performed in a van a few yards from the contam inated 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 by both field-portable and laboratory immunoassays using various extraction methods (8). The SITE studies served to advance regulatory and technical accep tance of immunoassay testing. And the resulting syn ergistic yet competitive relationship between re searchers, 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 Technol ogy. Whereas the SITE program's major focus is on innovative treatment and remediation technolo gies, the Consortium will be dedicated exclusively to development, demonstration, and evaluation of mon itoring and characterization technologies. The Con sortium can serve as the next step in the evaluation
A technician samples for immunoassay analysis, which will yield near-real time data on a potentially contaminated site. Photo by EPA, Characterization Research Division.
Interest in field-portable environmental immu nochemical methods is great among the on-site an alytical community. This interest has been ex pressed at the annual Immunochemistry Summit meetings, sponsored by the Immunochemistry Pro gram at the EPA National Exposure Research Labo ratory, Characterization Research Division-Las Ve gas (CRD-LV). The simultaneous emergence of electronic BBSs and their widespread use has led to many requests for the establishment of a BBS for en vironmental immunochemistry. This will be a pre lude to an electronic BBS encompassing all field an alytical technologies. CRD-LV is developing a pilot BBS for environ mental immunochemistry methods, including key contacts, literature citations, and case studies. The VOL. 29, NO. 7, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY • 3 1 3 A
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 PCBs— made field screening methods necessary. Using two immunoassays and one ion-specific electrode 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 immunoassay. These differences in sample p r e p a r a t i o n s h o u l d be c o n s i d e r e d w h e n comparing methods.
Ongoing research
pilot BBS is expected to be released this summer on an existing EPA BBS, possibly CLU-IN or the Office of Research and Development (ORD) system. To provide data for the BBS and for information about the expected date of release, contact Jeanette Van 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 Center— then called the Environmental Monitoring Systems 3 1 4 A • VOL. 29, NO. 7, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
Vendors are developing and evaluating immunoassays for an ever-expanding list of analytes of environmental concern (see p. 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 (9,10). 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 w h e n d e v e l o p i n g i m m u n o a s s a y s . The crossreactivity of the antibody between the analyte of concern and other similar compounds can confuse the issue. Cross-reactivity in 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 curve, sample matrix effects can be considered minimal. This quick quality control procedure can determine if sample cleanup steps are necessary. Though streamlined sample cleanup steps are highly desirable 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
cross-reactivity of metabolites and decomposition products, structural homoiogs, and other compounds Semiquantitative results from two field-portable immunoassays likely to be encountered in the sam Two field portable immunoassays were used at the Allied Paper/Portage Creek/Kalamazoo ple matrix (2). Pre-analytical proce River Superfund Site on known concentrations of performance evaluation materials in soil, dures such as sample drying, extrac sediment, and paper pulp samples to prove their capabilities. Semiquantitative results are tion, and cleanup are important in shown below. preparing samples fo r i m m u n o Performance chemical analysis (β). evaluation Average Supercritical fluid extraction cou material Number of number of false number of false Number of pled with enzyme-linked immu concentration, samples negatives, positives, immunoassays mg/kg analyzed immunoassay immunoassay in correct range nosorbent assay (SFE-ELISA) has achieved higher extraction efficien 0.5 12 0 cies with lower organic solvent use. 1.5 12 0 This technology was used to success 8.0 12 3 fully determine PCB concentrations 25 10 1 in soil samples from a Superfund site 45 10 0 (8, 12). Current SFE-ELISA research 100 6 0 Totals 62 4 focuses on multiresidue methods to determine pesticide residues in food and other matrices. vironmental Monitoring Methods Index, available ORD began investigating the use of immunoas through the National Technical Information Ser says for environmental monitoring applications in vice (73). This recognition of field methods is the next 1987. CRD-LV, an ORD laboratory, collaborates with step in regulatory acceptance of innovative meth researchers in the Superfund Office, the EPA Office ods. 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 Pro the enhancement of the image of field methods. gram's goals are based on input from these and other EPA is not the only regulatory agency participat client EPA program offices. Superfund, OPR and OW ing in the development of environmental immuno were the first program offices to recognize the ad assays. Other federal and state agencies rely on im vantages of rapid, portable, easy-to-use immunoas munoassays 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. Re widespread, ranging from blood and urine testing for cently, OW tested specific analytes in grab samples from commercial laundry effluent. In these diffi cult matrices, they have found good correlation be tween a commercial immunoassay kit and GC anal Key terms and definitions ysis (data unpublished). The OPP work focuses on Antibody. A protein that selectively recognizes and analysis of pesticide residues in soil, crops, and other binds to a target analyte or group of related ana matrices to assess occupational and nonoccupa lytes. tional exposure. As more efficacious pesticides with Antigen. A hapten-protein conjugate or other targe extremely low biologically active levels enter the mar molecule that can bind to a selective antibody. ket, analytical methods with commensurately lower Conjugate. A compound, usually a protein, codetection levels must evolve. valently bound to a hapten or a label, such as an The EPA program offices typically rely on analyt enzyme. ical methods developed for their specific applica Hapten. A small molecule that can induce antibody tions. The individual offices are evaluating integra production when covalently bound to a carrier tion of i m m u n o a s s a y s into their m o n i t o r i n g molecule. A hapten can react with the specific programs. In 1992, the Office of Solid Waste (OSW) antibodies produced in response to the haptenbegan including immunoassays in its guidance doc carrier conjugate. ument, SW-846. This compendium of analytical and Immunoassay. A physical assay based on the re test methods approved by OSW is used in determin versible interaction of a specific antibody with a ing regulatory compliance under the Resource Con target analyte(s). servation and Recovery Act; it is not an exhaustive Immunogen. A hapten-carrier conjugate or other list of Agency-wide required methods. large molecule used for the production of selective The EPA Environmental Monitoring and Mea antibodies. surement Council was chartered to coordinate meth Monoclonal antibody. A homogeneous antibody ods research, foster consistency in monitoring meth population derived from one specific antibodyods, and explore the feasibility of a national producing cell. laboratory accreditation program. To address accep Polyclonal antiserum. A heterogeneous population tance issues on an interagency level, the Consor of antibodies varying in selectivity and affinity for a tium on Site Characterization Technology was formed target analyte that is derived from several anti to provide a sound scientific basis for verifying tech body-producing cells. nologies. Publication of a list of field-portable tech nologies is anticipated later this year in the EPA EnVOL. 29, NO. 7, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY • 3 1 5 A
Immunoassay suppliers BioDesign International 105 York Street Kennebunk, ME 04043 (207)985-1944
Immunosystems, Inc. 4 Washington Avenue Scarborough, ME 04074 (207) 883-9900
BioNebraska, Inc. 3820 NW 46th Street Lincoln, NE 68524
Millipore Corporation 50 Ashbury Road Bedford, MA 01730 (800) 645-5476, x8079
(402)470-2100x101 (800) 786-2580 Bio-Tek Instruments, Inc. Highland Industrial Park P.O. Box 998 Winooski, VT 05404-0998 (802) 655-4040
480 Democrat Road, P.O. Box 70 Gibbstown, NJ 08027-0070 (800) 222-0342 Ensys Environmental Products, Inc. P.O. Box 14063 Research Triangle Park, NC 27709 (919)941-5509 Idetek, Inc. 1245 Reanwood Avenue Sunnyvale, CA 94089 (408) 745-0544 Idexx Laboratories, Inc. Corporation-Environmental 1 Idexx Drive
Neogen Corporation 620 Lesher Place Lansing, Ml 48912 (517)372-9200 New Horizons Diagnostics Corp. 9110 Red Branch Road (800)888-5015 Ohmicron Diagnostics, Inc. 375 Pheasant Run Newtown, PA 18940 (215)860-5115 Quantix 700 Route 130 North Suite 203 Cinnaminson, NJ 08077 (609) 786-3080 Strategic Diagnostics 128 Sandy Drive Newark, DE 19713 (302) 456-6789
Westbroc'ik, Mb 04092 (207) 856-0300 This list of vendors is not intend ed to be complete, and the mention of vendeirs here does not imply IEPA endorsement of any or all of their pro ducts.
Analytes for which immunoassays have been developed Pesticides Alachlor Aldicarb Atrazine Benomyl Capta η Carbendazim Carbofuran Chlorpyrifos methyl DDT Imazaquin Ivermectin Lindane Metalaxyl Metolachlor Paraquat Procymidone Triazines Trifluralin 2,4-D
Inorganics Cadmium Indium Lead Mercury Organics Benzenertoluene/ethylbenzene/ xylene compounds Diesel Fuel Gasoline Polyaromatic hydrocarbons Pentachlorophenol Polychlorinated biphenyls Trinitrotoluene Other c o m p o u n d s Antibiotics Hormones Mycotoxins Vitamins
Immunoassays for analytes listed may I ι commercially available or still in development.
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various pharmaceuticals and biomarkers of expo sure to the analysis of raw and processed agricul tural commodities for pesticide residues. The U.S. Geological Survey (USGS) and the U.S. Army Corps of Engineers have responsibility for mon itoring 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 Missis sippi during the Great Midwestern Flood of 1993 (14). Immunoassays are being used by state and fed eral agencies to ensure that the food supply is not contaminated with potentially hazardous chemi cals. The U.S. Food and Drug Administration uses im munoassays to check for mycotoxins, antibiotics, and pesticides in food commodities. The U.S. Depart ment of Agriculture, through their Food Safety and Inspection Services, collects and analyzes samples of meat and poultry.
Comparability and acceptability The comparability of results within and between kits and manufacturers is critical for successful incor poration of immunoassays into a larger study. Fre quently, immunoassays are more sensitive than GC methods and, although this sensitivity is a benefit, it could lead to difficulties when comparing data. Methods are difficult to compare directly because im munoassays can have greater sensitivity but can be limited by variations attributable to matrix effects. Simple or abbreviated extraction procedures re quire caution. In one study, splits of soil samples con taining PCBs were extracted by two different proce dures, simple m e t h a n o l shake extraction and conventional Soxhlet extraction. Detection of both extracts was accomplished by immunoassay. Soxhlet extraction proved more efficient for more contam inated samples. A 96-well microplate immunoas say provided quantitative PCB results (8). Kits should include information on extraction efficiencies over several concentration ranges for a variety of matri ces as part of their quality control measures. Scientists who have good experiences with im munoassays 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 oversell a promising tech nology is great. Responsible environmental scien tists are encouraging cautious evaluation of innova tive methods, warning against acceptance based solely on enthusiasm and potential. Acceptance can be accelerated without compromising technical va lidity, such as through performance-based meth ods to fulfill specific data quality objectives. In many monitoring situations, performance-based meth ods can be used without compromising technical va lidity or legal defensibility. Immunoassay test kits often are regarded as sim pler and easier to learn than more complex analyt ical procedures such as GC and MS. However, the ease-of-use issue is not without drawbacks. As im munoassays become more readily available, results might be interpreted erroneously. The regulatory community, chemical registrants, the vendor com munity, and organizations such as the AEIC want to
increase the judicious use of i m m u n o a s s a y s by train ing test kit users, p u b l i s h i n g guidelines for i m m u n o a s s a y use, a n d fostering inclusion of u n i f o r m in formation in i m m u n o a s s a y test kits. T h e increasing public a w a r e n e s s of t h e availability a n d u s e of im m u n o a s s a y s is a n a r e a of p r o m i s e a n d c o n c e r n : p r o m i s e that t h e technology will b e applied to m a n y m o r e s c r e e n i n g s i t u a t i o n s in t h e future a n d c o n cern that d a t a from t h e s e u s e s m i g h t b e misinter p r e t e d or m i s u s e d . The EPA CRD-LV I m m u n o c h e m i s t r y Program has sponsored annual Immunochemistry Summit meet ings since 1992. T h e s e m e e t i n g s are held in Las Ve gas a n d i n c l u d e f o r m a l p a p e r s , p o s t e r p r e s e n t a t i o n s , p a n e l d i s c u s s i o n s , a n d a m u l t i t u d e of o p p o r t u n i t i e s for informal n e t w o r k i n g . T h e S u m m i t M e e t i n g f o r m a t w a s d e s i g n e d to i n c l u d e re searchers a n d regulators from various g o v e r n m e n t agencies, c h e m i c a l m a n u f a c t u r i n g c o m p a n i e s , u n i versities, a n d test kit a n d i n s t r u m e n t m a n u f a c t u r ing c o m p a n i e s . S u m m i t I provided a u n i q u e envi r o n m e n t for t e c h n i c a l n e t w o r k i n g b e t w e e n t h e s e groups. O n e of t h e results w a s t h e f o r m a t i o n of t h e AEIC. This organization is n o w chartered, has h a d sev eral m e e t i n g s , a n d c o n t i n u e s to establish i n d u s t r y standards. Key discussion points at t h e S u m m i t meetings in clude research trends, regulatory a c c e p t a n c e , c o m m u n i c a t i o n issues, a n d d a t a quality criteria. I m m u n o c h e m i s t r y S u m m i t IV will take place August 2 - 3 . A m o n o g r a p h of t h e p a p e r s p r e s e n t e d at I m m u n o c h e m i s t r y S u m m i t III a n d t h e u p c o m i n g S u m m i t IV will b e p u b l i s h e d t o g e t h e r in early 1996.
Note EPA, through its Office of Research and Development (ORD), partially funded and collaborated in the research de scribed here. It has been subjected to the Agency's peer re view and has been approved as an EPA publication. Men tion of trade names or commercial products does not constitute endorsement or recommend them for use.
References (1) Berson, S. A. et al. /. Clin. Invest. 1956, 35, 170-90. (2) Van Emon, J. M.; Seiber, J. N.; Hammock, B. D. In Ad vanced Analytical Techniques; Sherma, J., Ed.; Academic Press: San Diego, CA, 1989; Vol. XVII. (3) Van Emon, I. M.; Gerlach, C. L. Environmental Lab. 1994, 6(2), 24-54. (4) Van Emon, I. M.; Lopez-Avila, V Anal. Chem. 1992, 64(2), 79A-87A. (5) "Superfund Innovative Technology Evaluation Program, Technology Profiles, Sixth Edition"; U.S. Environmental Pro tection Agency: Washington, DC, 1993; EPA/540/R-93/ 526. (6) Van Emon, J. M.; Gerlach, R. W. Bull. Environ. Contam. Tox icol. 1992, 48, 635-42. (7) White, R. J.; Gerlach, R. W.; Van Emon, J. M. "An Immu noassay for Detecting Gasoline Components"; U.S. Envi ronmental Protection Agency: Washington, DC, 1992; EPA/ 600/X-92/116. (8) Johnson, J. C; Van Emon, I. M. "Development and Eval uation of a Quantitative Enzyme-Linked Immunosor bent Assay (ELLSA) for Polychlorinated Biphenyls"; U.S. Environmental Protection Agency: Washington, DC, 1994;
The increasing popularity of field immunoassay analysis is, in large part, due to the highly portable equipment and minimal setup requirements. Photo by EPA, Characterization Research Division.
EPA/600/R-94/112. (9) Blake, D. A. et al. Anal. Biochem. 1994, 217, 70-75. (10) Wylie, D. E. et al. Anal. Biochem. 1991, 194, 381-87. (11) Gee, S. J.; Hammock, B. D.; Van Emon, J. M. "A User's Guide to Environmental Immunochemical Analysis"; U.S. Environmental Protection Agency: Washington, DC, 1994; EPA/540/R-94/509. (12) Lopez-Avila, V; Charan, C; Van Emon, ]. M. Environ. Test. Anal. 1994, 3(3), 34-39. (13) Environmental Monitoring and Management Council. En vironmental Monitoring Methods Index, Version 1.0, Us ers Manual; U.S. Environmental Protection Agency: Wash ington, DC, 1992; EPA/821-B-92-001. (14) Goolsby, D. Α., Battaglin, W. Α.; Thurman, Ε. Μ. "Occur rence and Transport of Agricultural Chemicals in Mis sissippi River Basin in July-August, 1993"; U.S. Geolog ical Survey, Washington, DC, 1993; Circular 1120-C. Jeanette M. Van Emon is director of the Immunochem istry Program at the EPA Exposure Research Labora tory, Characterization Research Division-Las Vegas. Clare L. Gerlach is the task leader for technical transfer at Lockheed Environmental Systems & Technologies Co., under contract with EPA.
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