Immunoassay Advances As Tool For Environmental Testing

Immunoassay technology, a quick and cost-effective method of detecting and measuring minute quantities of substances in the human body, has now transitioned from the clinical and biological research arenas to environmental investigations. The technique, which exploits the capacity of a mammalian antibody to latch onto a particular chemical with great specificity, is used in at-home pregnancy tests, workplace drug-screening programs, and AIDS testing. Within the last year, however, several immunoassay-based tests have been introduced that detect pesticides and other contaminants in the environment. Most immunoassay tests that have been adapted for environmental research are enzyme-linked immunosorbent assays (ELISAs), which use an enzyme as a label for measuring the amount of pesticide in a sample. While these tests display an accuracy comparable to that of traditional lab-based analytical methods, such as gas chromatography (GC), their low cost, speed, and portability (most are marketed as test kits that can be used in the field) have made them attractive to environmental chemists in a variety of fields. The first commercially available immunoassay kit for detecting pesticides was developed by biochemist Bruce Ferguson, founder and chief executive officer of ImmunoSys-tems Inc., based in Scarborough, Maine. "It all started around 1986, when several people asked whether I thought immunoassays could be used to detect pesticides," recalls Ferguson. Therapeutic drugs or drugs of abuse--common targets for clinical immunoassays--are structurally similar to pesticides, he says, and so he thought such a goal should be attainable. In retrospect, Ferguson adds, while developing the products was relatively easy, the difficult part was convincing environmental chemists that this alien biotech methodology was a worthwhile technique. Of the 14 tests he has gone on to develop, "it was only the first 13 that were tough to sell." Ferguson's first immunoassay kit, initially offered in 1988, detected atrazine, a common herbicide. Other tests since developed by his company include those for the pesticides carbofuran, aldicarb, 2,4-D, and alachlor. Millipore Corp. of Bedford, Mass., has exclusive marketing rights to ImmunoSystems' products, marketing them under the EnviroGard trademark. Like other enzyme-based immunoassays, these environmental assays function in the following way: The kit includes a test tube or microtiter plate coated with antibody specific to the compound of interest, such as atrazine. Sample is added to the test tube, along with a known amount of labeled atrazine-enzyme conjugate supplied with the kit. This labeled antigen competes with the unlabeled sample antigen for a limited number of antibody binding sites. The immobilized antibodies bind to the unlabeled (sample) atrazine and the labeled atrazine in direct proportion to their relative concentrations in the tube. When the unbound antigen is washed away, reagents are added that give rise to a color change. Since the color change results from the labeled enzyme conjugate, darker color means that more of the labeled atrazine has stayed behind, indicating that less sample atrazine was present. Hence, the lighter the color, the higher the concentration of atrazine in the sample. The resulting color is then compared to standards containing known concentrations of the pesticide. Results are sometimes expressed qualitatively (yes, the pesticide is present, or no, it is not) or semiquantitatively (the standards can be used to delineate upper, middle, and lower ranges of the assay). For quantitative test results, a spectrophotometer can be used to measure the color change. Within the last two years, the EnviroGard test kits have been joined by products from a handful of other companies. Ohmicron of Newtown, Pa., offers pesticide detection kits--for lab and field use--that use magnetic particles in solution as the solid phase that immobilizes the antibody. This innovation improves the precision of the tests, according to Bob Sandberg, the company's vice president of sales and marketing. In other kits, the test tubes and microtiter plates have to be carefully coated individually, to ensure that the coating is the same from tube to tube--a difficult and time-consuming process. But magnetic particles can be dispersed in solution, says Sandberg, "so the amounts are easy to control with precision." Ohmicron's RaPID Assays System includes tests for eight commonly used pesticides, and the company intends to add two more to the line by the end of June, he says. EnSys Inc., headquartered in Research Triangle Park, N.C., specializes in kits for the onsite detection of organic environmental contaminants, including PCBs (polychlorinated biphenyls), PCP (pentachloro- phenol), and TPHs (total petroleum hydrocarbons). Another company, Agri-Diagnostics Associates of Cinnaminson, N.J., markets tests for pesticides and crop diseases. ImmunoSystems has recently added a PCB test to its line, as well. Mike Thurman, a research hydrologist in the water resources division of the United States Geological Survey in Lawrence, Kans., has used pesticide immunoassays extensively in his studies of surface and groundwater pesticide contamination. He says that when he first tried Ferguson's atrazine kit back in 1987, before it hit the market, he was struck by the utility and low cost of the technique. Thurman, whose background is in gas chromatography/mass spectrometry (GC/MS) analysis, says that the kit's test results were comparable to those obtained with GC/MS; but at less than $20 per sample, it cost only about one-tenth as much as the latter technique. Thurman says that the pesticide immunoassays are best suited to the rapid screening of a large number of samples. The samples that test positive, or that test above a certain threshold, can then be verified by standard lab techniques, such as GC/MS. This screening can vastly cut costs, since only a portion of the total samples goes through the full analysis. "This is a direct analogy to clinical use," he points out. For example, in workplace drug screening programs, specimens that come up positive for cocaine are verified by further analysis. Pesticide contamination in drinking water has become a sensitive issue in many parts of the U.S., notes Thurman, particularly in the agriculturally dominated Midwest. In many states, he says, there are even efforts to ban certain pesticides. Partially in response to these grumblings, chemical companies have been cooperating with large-scale, government-funded testing programs to assess the effects of their products on water supplies--testing programs that are beginning to rely heavily on immunoassays. For example, says Thurman, with help from Ciba Geigy Corp. of Greensboro, N.C., makers of atrazine, the state of Wisconsin recently completed a large groundwater survey in which they took some 2,000 samples. As in Thurman's own work, positive immunoassay results were verified by conventional laboratory techniques. Another bustling area in environmental testing is the remediation of toxic waste sites, where organic chemicals such as PCBs often reside. According to Mike Taylor, vice president of Roy F. Weston Inc., an environmental consulting firm based in West Chester, Pa., many such sites--including those targeted by EPA's Superfund program--are just beginning to be cleaned up. Although Taylor has evaluated PCB immunoassay tests and found them useful for onsite screening of soil samples for PCB content, he doesn't advocate their use in site remediation because "I don't believe in screening." He explains that "for a little more money, you can get the full test done by approved methods." He says that the immunoassay tests for PCBs have not been promulgated as an approved methodology by the Environmental Protection Agency, and although they may be a useful technique, without EPA's blessing, the results may not be accepted by other agencies or state or local authorities. Taylor says that if EPA does approve immunoassays, "I'd be inclined to switch over, because of the [reduced] cost and less complexity." He adds, however, that "in my experience, the myth that makes them look good is that you don't need someone fully trained. That's not the case." He points out that the person doing the testing needs to understand how the kits work, how to interpret the results, and how to handle the solvents used, particularly in the tests for organic contaminants. Otherwise, he says, "they may be missing the boat completely." A spokesperson for EnSys shares Taylor's concerns. "This is not an off-the-shelf product," says Ashling Scallen, the company's senior product manager. "People do need minimal training." ImmunoSystem's Ferguson argues that the technique's current lack of recognition by EPA does not preclude its use. "There is still a valid place for these kits for people willing to screen and monitor environmental samples," he says. "That's because if these kits give positive results, you should confirm the results by an appropriate technique." Furthermore, he points out, a number of agencies--including EPA, the Food and Drug Administration, and the U.S. Department of Agriculture--"are all setting up programs to validate and/or recognize these pesticide immunoassays." EPA's Jeanette Van Emon, technical program manager for the immunochemistry program at the agency's Environmental Monitoring Systems Laboratory in Las Vegas (EMSL-LV), says that, in fact, EPA is very interested in immunoassay technology's promise in environmental analysis. EMSL-LV is currently conducting a number of investigations that assess the performance of immunoassay kits in several applications, such as monitoring the progress of cleanup activities on Superfund sites and assessing the persistence of pesticides on foliage in agricultural fields to determine when it's safe for workers to reenter the field after a pesticide spraying. In January, EPA director William Reilly presented Van Emon with a Science Achievement Award, given jointly by the agency and the American Chemical Society, in recognition of her ongoing work with environmental immunochemistry. Indications are that the environmental immunoassay market will continue to expand rapidly. Van Emon says that companies that currently specialize in clinical immunoassay diagnostics are eyeing the environmental sector with great interest, and many are poised to enter the market. Ferguson expects that the market will experience the same growth as the clinical diagnostic market that preceded it: "Why shouldn't immunoassay technology find success in the environmental arena, as it has in the medical?" Along with the pesticide, PCP, and PCB tests now available, Ferguson anticipates the development of a wide range of tests for assorted industrial chemicals, such as solvents and dyes. In fact, while immunochemists may cringe at the idea, "my general feeling is, you can make an antibody to anything," he says. "But some things are harder than others."

Immunoassay Advances As Tool For Environmental Testing

Interested in reading more?

Become a Member of