New technologies expand scope of immunoassays to pesticide residues Pacifichem '95 Honolulu A. Maureen Rouhi, C&EN Washington ew ways to prepare antibodies, as well as novel sensing and cleanup systems, are increasing the usefulness of immunoassays in agrochemical analysis, according to scientists at an agrochemistry symposium held last month at Pacifichem '95. Based on the antibody-antigen reaction, immunoassays were developed initially for medical diagnostics. But they are now rapidly gaining acceptance for analysis of trace contaminants, including pesticides, according to Shirley J. Gee, a staff research associate with the department of entomology at the University of
N
doma Facility at the University of California, Berkeley. Hybridoma technology produces antibodies of defined specificity in virtually unlimited amounts, Karu said, but it is expensive, time-consuming, and labor intensive. It also requires use of live animals.
California, Davis, and one of four coorganizers of the sessions on immunoassays. Assays are now available for diverse applications, from monitoring drinking water quality to detecting pesticide metabolites in urine and other biomarkers of exposure to hazardous compounds, Gee told C&EN. A major hurdle in developing a new assay is generating the best antibody for the analyte. Commercial assay kit manufacturers that previously used antisera increasingly are using Hall (right) and coworkers—microbiologist Hung Lee (left) monoclonal antibodies and graduate student Steven R. Webb—examine a DNA (hybridomas), said Alex- sequencing gel of a recombinant antibody fragment. ander E. Karu, director of the College of Natural Resources Hybri Researchers are overcoming these limitations through new technologies. For example, using recombinant DNA procedures, Karu and coworkers are preparing antibody fragments. These pieces retain the binding properties of the intact antibody and can be expressed in useful amounts by easy-to-grow organisms such as Escherichia coll
Model based on the DNA sequence of a recombinant antibody shows how the antibody specifically binds the (S)-isomer of the imidazolinone herbicide imazethapyr. A double pocket in the antibody's light chain (fuchsia) accommodates the isopropyl group, and a smaller single cavity in the heavy chain (blue) takes in the methyl group on the imidazole ring. The model was prepared by Tina E. Chin at the University of California, Berkeley, and Victoria A. Roberts of Scripps Research Institute, La folia, Calif. Courtesy of Alexander E. Karu, with permission from the Agricultural Products Research Division, American Cyanamid Co.
But methods for preparing recombinant antibodies are not yet routine, Karu explained. Although his group has prepared recombinant antibodies for polynuclear aromatic hydrocarbons and phenylurea and imidazolinone herbicides, he stressed that one is likely to encounter problems in every step of the process, from gene amplification to cloning, selection, and expression. Karu also gave an overview of what could be the next advance—deriving antibodies from semisynthetic combinatorial libraries of antibody fragments. These libraries are many orders of magnitude more diverse than the mammalian antibody repertoire, some containing up to 1010 to 1012 antibodies, Karu told C&EN. "Selecting antibodies from such libraries could make it possible to derive new antibodies without using live animals, as well as eliminate the need for gene cloning," he noted. However, a problem with such libraries is that the JANUARY 22,1996 C&EN
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SCIENCE/TECHNOLOGY vast majority of members have low or no affinity for the antigen. J. Christopher Hall, a professor at the department of environmental biology at the University of Guelph, Ontario, also reported success with recombinant antibodies. Using a technique called phage display to screen recombinant antibodies cloned from a hybridoma, Hall and his coworkers have prepared a recombinant antibody fragment with specificity for and affinity to cyclohexanedione herbicides similar to those of the parent antibody. "Our work proves recombinant techniques can be a less expensive route to specific immunoassays/' said Hall. Advances in sensing technologies also were discussed in Honolulu. For example, the research of Rosie B. Wong, principal research biochemist at the agricultural products research division of American Cyanamid, Princeton, N.J., focuses on rapid systems with greater precision and higher sensitivity, as well as automated systems that are less prone to operator errors. Using an antibody against an imidazolinone herbicide, prepared in collaboration with Karu, Wong has compared commercial sensors with respect to detection limits, precision, consistency, responses to various matrices, and compatibility with automation. Most flow systems can be applied to environmental matrices, although an optical system based on surface plasmon resonance gives the best sensitivity at 0.1 pg per L, she said. Surface plasmon resonance refers to a shift in the angle of reflection of incident light due to differences on the surface caused by molecular binding, she explained. Still, scientists keep pushing levels of detection to even lower concentrations. For example, John S. Vogel, a senior scden-
The accelerator mass spectrometer at Lawrence Livermore National Laboratory occupies an entire building. tist at Lawrence Live r m o r e National Laboratory, and his colleagues have developed extremely sensitive assays by labeling antigens and antibodies with long-lived isotopes like carbon-14 (halflife of 5,730 years), which they rapidly detect by accelerator mass spectrometry. Vogel's group has an assay that can detect the herbicide atrazine in the attomole (10~18 mole) range. At this level, it is possible to detect mutagen-DNA adducts in only 1,000 mammalian cells or carcinogenic metabolites from a few grams of cooked meat, Vogel explained. But with current accelerator mass spectrometers occupying whole buildings, the technology is far from becoming widely available. "Our goal is to develop procedures that can be used with the smaller and less expensive spectrometers that are now being designed," Vogel said. In Germany, Petra M. Kramer, a research scientist at the Institute for Biotechnology Research in Braunschweig, has been studying on-line and continuous monitoring of pesticide residues in groundwater. Her work was spurred by a 1980 European directive setting a limit of 0.1 pg per L for pesticides in drinking water. "The methods that are good enough
to detect at these very low levels are primarily chromatographic," Kramer said. "Not only are they costly and time-consuming but they also require large volumes of solvents, creating a waste disposal problem," she explained. Kramer has been studying flow injection immunoanalysis (FIIA) as an environmentally friendly technique that is sensitive and specific enough to comply with the European regulation. In the system she and her associates are developing, analytes compete for binding sites on immobilized antibodies in a reactor. Pumps deliver samples and reagents to the reactor at specified intervals to allow completion of all steps, including the problematic one of regenerating the column. The method is still very much a work in progress. Working with a German company—Meta GmbH, based in Altenberge—to build a prototype of the equipment, Kramer's group has improved the procedure so it is now pos-
Immunoassay symposium participants included (from left) Gee, Stanker, Kramer, and Altstein. 26 JANUARY 22, 1996 C&EN
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sible to regenerate the column several hundreds of times with only a minor decline in the signal. Sample cleanup is another area of interest. Agrochemical residues are found in complex matrices, such as food, soil, sediments, and animal tissues. Often, isolating the analyte from the muck can be as demanding as detecting it. One of the most convenient cleanup techniques is immunoaffinity chromatography, according to Miriam Altstein, a biochemist from the Institute of Plant Protection at the Volcani Center in Bet Dagan, Israel. The method uses antibodies immobilized on a support to bind the analyte while contaminants are washed out. Then the analyte is displaced from binding for subsequent analysis. Altstein and David Avnir, a chemist from Hebrew University of Jerusalem, and their coworkers have been using ceramic technology for immunoaffinity chromatography. They have incorporated antibodies to nitroaromatic compounds and the herbicide atrazine into ceramic matrices by using the sol-gel process. Although trapped, the antibodies retain their activity, binding free antigens and concentrating them from aqueous solutions. The ceramic matrix has many desirable properties—such as stability, inertness, and porosity—that make it an excellent support, Altstein noted. Research biologist Larry H. Stanker and his coworker Mark T. Muldoon at the Food Animal Protection Research Laboratory of the Agricultural Research Service, College Station, Texas, are taking a different approach, called molecular imprinting. Impressions—or "prints"—of a chemical are created in a polymer when its molecules are in the mixture while the polymer is forming. When the print molecules are removed later, the polymer is left with pockets that recognize the chemical. Imprinting can be used to clean samples before analysis by all sorts of methods, including immunoassays, said Stanker, who was also a coorganizer of the immunoassay sessions. "It allows us to remove extraneous materials in a short time," he said. With the new technologies, "antibodies and immunoassays will continue to provide new avenues in agrochemical analysis" said coorganizer Gee. 'Ί hope the symposium was successful in stimu lating even more ideas." •
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