Immunoassay pesticide applicators

The ability to perform quantitative assays on complex mixtures with little sample clean-up is perhaps the most attractive feature of immunoassays for application to agricultural chemistry. A large portion of the cost and labor involved in pesticide residue analysis is invested in sample extraction and clean-up steps to remove substances which may interfere with subsequent chemical analysis. Since most preparatory steps are not required prior to performing an immunoassay, samples can be analyzed much less expensively. This will permit the vast number of data points required for pesticide registration to be gathered in a more timely and cost-effective manner. Studies which were prohibitively expensive because they would have required large numbers of expensive assays can be completed using immunoassay procedures. Such studies may include analysis of pesticide movement from application areas and the rate of dissipation of pesticide from crop tissue, soils, and processed foods. 

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

The development of sensitive and inexpensive immunoassays for low molecular weight pesticides has been an important trend in environmental and analytical sciences during the past two decades. 0.27-29 jq design an immunoassay for a pesticide, one can rely on the immunoassay literature for agrochemicals, " but many of the innovations in clinical immunoanalysis are also directly applicable to environmental analysis. - Conversely, the exquisite sensitivity required and difficult matrices present for many environmental immunoassay applications have forced the development of technologies that are also useful in clinical immunoassay applications. In the following discussion we will describe widely accepted procedures for the development of pesticide immunoassays. 

In the following discussion, the detection of pesticides and veterinary dmgs in food animals by immunoassay will be described. Discussion will be organized by compound class, the specific analyte, and, finally, the tissues examined. The general principles described in the first part of this review provide the rationale in the applications described in the following pages. 

The use of immunoassays for the determination of pesticides and veterinary medicines in food animals has increased since the early 1990s. The advantages of simple analysis, quick results, and high throughput make immunoassays a powerful technique for problematic matrices commonly encountered in animal agriculture. Careful development and validation are required to obtain accurate results, however. This review has demonstrated that most immunochemical techniques have been designed for use with milk samples, but a number of applications have also been developed for liver and muscle samples. The development of immunoassay techniques for residue analysis in eggs has clearly not been pursued to the extent of other edible tissues. 

V. Lopez-Avila, C. Charan, and J. van Emon, Supercritical fluid extraction-enzyme-linked immunosorbent assay applications for determination of pesticides in soil and food, in Immunoassays for Residue Analysis Food Safety (R.C. Beier and L.H. Stanker eds), ACS Symposium Series 621, American Chemical Society, Washington (1996). 

Environmental applications of HRP include immunoassays for pesticide detection and the development of methods for waste water treatment and detoxification. Examples of the latter include removal of aromatic amines and phenols from waste water (280-282), and phenols from coal-conversion waters (283). A method for the removal of chlorinated phenols from waste water using immobilised HRP has been reported (284). Additives such as polyethylene glycol can increase the efficiency of peroxidase-catalyzed polymerization and precipitation of substituted phenols and amines in waste or drinking water (285). The enzyme can also be used in biobleaching reactions, for example, in the decolorization of bleach plant effluent (286). 

Hennion M.-C. (1998). Applications and validation of immunoassays for pesticides analysis. 

The application of immunoassays to the determination of various urea pesticides have been reported (181,182), and this technique has a great potential for residue analysis by using rapid, simple, and cost-effective tests (183,184). 

Dombrowski, T.R., E.M. Thurman, and G.B. Mohrman (1997). Evaluation of immunoassay for the determination of pesticides at a large-scale groundwater contamination site. In D.S. Aga and E.M. Thurman, eds., Immunochemical Technology for Environmental Applications. ACS Symposium Series 657. Washington, DC American Chemical Society, pp. 221-233. 

Several qualitative and quantitative immunochemical methods and their application to the analysis of environmental samples have been described for OP insecticides, a family that includes widely used pesticides such as azinphos-ethyl/methyl, dichlorvos, fenitrothion or fenthion, malathion, mevinphos, and parathion. Mercader and Montoya202 produced monoclonal antibodies against azinphos-methyl and developed an ELISA that was used for the analysis of water samples from different sources, reaching detectability levels near 0.05 pg I. Watanabe et al.203 reported the production of polyclonal antibodies and ELISA procedures to analyze fenitrothion in river, tap, and mineral water (LOD = 0.3 pg L ). Banks et al.204 produced polyclonal antibodies against dichlorvos, an organophosphate insecticide used for stored grain, which also cross-reacts with fenitrothion. Nishi et al.205 reported the first immunoassay for malathion. Residues of this insecticide have... 

The expense of an analytical procedure depends upon much more than the cost of the final analysis. Much of the expense of an assay is related to sample preparation, and for many applications immunoassays have tremendously reduced the time needed for sample preparation. Another consideration is the amount of time needed for the development of an assay. The additional expertise which must be developed in an analytical laboratory before immunoassays can be used with confidence may seem formidable, and waiting for an animal to develop antibodies may lead to unacceptable delays in assay development. On the other hand, once a usable antibody titer is obtained, the development of a workable assay is usually straightforward. It is also likely, if immunoassays become accepted for some aspects of pesticide analysis, immunoassay kits or at least critical reagents will become commercially available. Such kits already exist for many pharmaceutical products and hormones, and numerous companies will supply antibodies to a user supplied hapten on a contract basis (83). 

Applicability. Parker (4) points out that one can assume that workable radioimmunoassays can be developed "with all except the smallest or most unstable molecules." Once a useful antibody titer is obtained, often only very small changes in a generalized procedure are needed to obtain a workable assay. Although immunoassays would appear to be generally applicable to pesticide analytical problems they may be most useful in solving specific problems which appear intractable when classical procedures are used. Immunoassays are often most sensitive and specific when... 

The application area of LC-MS is rapidly growing. LC-MS is now regularly used for the analysis of many different types of compound drugs and metabolites, herbicides-pesticides and metabolites, surfactants, dyes, saccharides, lipids-phospholipids, steroids, and many others. In our opinion, the area that profits more from the development of LC-MS is bioanalysis natural products, proteins, peptides, nucleosides, and metabolic studies. Despite the current trends toward immunoassays-biospecific assays and capillary electrophoresis, LC-MS is an extremely powerful analytical technique that is considered complementary to the above mentioned, rather than competitive. 

Actual and perceived food safety concerns have necessitated an increase in the monitoring of foods for such natural contaminants as aflatoxins and for residues of pesticides. Immunoassays can provide rapid, simple, and relatively inexpensive methods for the detection of analytes with specificity and with sensitivities directed at the levels of concern. Particularly for aflatoxins, they are rapidly assuming a significant role in the monitoring of foods. However, the misuse of these techniques can potentially compromise any food safety improvement that may result from increased surveillance. Experiences of the Division of Contaminants Chemistry in the development, validation, and applications of immunoassays for natural toxins is discussed. 

We have also applied ELISA to several biological pesticides including the endotoxin of Bacillus thurineiensis kurstaki (Btk). In this application to a macromolecular analyte, we have used a double antibody sandwich ELISA for Btk to measure the amount of ELISA reactive material in formulations of the pesticide. Figure 7 shows the use of an ELISA standard curve of gel purified Btk endotoxin to measure the immunoreactive material in dilutions of two Btk formulations. It has been demonstrated that ELISA can serve as a quick quality control check for formulations of Bacillus thurineiensis lsraelensis (44). Such examples indicate that immunoassays will be increasingly important as biologicals and products of recombinant DNA research impact our field (M) ... 

Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or field situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful in residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and field testing of the final assay. 

Emphasis will be placed on the justification of and the resources required for the successful incorporation of immunochemical technology into an existing analytical laboratory. Special attention will be given to aspects of immunochemical and related technology not covered in other recent reviews. Present use of immunoassay for pesticide analysis will be described and future potential applications and problems will be discussed. 

Applications of immunoassay to pesticide chemistry have been described which address some difficult problems in analysis by classical methods. These include stereospecific analysis of optically active compounds such as pyrethroids (38), analysis of protein toxins from Bacillus thuringiensis (5,37), and compounds difficult to analyze by existing methods, such as diflubenzuron (35) and maleic hydrazide (15 also Harrison, R.O. Brimfield, A.A. Hunter, K.W.,Jr. Nelson, J.O. J. Agric. Food Chem. submitted). An example of the excellent specificity possible is seen in assays for parathion (10) and its active form paraoxon (3). Some immunoassays can be used directly for analysis without extensive sample extraction or cleanup, dramatically reducing the work needed in typical residue analysis. An example of this is given in Figures 2 and 3, comparing the direct ELISA analysis of molinate in rice paddy water to the extraction required before GC analysis. 

Assay application. At this point major differences appear between the historical use of clinical immunoassays and the potential applications of environmental and pesticide immunoassays. Most clinical assays have been applied to simple or well defined and consistent matrices such as urine or serum. In contrast, most matrices likely to be analyzed for pesticides are more complex, less well defined, and more variable. The potential for serious problems with matrix effects in the environmental field is far greater than most clinical immunoassays have encountered. The application of immunoassays to environmental analysis requires sampling strategies, cleanup procedures, and data handling fundamentally similar to those presently in use in any good analytical lab. The critical factor in the success of immunochemical technology will likely be competence... 

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