Pesticide immunoassays examples

Conjugation to carriers. A wide range of proven methods are available for conjugation of haptens to their carriers, most of them using common commercially available reagents. These have been summarized previously (11,14) and many examples of their use in pesticide immunoassay development exist (3.10.12.15.19.32.35.36.38). 

Example 6-2 The following standard addition plot was obtained for a competitive electrochemical enzyme immunoassay of the pesticide 2,4-D. A ground water sample (diluted 1 20 was subsequently assayed by the same protocol to yield a current signal of 65 nA. Calculate the concentration of 2,4-D in the original sample. 

Table 4 Examples of immunoassays developed for pesticides and their metabolites...

Immunoaffinity procedures can be performed either on-line or off-line, and can be coupled to chromatographic systems [ 118,119] or even to immunoassays [120]. Many examples can be found in the literature regarding the use of immunoaffinity extraction of drugs and pharmaceuticals from biological matrices, as well as of organic pollutants such as pesticides from environmental samples [115,121-124]. 

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). 

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) ... 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

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. 

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. 

This table illustrates one of the major impediments to the rapid assimilation of immunochemical technology into pesticide residue analysis labs. Because of the amount and variety of work involved, new method development costs may be high when compared to routine chromatographic methods. However, the low cost per run allows for rapid recovery of the initial investment with sufficiently high sample loads. For example, the cost of reagents and supplies for an ELISA for diflubenzuron was estimated to be 0.20/sample as compared with 4 for HPLC or 11 for GC (35). In addition to the lower reagent and supply costs, the major economic advantage of immunoassay is the dramatic decrease in labor costs. 

Once analytical capability and rapid turn around time are available locally or on the farm, many crop management decisions will be guided by this type of analysis. One example available commercially allows state inspectors to rapidly check that pesticide containers have been properly rinsed before they are discarded. Certain states require a deposit on all restricted pesticide containers. Deposits are refunded only if the container being returned passes a rapid assay which confirms that the container has been properly rinsed. The tests currently used for this on-site analysis are based on immunoassays as well as a colorimetric cholinesterase inhibition assay (26). 

Commercial Immunoassay Kits. Most commercially available immunoassay kits have been developed for determination of pesticides in water. At a minimum, appropriate residue extraction procedures must be developed before these kits can be applied to pesticide residue analysis of foods. Encouraging results have been obtained in preliminary FDA evaluations of several kits (12). For example, in studies of a kit for detection of triazine herbicides, a typical residue extraction solvent (acetonitrile) was used and then diluted with water to levels tolerated by the immunoassay. Visual comparison of color developed for extract, standard, and reagent blank was made for qualitative analysis. Spectrophotometric readings of the color were made for quantitative analysis. 

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