Enzyme-linked immunosorbent assay pesticides

H.A. Moye, Enzyme-linked immunosorbent assay (ELISA), in Pesticide Residues in Foods Methods, Techniques, and Regulations, W.G. Fong, H.A. Moye, J.N. Seiber, and J.P. Toth (eds), Wiley, New York, Chapt. 6 (1999). 

The need to understand the fate of pesticides in the environment has necessitated the development of analytical methods for the determination of residues in environmental media. Adoption of methods utilizing instrumentation such as gas chro-matography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), liquid chromatography/tandem mass spectrometry (LC/MS/MS), or enzyme-linked immunosorbent assay (ELISA) has allowed the detection of minute amounts of pesticides and their degradation products in environmental samples. Sample preparation techniques such as solid-phase extraction (SPE), accelerated solvent extraction (ASE), or solid-phase microextraction (SPME) have also been important in the development of more reliable and sensitive analytical methods. 

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). 

Most of the analytical methods for the analysis of pesticides in food are based on instrumental approaches based on chromatography coupled to mass spectrometry. However, a great effort of development has been paid to develop rapid screening methods based on biological methods, such as, enzyme linked immunosorbent assays (ELISA). 

Hernandex, F., R. Serrano, M. Miralles, and N. Font (1996). Gas and liquid chromatography and enzyme linked immunosorbent assay in pesticide monitoring of surface water from the western Mediterranean. Chromatographia, 42(2/3) 151-185. 

The enzyme-linked Immunosorbent assay (ELISA) is a rapid Immunochemical procedure which can be used for trace analysis. We have applied the procedure to paraquat and other compounds difficult to analyze by the more classical methods. The Immunoassay for paraquat shows the practicality of the method for fortified and actual residue samples, and Is being compared with a gas chromatography procedure. Our work with the ELISA Illustrates that the Immunochemical technology can be used to solve problems encountered In pesticide residue analysis. 

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. 

Indeed, the promise of IA has led to workable enzyme-linked immunosorbent assays (ELISAs) for a variety of pesticides, including thiolcarbamates, triazoles and triazines, substituted ureas and sulfonylureas, bipyridilium compounds, and other groups of chemicals(2) (Table I). Additionally, radioimmunoassay (RIA) methods exist for chlorinated hydrocarbons, organophosphates, phenoxy adds and other chemical classes. 

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. 

Immunoassay techniques are based on the antigen-antibody interaction. These techniques involve a competitive reaction between antigen molecules of the target molecules and labeled antigen molecules for a limited number of antibodies. Enzyme-linked immunosorbent assays (ELISA) in which antibodies are immobilized on a solid phase are the most popular for pesticide detection. As pesticides are small molecules, in order to synthesize antibodies, pesticide derivatives (haptens) must be synthesized and coupled to carrier proteins. ... 

Stocklein WFM, Rohde M, Scharte G et al. Sensitive detection of triazine and phenylurea pesticides in pure organic solvent by enzyme linked immunosorbent assay (ELISA) Stabilities, solubilities and sensitivities. Anal Chim Acta 2000 405 255-265. 

Depending on the label, lAs are classified in different groups. Radioisotopes are used in RIAs, enzymes in enzyme-linked immunosorbent assays (ELISAs) or EIAs, fluorophores in FIAs or PFIAs and chemiluminescent compounds in CLIAs. Additional types of lA exist, but are not very common in pesticide analysis. A more detailed description of these lAs can be found in Gosling. 

The procedures for production of specific antibodies and their application in a competitive inhibition ELISA (Enzyme-Linked Immunosorbent Assay) are discussed in detail in the preceding chapter (Vanderlaan et al., this volume). In addition, other comprehensive overviews of the immunoassay development process in the pesticide field are available in general, a... 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

As mentioned above, GC and LC techniques (and GC-MS and LC-MS in particular) represent the major determinative approaches used in current MMRMs. Other, much less widely applicable and applied techniques include (1) capillary electrophoresis and capillary electrochromatography (2) thin-layer chromatography and (3) an array of immunoassays, such as immunosensors and enzyme-linked immunosorbent assay (ELISA). Immunoassays are, however, relatively useful for sensitive and rather rapid and inexpensive screening (followed by a confirmatory method in the case of a positive response) of selected pesticides for which ELISA kits or sensors are available. 

Nunes GS, Toscano lA, and Barcelo D (1998) Analysis of pesticides in food and environmental samples by enzyme-linked immunosorbent assays. Trends in Analytical Chemistry 17 79-87. 

The separation or complexation of humic substances and/or heavy metals is successful in favorable cases, e.g., by the addition of bovine serum albumin (BSA) in a trinitrotoluene enzyme-linked immunosorbent assay (ELISA) [18]. In the. screening of soil samples for pesticides, persistent centrifugation at 17 000 rpm, and simple dilution with bidi.stilled water in the ratio 1 10 was sufficient [19]. 

The ICMs used for pesticide analysis include immimoassays (lAs) and the use of antibodies for sample preparation (e.g., for SPE and the cleanup of samples) [153], detection in flow-injection analysis, and biosensors. The earliest ICMs to be developed for pesticides analysis were lAs. There are various t) es of lAs, but the most frequently used in this context is the enzyme-linked immunosorbent assay (ELISA) [185]. ELISA is a heterogeneous assay because the antibodies or antigens are immobilized on a solid phase. Table 18.3 lists selected ELISA methods for the determination of pesticides in water samples [186-190]. Bjamason et al. have proposed an enzyme flow immunoassay (EFIA) using a protein G column for the determination of triazine herbicides in surface and wastewaters with a linear range between 0.1 and 10 pg/L [191]. 

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