Competitive Enzyme Linked Immunosorbent Assay ELISA

Of the several available IA techniques, the most popular is the ELISA. Two different ELISA formats have been used for the analysis of mycotoxins, both of which involve the separation of free toxin in one phase from the bound toxin in another phase (130). In direct competitive (DC) ELISA, the mycotoxin-specific antibody is immobilized in the wells of a microassay plate or on the wall of an assay tube. Sample extracts and mycotoxin standard solutions are pipetted into the antibody-coated wells or tubes with a fixed amount of enzyme-labeled mycotoxin. The plates or tubes are incubated between five minutes and more than an hour, which allows the labeled and unlabeled mycotoxin to compete for binding sites on the antibodies. The unreacted material is then washed away and the amount of labeled an alyte bound by the immobilized antibody is quantified by addition of an enzyme substrate that forms a colored product. The amount of color formed is inversely proportional to the amount of unlabeled mycotoxin in the sample (136). To avoid sample matrix interferences that can occur with DC ELISA, the sample can either be diluted or subjected to a cleanup step.

In indirect competitive (IDC) ELISA the mycotoxin is coupled to a carrier protein such as bovine serum albumin and bound to the wells of a microtiter plate or assay tube (136). The sample extract is added to the microassay well followed by addition of a fixed amount of mycotoxin specific antibody. The antibody in solution partitions between the free mycotoxin in the sample and the toxin bound by the microassay plate. The antibody not bound to the microassay plate is washed away and quantified by addition of a second antibody that specifically binds the first antibody. An enzyme substrate is added, and the amount of color formed is proportional to the amount of analyte in the sample extract. A major advantage of IDC ELISA is that it requires much less antibody than DC ELISA (130). One drawback of IDC ELISA is that it requires a longer analysis time than DC ELISA (131).

Several studies have investigated the accuracy and precision of ELISA by comparing the technique with other analytical methods. In general, excellent correlations have been found between results obtained by ELISA and TLC, HPLC, or GC for aflatoxins (147,148), deoxynivalenol (149), and fumonisins (150,151). However, fumonisin results obtained by ELISA can be higher than those determined by chromatographic methods (151-153). This phenomenon is due to the presence of structurally related compounds that cross-react with the antibodies. Use of antibodies of higher affinity to the analyte may prevent this problem (131).

In recent years, ELISA kits have been made available from commercial sources for quantitative and semiquantitative analysis of aflatoxins, deoxynivalenol, fumonisins, ochratoxin, and zearalenone in food and feed (129). The kits usually are self-contained and include all the reagents: standards, controls, buffers, and substrates. Several commercial ELISA kits have been approved as AOAC official methods for aflatoxin and zearalenone (32,129). A membrane-based visual dipstick enzyme immunoassay was developed for analyzing up to five mycotoxins simultaneously (134).

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