No abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid quantitation of T-2 toxin, a tricothecene mycotoxin produced by members of the genus Fusarium. T-2 toxin was first converted to the T-2 hemisuccinate (T-2 HS) and then conjugated by the water-soluble carbodiimide method to either bovine serum albumin for use as an immunogen or to horseradish peroxidase for use as an enzyme marker. T-2 antiserum was air-dried onto polystyrene microtissue culture plates and the ELISA conducted by simultaneously incubating standards of T-2 toxin and the T-2 HS-peroxidase conjugate. Competition curves were prepared by determining total bound enzyme. The ELISA took about 2 hr to complete and allowed minimal detection of T-2 at levels of 2.5 pg/assay. Average recoveries from samples of wheat flour spiked with T-2 toxin in the 1.0-30 ppb range were 95 • 25% and those for corn meal spiked in the 5.0-30 ppb range were 98 + 19%. The results suggested the ELISA is a simple and convenient alternative for the screening ofT-2 toxin in food and feeds. 940A / JAOCS December 1981

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B,,-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B28-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B, and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B, and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B, quantitation were 100 and 10 pg per assay, respectively.

Hemisuccinate (HS) and hemiglutarate (HG) of aflatoxin B2a (afla B2a) were prepared by refluxing afla B2a with the corresponding anhydride and 4‐N, N‐dimethylaminopyridine in tetrahydrofuran. Two epimers of the respective HS or HG which show different chromatographic behavior and physiochemical properties were isolated and characterized. Afla B2a‐HS hydrolyzes very rapidly in aqueous solution and was not used for further study. Afla B2a‐HG hydrolyzes at a much slower rate and was selected for the coupling to protein. Using the mixed anhydride method, as much as 12 moles of afla B2a‐HG were conjugated to each mole of bovine serum albumin (BSA). The antibody obtained from rabbits immunized with afla B2a‐HG BSA is most specific to afla B1 and shows little cross reaction with afla G1 and aflatoxicol. The lower limit for detection of afla B1 by radioimmunoassay using this antibody is in the range of 30–50 pg per assay.

We describe an affinity-column-mediated, enzyme-linked immunometric assay that is highly sensitive and adaptable to automation. Digoxin is the model test analyte. A comparison of digoxin with its analog, ouabain, for use as the immobilized ligand on the affinity column showed ouabain to be superior. We also report the effect of column elution rate. Antibody-enzyme conjugates prepared with the monovalent Fab'-fragment and the divalent F(ab')2-fragment coupled to beta-galactosidase are compared in terms of their overall assay performance. Although the monovalent Fab'--beta-galactosidase conjugate yields a more sensitive assay and dose-response curves that are linear over a wider range, the divalent F(ab')2--beta-galactosidase conjugate provides an assay with adequate sensitivity and extremely good precision, and is generally easier to synthesize reproducibly. This fully automated, rapid, and precise assay for digoxin is compatible with the Du Pont aca discrete clinical analyzer.