Characterization of an enzyme linked immunosorbent assay for Aflatoxin B(1) based on commercial reagents

Two indirect ELISA have been investigated for the determination of Aflatoxin B(1), employing only reagents commercially available, whose composition is not exactly known. In both cases the antigen (Aflatoxin B(1)-BSA) was coated to the solid phase (polystyrene microtiter plates). In one procedure the specific antibody was a conjugate with peroxidase, while in the other one it was not conjugated, and a second antibody labelled with alkaline phosphatase was used. A simple model was employed to characterize the equilibria, which is of help also if the exact composition of the immunoreagents is not known, and allows to predict the shape and position of the competition curve. The factors which determine the dynamic range were found to be the affinity constant the complex in the solid and the amount of antigen in the solid, and the affinity constant of the complex in solution phase. Useful aspects of the antigen-antibody complexation equilibria in the solid phase were investigated by ELISA at zero concentration of antigen in solution, obtaining c (s)c ( *) and K'f(n)(T). The equilibria in solution were studied by competition ELISA, obtaining K, the affinity constant of the antigen-antibody complex in solution. Similar results were obtained with the two procedures, for instance the affinity constant in solution was 2 x 10(8). A procedure for the determination of Aflatoxin B(1) in food samples was developed.