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 Afl atoxin B-1, employing only reagents commercially available, whose comp osition is not exactly known. In both cases the antigen (Aflatoxin B-1 -BSA) was coated to the solid phase (polystyrene microtiter plates). I n one procedure the specific antibody was a conjugate with peroxidase, while in the other one it was not conjugated, and a second antibody l abelled with alkaline phosphatase was used. A simple model was employe d to characterize the equilibria, which is of help also if the exact c omposition of the immunoreagents is not known, and allows to predict t he shape and position of the competition curve. The factors which dete rmine the dynamic range were found to be the affinity constant the com plex in the solid and the amount of antigen in the solid, and the affi nity constant of the complex in solution phase. Useful aspects of the antigen-antibody complexation equilibria in the solid phase were inves tigated by ELISA at zero concentration of antigen in solution, obtaini ng c(s)c and K'f(T)(n). The equilibria in solution were studied by co mpetition ELISA, obtaining K, the affinity constant of the antigen-ant ibody 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. (C) 1997 Elsevier Science B.V.