PRODUCTION AND CHARACTERIZATION OF ANTIIDIOTYPE AND ANTI-ANTI-IDIOTYPE ANTIBODIES FROM A MONOCLONAL-ANTIBODY AGAINST AFLATOXIN

Anti-idiotype antibodies (anti-id, Ab2) for aflatoxin (AF) were generated by immunizing rabbits with affinity-purified mouse monoclonal antibody (mAb) 575G4H7 (Ab1) against the toxin. The affinity-purified polyclonal Ab2 antibodies (pAb2) were subjected to various anlayses and were used to generate polyclonal anti-anti-idiotype antibodies (pAb3) in the ascites of BALB/c mice. In a biotin-avidin-amplified ELISA, the concentration causing 50% inhibition (ID50) Of binding of Ab1 to the solid-phase AFB(1)-bovine serum albumin (BSA) by pAb2 was found to be 0.76 mu g/mL. Inhibition of binding of Ab1 to the solid-phase Ab2 by free AFB(1) (ID50 = 1.16 mu g/mL) was less effective than in the AFB(1)-BSA-based ELISA. Radioimmunoassay (RIA) revealed that ID50 values of binding of tritiated AFB(1) to Ab1 by free AFB(1) and pAb2 were 100 ng/mL and 100 mu g/mL, respectively. Both indirect ELISA and RIA analysis showed that pAb3 has characteristics similar to those of other anti-AFB antibodies with high specificity and sensitivity for AFB(1) detection. In ELISA, the ID50 values of binding of pAb3 to the solid-phase AFB(1)-BSA by aflatoxins B-1, B-2, G(1), G(1), and B-3 were found to be 0.38, 7.7, 4.42, 162, and 225 ng/mL, respectively. In RIA, the ID50 values of binding of pAb3 to the marker ligand [H-3]AFB(1) by aflatoxins B-1, B-2, G(1), G(2), and B-3 were found to be 0.88, 79, 34, 850, and 307 ng/mL, respectively. The overall analytical recoveries of AFB(1) (10-100 ppb) added to the white and yellow corn in the pAb3-based ELISA were found to be 84.1 and 85.4%, respectively.