ANTIIDIOTYPE AND ANTI-ANTI-IDIOTYPE ANTIBODIES FOR AFLATOXIN FROM LAYING HENS

Anti-idiotype (anti-id) antibodies (IgY2) for aflatoxin (AF) were obta ined from the egg yolks of laying hens immunized with affinity-purifie d rabbit polyclonal anti-aflatoxin B-1 (AFB(1)) carboxymethyloxime-bov ine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity puri fied and then subjected to various analyses. Inhibition of the binding of pAb1 to the solid-phase AFB(1)-BSA by IgY2 and the binding of pAb1 to the solid-phase IgY2 by free AFB(1) were demonstrated in a biotin- avidin amplified enzyme-linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID50) of the binding of pAb1 to AFB(1)-BSA was found to be 2.45 mu g/assay. The ID50 concentr ation of the binding of pAb(1) to IgY2 by free AFB(1) was found to be 0.30 mu g/assay. Inhibition of the binding of AFB(1)-horseradish perox idase (HRP) to the solid-phase pAb1 by IgY2 (ID50 = 9.65 mu g/assay) w as also demonstrated in rite direct ELISA. Egg yolk anti-anti-id antib odies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti-AFB(3)-hemisuccinate-BSA monoclonal antibody. IgY3 was su bjected to affinity chromatography purification with Sepharose gel a r m eh with AFB(2)-carboxymethyloxime, and then subjected to various ana lyses. ELISA analysis revealed that IgY3 has characteristics similar t o other anti-AFB antibodies induced in various experimental animals. I n the direct ELISA, the ID50 of the binding of AFB(1)-HRP to solid-pha se IgY3 by AFB(1) was found to be 0.12 ng ml(-1) In the indirect ELISA , the ID50 of the binding of IgY3 to solid-phase AFB(1)-BSA by AFB(1) was found to be 2.2 ng ml(-1). The IgY3-based ELISA analysis showed hi gher sensitivity than tl?at of the egg yolk antibodies directly agains t AFB-protein conjugates (IgYI). A good correlation was found for the data obtained from IgY3-based and pAb1-based ELISAs in the analysis of AFB in the fungal culture filtrates.