Anti-idiotype and anti-anti-idiotype antibodies for aflatoxin from laying hens

Anti-idiotype (anti-id) antibodies (IgY2) for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity-purified rabbit polyclonal anti-aflatoxin B-1 (AFB(1)) carboxymethyloxime-bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified 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 concentration 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 peroxidase (HRP) to the solid-phase pAb1 by IgY2 (ID50 = 9.65 mu g/assay) was also demonstrated in rite direct ELISA. Egg yolk anti-anti-id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti-AFB(3)-hemisuccinate-BSA monoclonal antibody. IgY3 was subjected to affinity chromatography purification with Sepharose gel a rm eh with AFB(2)-carboxymethyloxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti-AFB antibodies induced in various experimental animals. In the direct ELISA, the ID50 of the binding of AFB(1)-HRP to solid-phase 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 higher sensitivity than tl?at of the egg yolk antibodies directly against 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.