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

Abstract

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 pAbl 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 (ID 50 ) of the binding of pAb1 to AFB 1 -BSA was found to be 2.45 µg/assay. The ID 50 concentration of the binding of pAb1 to IgY2 by free AFB 1 was found to be 0.30 µg/assay. Inhibition of the binding of AFB 1 -horseradish peroxidase (HRP) to the solid-phase pAbl by IgY2 (ID50 = 9.65 µg/assay) was also demonstrated in the 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 armed with AFB 2 -carboxymethytoxime, 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 ID 50 of the binding of AFB 1 -HRP to solid-phase lgY3 by AFB 1 was found to be 0.12 ng ml -1 . In the indirect ELISA, the ID 50 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 that of the egg yolk antibodies directly against AFB-protein conjugates (IgY1). 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.