Anti‐idiotype and anti‐anti‐idiotype antibodies generated from polyclonal antibodies against aflatoxin b₁

Abstract

Polyclonal anti-idiotype antibodies pAb2) for aflatoxin were generated from mice ascites after immunization with affinity-purified rabbit polyclonal antibody (pAbl) against aflatoxin B 1 (pAFB 1 ). After affinity chromatography purification, pAb2 were subjected to various analyses, and were used to generate anti-anti-id antibodies (pAb3) in the ascites of Balb/c mice. ELISA analyses revealed that the anti-id antibodies bound specifically to the original pAb1, but not to other types of monoclonal antibodies or normal mouse IgG. The inhibition of binding of AFB 1 to pAb1 by pAb2 was demonstrated in both regular and biotin-avidin amplified enzyme-linked immunosorbent assay (ELISA) systems in which AFB 1 -bovine serum albumin (BSA) was coated on to the microtiter plate. The concentrations causing 50% inhibition (ID 50 ) of the binding of pAbl to AFB 1 -BSA by pAb2 were found to be 6.5 and 1.7 µg/assay respectively. Inhibition of the binding of pAb1 to the solid-phase pAb2 by free AFB 1 (ID 50 = 0.63 µ/assay) was also demonstrated in the ELISA. pAb3 were found to have similar characteristics to the original pAb1. In the direct ELISA, the ID 50 of binding of AFB 1 -horseradish peroxidase to the solid-phase pAb3 by AFB 1 was found to be 0.20 ng ml -1 . In the indirect ELISA, the ID 50 of the binding of pAb3 to the solid-phase AFB 1 -BSA by AFB 1 was found to be 1.84 ng ml -1 Analysis of a naturally contaminated corn sample for AFB 1 by the pAb3-based ELISA gave a result similar to that obtained from the regular pAb1-based ELISA (19.7 and 21.8 µg kg -1 respectively).

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

“…Such properties are similar to recent observations for the anti-id antibodies generated in rabbits and mice (Hsu & Chu, 1994, 1995, as well as data obtained from studies for the anti-id antibodies of several other hapten-type toxins and insecticides (Chanh et al, 1990;Shestowsky et al, 1992;Spinks et al, 1993). Whereas IgY2 appear to have little use in the immunoassay of aflatoxins, IgY2 could be used as a good immunogen in generating the Ab3 as in previous studies (Hsu & Chu, 1994, 1995. In the present study, IgY3 were generated in chickens after immunization of rabbit pAb2 that were induced by immunizing with anti-AFB 3 -HS-BSA monoclonal antibodies.…”

“…In the present study, IgY3 were generated in chickens after immunization of rabbit pAb2 that were induced by immunizing with anti-AFB 3 -HS-BSA monoclonal antibodies. Similar to the pAb3 obtained from rabbits (Hsu & Chu, 1994) and mice (Hsu & Chu, 1995) after immunizing with pAb2, IgY3 obtained from this study have also been shown to be useful for the ELISA of aflatoxins. The sensitivity of the present IgY3-based ELISA for AFBi was found to be comparable with most of the pAbl-and mouse pAb3-based ELISAs.…”

“…The specific binding of IgY2 to pAbl was analyzed by a biotin-streptavidin amplified competitive ELISA as previously described (Hsu & Chu, 1995). In this assay, microtiter plate wells were coated with AFB,-BSA (2 fig ml" 1 , in 0.01 M-PBS, 0.1 ml/ well).…”

“…Some Ab2 have been shown to mimic effectively the biological functions of the hapten (Nisonoff, 1991). Recently, anti-id antibodies for aflatoxins from rabbits and mice have been obtained and characterized (Hsu & Chu, 1994, 1995. To test whether anti-id and anti-anti-id antibodies could be generated from the egg yolks of laying hens, the rabbit idiotype antibodies against AFBi-carboxymethyloxime (CMO)-bovine serum albumin (BSA) and rabbit anti-id antibodies against anti-AFB 3 -hemisuccinate (HS)-BSA monoclonal antibodies used in previous studies (Chu et ah, 1987;Hsu & Chu, 1994) were chosen as pAbl and pAb2 in generating anti-id antibodies (IgY2) and anti-anti-id antibodies (IgY3) respectively.…”

Recent studies of mycotoxins

“…This situation has been alleviated to some extent by the development of immunological methods and the production of monoclonal antibodies (Morgan and Lee 1990). In efforts to increase the sensitivity of these methods Hsu and Chu (1995) generated anti-idiotype and anti-anti-idiotype antibodies from polyclonal antibodies against aflatoxin B 1 . Immunological reagents can be used in two modes, for direct analysis by ELISA after extraction, or in the clean-up stages in the form of affinity columns.…”