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

Abstract

Polyclonal anti‐idiotype antibodies (anti‐id, pAb2) for aflatoxin were generated from mice ascites after immunization with affinity‐purified rabbit polyclonal antibody (pAbl) against aflatoxin B₁ (pAFB₁). 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 AFB₁ to pAb1 by pAb2 was demonstrated in both regular and biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) systems in which AFB₁‐bovine serum albumin (BSA) was coated on to the microtiter plate. The concentrations causing 50% inhibition (ID₅₀) of the binding of pAbl to AFB₁‐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 AFB₁ (ID₅₀ = 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 ID₅₀ of binding of AFB₁‐horseradish peroxidase to the solid‐phase pAb3 by AFB₁ was found to be 0.20 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of pAb3 to the solid‐phase AFB₁‐BSA by AFB₁ was found to be 1.84 ng ml^‐1 Analysis of a naturally contaminated corn sample for AFB₁ 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).

Keywords:

• Aflatoxin
• antibody
• anti‐idiotype
• immunoassay