Spectral characteristics of fluorescence and circular dichroism of aflatoxin B1 reaction with its anti-idiotypic antibody

Abstract

Aflatoxin B1 (AFB1) is a toxic secondary metabolite and sensitive methods for its analysis have been developed. In our lab, a number of works have been carried out, including exploitation of detection methods and production of anti-idiotypic antibody (Ab2) against Fab fragment of anti-AFB1 antibody (Ab1). In this paper, Ab2 was generated upon the immunization of mice with F(ab′)2 fragment, which was specific to AFB1 and obtained by pepsin digestion of Ab1. The characteristics of Ab2 was primarily investigated by indirect competitive enzyme-linked immunosorbent assay (icELISA), which indicated that Ab2, might bear an internal image of antigen AFB1 and was able to combine to F(ab′)2 in competition with AFB1, and the concentration of Ab2 to cause 50% inhibition of binding (IC50) was 131.8μg/mL. In addition, fluorescence and circular dichroism studies were designed to explore the mutual relationship among AFB1, F(ab′)2 and Ab2. The fluorescence spectroscopy implied that both AFB1 and Ab2 act as a quencher upon F(ab’)2, and the Ab2 could compete with AFB1 when both of Ab2 and AFB1 reacted with F(ab′)2. The circular dichroism (CD) spectrum suggested that both the binding of Ab2 and AFB1 on F(ab′)2 brought secondary conformation change of F(ab′)2, especially in the changes of α helix and β sheet. The research performed would provide unique insight into the comprehension of interaction among AFB1, F(ab′)2 and Ab2 as well as offer structural information for substitution researches of toxic antigen like AFB1.