High Affinity Antibodies for the Development of a Specific Immunoassay for the Determination of Bongkrekic Acid in Food

Bongkrekic acid (BA) is a fatal bacterial toxin produced by Burkholderia gladioli pathovar cocovenenans (B. gladioli cocovenenans). Rapid and sensitive monitoring of BA occurrence in food and immediate treatment after poisoning by effective antidotes is highly demanded for food and public safety. In this work, a novel monoclonal antibody (mAb) 2A9 against BA with a 50% inhibition concentration (IC50) of 1.7 μg/L was prepared and employed to establish an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for BA determination, with limits of detection ranging from 0.2 to 0.5 μg/kg in rice, tremella, wet rice noodles, and black fungus. Furthermore, the single-chain variable fragment (scFv) of mAb 2A9 was prepared to provide insights into its potential drug developability of as antidotes. Molecular recognition analysis revealed that the key residues involved in BA binding were GLY-103 and ILE-38 in the heavy chain and PHE-101 in the light chain, with hydrophobic interactions playing a predominant role. Virtual mutagenesis further suggested that VAL-99 in the heavy chain and GLN-95 in the light chain represent promising mutation sites for evolving scFvs with enhanced affinity to BA. In addition, the potential bioconjugation sites among the three carboxyl groups of BA were identified based on antibody-BA recognition analysis.