Amide-containing neoepitopes: the key factor in the preparation of hapten-specific antibodies and a strategy to overcome

Abstract

For a long time, people have suffered from uncertainty, complexity, and a low success rate in generating and screening antibodies against small molecules, which have become the core bottlenecks of immunochemistry. Here, the influence of antigen preparation on antibody generation was investigated at both molecular and submolecular levels. Neoepitopes (amide-containing neoepitopes) formed in the preparation of complete antigens are one of the most important factors limiting the efficiency of generating hapten-specific antibodies, which was verified by different haptens, carrier proteins, and conjugation conditions. Amide-containing neoepitopes present electron-dense structural components on the surface of prepared complete antigens and, therefore, induce the generation of the corresponding antibody with much higher efficiency than target hapten. Crosslinkers should be carefully selected and not overdosed. According to these results, some misconceptions in the conventional anti-hapten antibody production were clarified and corrected. By controlling the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) during the synthesis of immunogen to limit the formation of amide-containing neoepitopes, the efficiency of hapten-specific antibody generation could be significantly improved, which verified the correctness of the conclusion and provided an efficient strategy for antibody preparation. The result of the work is of scientific significance in the preparation of high-quality antibodies against small molecules.

Cross‐reaction mediated by distinct key amino acid combinations in the complementary‐determining region (CDR) of a monoclonal antibody

“…Consistent with prior research, peptides were conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) carrier proteins 14,15 using 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide (EDC) activators for immunization. We used EDC to activate the carboxyl terminals of three polypeptides, coupled with the carrier proteins BSA and KLH 16 . The cross‐linking of the polypeptides to the carriers was outsourced to Shanghai Qiangyao Biological Company.…”

“…We used EDC to activate the carboxyl terminals of three polypeptides, coupled with the carrier proteins BSA and KLH. 16 The cross-linking of the polypeptides to the carriers was outsourced to Shanghai Qiangyao Biological Company.…”