Effect of hapten structures on development of novel antibody against capsaicin and dihydrocapsaicin

Abstract

• Three novel capsaicinoids haptens were synthesized, and the antibody-eliciting properties of four hapten-protein conjugations were investigated. • Novel polyclonal antibodies against capsaicinoids were prepared, and the important role of molecular structure and electrostatic potential in producing specific antibodies was verified. • A competitive indirect ELISA (icELISA) was established for capsaicinoids detection in spicy foods. Capsaicinoids content as a basis for the evaluation of spicy taste has attracted much attention. Antibody, as an important component of immunoassay, plays an important role in the detection of capsaicinoids levels. The molecular structure of the hapten is the key factor affecting the quality of small molecule antibodies. In our study, six capsaicinoids haptens were selected with different junction arm of length and structure to analyzed immune effect by geometric structure and electrostatic potential (ESP). Among them, three haptens were capsaicinoids structural analogues, and others were designed and synthesized. The results showed that the retention of amide bond (–NH–CO–) and hydroxyl group in the benzene ring of capsaicinoids hapten as well as rigid spacer arm that linked proteins to hapten had a positive effect on the affinity of the antibody. It provided a theoretical basis for rapid screening of small molecular substance haptens. To test our hypothesis, four kinds of haptens were used to synthesize antigens and immunize animals. Comparing 28 antiserum/coating conjugates combinations tested by indirect competitive enzyme-linked immunosorbent assay (icELISA) method for capsaicinoids (i.e., capsaicin and dihydrocapsaicin), the homologous combination of Pa-B2/OVA-hapten B had been shown to be most suitable for the determination of capsaicin and dihydrocapsaicin, which was consistent with the ESP analysis. In short, ESP analysis can speed up haptens screening and reduce the workload while producing high-quality antibodies. Thus, an icELISA based on hapten B polyclonal antibody was developed with IC 50 value of 0.36 μg mL −1 and the limit of detections (LODs) of 20 µg kg −1 in spicy food samples. Moreover, these results were also validated by high performance liquid chromatography (HPLC), and showed good agreements.