In view of the toxicological hazard and important applications in analgesics and cancer chemotherapeutics of αB-CTX, it is urgent to develop an accurate, effective and feasible immunoassay for the determination and analysis of αB-CTX in real samples. In this study, MBP-αB-CTX4 tandem fusion protein was used as an immunogen to elicit a strong immune response, and a hybridoma cell 5E4 secreting IgG2b against αB-CTX was successfully screened by hybridoma technology. The affinity of the purified 5E4 monoclonal antibody (mAb) was 1.02 × 108 L/mol, which showed high affinity and specificity to αB-CTX. Epitope 1 of αB-CTX is the major binding region for 5E4 mAb recongnization, and two amino acid residues (14L and 15F) in αB-CTX were critical sites for the interaction between αB-CTX and 5E4 mAb. Indirect competitive ELISA (ic-ELISA) based on 5E4 mAb was developed to detect and analyze αB-CTX in real samples, and the linear range of ic-ELISA to αB-CTX was 117-3798 ng/mL, with a limit of detection (LOD) of 81 ng/mL. All the above results indicated that the developed ic-ELISA had high accuracy and repeatability, and it could be applied for αB-CTX detection and drug analysis in real samples.
Keywords: ELISA; epitope; hybridoma; monoclonal antibody; αB-conotoxin VxXXIVA.