Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody
Hengkun Tang,
Haimei Liu,
Yehong Gao,
Rui Chen,
Mingke Dong,
Sumei Ling,
Rongzhi Wang,
Shihua Wang
Affiliations
Hengkun Tang
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Haimei Liu
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Yehong Gao
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Rui Chen
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Mingke Dong
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Sumei Ling
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Rongzhi Wang
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Shihua Wang
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
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.
