title: Preparation and identification of monoclonal antibodies against ω-conotoxin MVIIA.
abstract: ω-Conotoxins MVIIA (ω-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against ω-conotoxin MVIIA. In this study, an MAb against ω-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a (+), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against ω-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against ω-CTX MVIIA was 7.33×10(9) L/mol, and the cross-reaction test showed that the MAb specifically bound ω-CTX MVIIA. The MAb could be used as a specific antagonist for ω-CTX MVIIA in the physiological study on the CaV channels in the nervous system.
authors.affiliation: Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, School of Life Sciences, Fujian Agriculture and Forestry University , Fuzhou, China .
authors.forename: Yanling
authors.initials: Y
authors.lastname: Yang
authors.forename: Yanling
authors.initials: Y
authors.lastname: Ma
authors.forename: Heng
authors.initials: H
authors.lastname: Li
authors.forename: Shihua
authors.initials: S
authors.lastname: Wang
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D020960:omega-Conotoxins / Q000235:genetics / Q000276:immunology*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D011993:Recombinant Fusion Proteins; D020960:omega-Conotoxins; C078452:ziconotide