Preparation Of Monoclonal Antibody Against ω-conotoxin MⅦA And Development Of Detection Method Based On Monoclonal Antibody

ω-CTX MⅦA,a peptide with 25 amino acid residues originally secreted by Conus magus,is a selective and reversible N-type voltage-gated calcium channel blocker,which could be used as an analgesic for pain.It has important applications in the treatment of neurological diseases and the identification of ion channel sub-types,and has become a research spot in the field of polypeptide chemistry,molecular biology and pharmacology.Conotoxin has high toxicity to humans and animals,and it also can cause convulsion,tremor,numbness and loss of activity,even death.In view of the important function and toxicity of the ω-CTX MⅦA,it is urgent to develop immunoassays based on monoclonal antibody(McAb)against ω-CTX MⅦA to detect the ω-CTX MⅦA in real samples.This study aimed to prepare a high affinity and specific McAb against ω-CTX MⅦA,and to develop ELISA and colloid gold strip method to achieve the fast detection of ω-CTX MⅦA.The purified fusion protein GST-ω-CTX MⅦA was used to immunize the Balb/c mice,and the resulted fusion protein Trx-ω-CTX MⅦA was applied for anti-serum titer determination and antibody screening.A cell named 2E5 was selected successfully by cells fusion of SP2/0 with spleen cells isolated from immunized mouse,whose anti-serum titer reaching 1:8000,and the isotype of McAb 2E5 belongs to IgG1.The ascites fluid harvested from mice was further purified by protein G affinity chromatography,and the purity of the purified IgG antibody was identified by SDS-PAGE.The resulted IgG antibody displayed high specificity to ω-CTX MⅦA antigen,with a high affinity of 2.79×109 L/mol.The result derived from indirect competitive ELISA(ic-ELISA)indicated that the IC50 of this method reached 1.306 μg/mL,and the limit of detection(LOD)was 0.138 μg/mL.The linear range to detect ω-CTX MⅦA was 0.198～7.219 μg/mL.The colloidal gold was prepared by sodium citrate reduction method and labeled with the purified anti-ω-CTX MⅦA antibody.After purification,the labeled colloidal gold was solidified on the release pad.The complete antigen Trx-ω-CTX MⅦA and goat anti-mouse IgG antibody was coated at the T-line and C-line of NC membrane,respectively.The colloidal gold test strips were assembled,and different concentrations of standard ω-CTX MⅦA samples were used to determine the LOD.The detection result demonstrated that the developed colloidal gold test strip has high specificity and accuracy,and the LOD of test strip was 1 μg/mL.The above results further showed that the monoclonal antibody obtained from this study can be used to develop ELISA and colloidal gold strip detection method,and it may be provided a reference for detection of ω-CTX MⅦA in real samples.