Development And Identification Of Monoclonal Antibodies Against Botulinum Toxin Type F

Botulinum neurotoxin（BoNT）is one of the most poisonous natural substances,which is a lethal toxin.BoNT can cause muscle relaxation paralysis through inhibiting the release of acetylcholine at the neuromuscular junction.Seven serotypes of BoNT have been described（A-G）,at least four of which have been reported to cause human botulism（BoNT/A,B,E,F）.There are no drugs can effectively treat BoNT.The purpose of this work is to prepare monoclonal antibodies against BoNT/F,which provides a certain experimental basis for the development of the effective therapeutic drugs against BoNT/F.In this study,monoclonal antibodies against BoNT/F were prepared by hybridoma technology and phage antibody library technology.Firstly,BALB/c mice were immunized with BoNT/FHc protein as immunogen.The titer of tail blood of mice immunized 3 times was detected by enzyme-linked immunosorbent assay（ELISA）.The spleen cells of mice with the highest titer of tail blood were fused with myeloma Sp2/0 cells under the action of polyethylene glycol（PEG）to prepare hybridoma cells.After 14 days of cell fusion,cells from the wells giving positive signals for antibody production were screened by indirect ELISA.Then the positive cells were cloned by limiting dilution.The subtype of the monoclonal antibody in the hybridoma cell supernatant were detected through antibody subtype identification kit.Monoclonal antibodies were prepared by intraperitoneal injection of hybridoma cells in mice,the antibodies in the ascites were purified by affinity chromatography on protein-G.Protein concentrations were determined with Lowry-kit,and the neutralization titer of purified monoclonal antibodies were determined by mice neutralization test.Then,the affinity constants of the monoclonal antibodies 1F4 with the highest titer in the supernatant of hybridoma cells and the monoclonal antibodies 1E2（A）with the highest neutralization titer were measured by indirect ELISA.The amino acid sequence of the variable region of the monoclonal antibodies was analyzed by IgBLAST.The variable region gene of mice antibody 1E2（A）was ligated into humanized plasmid by enzyme digestion to construct chimeric humanized plasmid.Finally,the phage antibody library was used to screen the monoclonal antibodies against BoNT/F.After that,the scFv gene was screened with BoNT/FHc as antigen and its sequence was analyzed.After BALB/c mice were immunized with antigen BoNT/FHc protein for three times,the titer of tail blood of mice all reached 1:16000,with the highest titer of1:32000 in mouse No.2.The fusion rate was 74.47%and the positive rate was 8.59%.Seven cell lines（1F4,1E2（A）,4B7（A5）,4B7（B2）,4B7（G3）,1E2（B）,4B7（F1））which could stably secrete monoclonal antibodies were obtained.The isotype of all antibodies was belonging to IgG1 andκ.The SDS-PAGE results showed that all the hybridoma cells produced antibodies in the abdominal cavity of mice,and the purity of the antibodies purified by protein G column was greater than 90%.The concentrations of the antibodies assayed by Lowry methods were 2.0 mg/mL,1.8 mg/mL,0.5 mg/mL,2.7 mg/mL,2.1 mg/mL,2.5 mg/mL,2.8 mg/m L respectively.The results of animal experiments showed that the neutralization titer of monoclonal antibody was 0LD₅₀/mg,1200 LD₅₀/mg,0 LD₅₀/mg,200 LD₅₀/mg,400 LD₅₀/mg,200 LD₅₀/mg,800LD₅₀/mg respectively.The affinity constant of monoclonal antibody 1F4 was 1.08×10^-8mol/L and the affinity constant of monoclonal antibody 1E2（A）was 8.45×10^-5mol/L.The CDR3 sequence of light chain variable region of antibody 1F4 was QQHYSTPWT,and the CDR3 sequence of heavy chain variable region was TRHGYFPSWFAY,the CDR3 sequence of monoclonal antibody 1E2（A）was confidential information.The chimeric humanized plasmid of 1E2（A）was successfully constructed under the action of molecular biology method.Spleen RNA extracted from mouse No.1 was reverse transcribed into cDNA.The variable region gene of antibody light and heavy chain around 300 bp was amplified,and the scFv sequence of 750 bp was amplified by PCR.A mouse phage single-chain antibody library with a library capacity of 1.2×10⁴ was obtained.The antibody library was screened with BoNT/FHc as antigen,and the FR1sequence of mice heavy chain variable region was obtained.In this study,seven hybridoma cell lines can stably secrete monoclonal antibodies were screened,the affinity constant of 1F4 was 1.08×10^-8 mol/L,and the neutralizing titer of monoclonal antibodies 1E2（A）was 1200 LD₅₀/mg,which provided a new method for the detection against BoNT/F,and it also laid the foundation for the treatment after BoNT/F poisoning.At the same time,a humanized plasmid of 1E2（A）was constructed,which provides related experimental date of plasmids and sequences for humanization and antibody expression of 1E2（A）.