Preparation Of Monoclonal Antibody And Multiple Antibody Against Clostridium Septicums Alpha Toxin And Preliminary Establishment Of DAS-ELISA

Clostridium septicum is widely distributed in nature environment such as soil,and is the main pathogen causing malignant edema in animals and humans.The bacteria infected sheep mainly caused rapid epidemic of sheep,and the sheep died quickly within 2-3 days,which caused great losses to the animal husbandry industry in China.Therefore,it is urgent to establish an early diagnosis method for C.septicum disease,such as braxy,in order to screen suspicious clinical samples quickly.The main virulence factor of C.septicum is alpha toxin,which has many biological activities such as hemolysis,lethality and necrosis.In this study,alpha toxin was taken as the main research object.Preliminary exploration of double antibody sandwich ELISA(DAS-ELISA)for detection of alpha toxin by preparing monoclonal antibodies and polyclonal antibodies,to provide technical support for early clinical diagnosis of C.septicum disease such as braxy.This study is mainly divided into three parts:1.Preparation of C.septicum natural alpha toxin and recombinant toxin proteinAfter the recovery and enrichment culture of C.septicum reference strain(ATCC 12464),the crude protein of C.septicum was extracted.The purified protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and the natural alpha toxin of 46 kDa was obtained.According to the CDs region sequence of C.septicum alpha toxin published on NCBI,the full length of alpha toxin gene was amplified,and the prokaryotic expression system of C.septicum alpha toxin was constructed.The soluble recombinant protein was expressed and purified under the optimum induction conditions(when OD600 nm was 0.4-0.6,IPTG with concentration of 0.5 mol/mL was added to the bacterial solution,and induced to express for 5 hours at 37℃).The final concentration of the protein was 4.28 mg/mL.Western blot analysis showed that the protein had good immunogenicity,which proved that the prokaryotic expression vector of C.septicum alpha toxin was successfully constructed.2.Preparation of monoclonal antibodies and polyclonal antibodiesThe purified recombinant alpha toxin protein was used to immunize New Zealand white rabbits,the positive serum was Purified by antigen affinity chromatography,the purified polyclonal antibody concentration was 0.98 mg/mL,The polyclonal antibody against the recombinant protein titer test result is 1:256000,and the test result of the natural alpha toxin titer is 1:128000.Besides,the results of SDS-PAGE and western-blot showed that the polyclonal antibody had high purity and good specificity.The BALB/c mice were immunized with the inactivated recombinant α-toxin protein.The spleen cells were prepared after the sera of the mice were transfected and the cells were fused.Eight strong positive cell lines were identified through hybridoma cell screening and subcloning experiments.The stability test showed that 6 cell lines could secrete monoclonal antibodies stably,among which 3E8,6B12 and 6F10 cell lines had strong ability to recognize natural alpha toxin.Two of the cells,3E8 and 6B12,were selected and intraperitoneally injected with BALB/c mice.The ascites was collected and purified.The indirect ELISA showed that the titer of the two monoclonal antibodies against the recombinant protein was1:256000.The results of the toxin titer test were 1:128000,and the concentrations of the two monoclonal antibodies were 2.27 mg/mL and 1.96 mg/mL,respectively.Besides,the purity and specificity of the monoclonal antibody were identified by SDS-PAGE and Western-blot methods.And the results show that the Monoclonal antibodies are highly purified and have good specificity.3.Preliminary construction of DAS-ELISA against C.septicum alpha toxinIn order to establish a highly efficient and sensitive DAS-ELISA assay,the optimal pairing antibody and antibody working concentration were initially explored.in this study,3E8 and 6B12 were used as detection antibodies respectively.The optimal working concentration of polyclonal antibody as capture antibody was determined to be 1.25 μg/mL by square titration experiment,which was the highest in 3E8.The optimum working concentration was 0.5 μg/mL and specificity of the method was validated under the optimized conditions.Those results showed that the method could only recognize recombinant alpha toxin and natural alpha toxin of C.septicum,and had no cross reaction with other proteins.In this study,a prokaryotic expression system of C.septicum alpha toxin recombinantprotein was successfully constructed.Under the optimal induction conditions,a large number of soluble target proteins were induced.Based on the recombinant protein of alpha toxin,high purity monoclonal antibodies and polyclonal antibodies against C.septicum alpha toxin with high potency,specificity and stability were successfully prepared.The optimum ligand and working concentration of antibody for the detection of alpha toxin by DAS-ELISA were also explored,and the specificity of the preliminary optimized DAS-ELISA method was verified.It provides an important clinical diagnosis method for the diseases caused by C.septicum in livestock and poultry breeding,and also provides technical support for the development of C.septicum vaccine and related drugs.