Establishment Of Blocking ELISA Antibody Detection Method For Clostridium Septicum ? Toxin

Clostridium septium is the main pathogen of human non-traumatic or spontaneous gas gangrene and malignant edema disease of pigs,horses,cattle,sheep,chickens,deer and other animals.Factors and immunogens have hemolytic activity and can cause body cell necrosis.Infection of sheep by Clostridium septium caused the rapid epidemic of sheep,and the disease course of sheep was mostly acute,and died quickly after the onset of disease.The mortality rate was high,which brought huge economic losses to China's animal husbandry.Vaccine immunization is the main means of preventing the rapid outbreak of sheep,due to the lack of in vitro antibody detection methods.The evaluation of the effect of this kind of vaccine after immunization mostly uses the method of injecting mice through the tail vein after serum-toxin neutralization,and determining the serum neutralizing antibody titer according to the death of the mouse.This method is cumbersome and difficult to use for the immunization of clinical large samples.To this end,this study intends to establish a monoclonal antibody against C.putrefium toxin to establish an ELISA detection method for C.putrefium toxin to meet the requirements of fast,simple and high-throughput.It is used for vaccine efficacy testing and after vaccine immunization.A prokaryotic expression system of C.putrefium recombinant alpha toxin was established,and 11 virulence genes of C.putrefium alpha toxin were deleted.The non-toxic mutant of C.putrefium recombinant toxin was expressed in vitro.The protein was mainly in soluble form.The expressed protein was purified to obtain the target protein with a concentration of 0.76 mg / mL,and the recombinant alpha toxin protein was identified as having good reactogenicity by Western blot.Extract the natural alpha toxin of C.putrefium,prepare the toxoid,immunize BALB / c mice with a dose of 30 mice MLD per mouse,measure the serum titer after three basic immunizations,select the higher titer mice for booster immunity and fusion,In vitro ELISA method was used to screen hybridoma cells by indirect ELISA method and subcloning of positive hybridoma cells,and finally obtained seven strong positive monoclonal hybridoma cell lines,which were tested,including 374,333,Five hybridoma cells 932,443 and 447 have a strong ability to recognize the natural alpha toxin of C.putrefium.After subtype identification,three strains 374,333 and 932 were IgG,and the rest were IgM or IgA.Two of the 333 monoclonal antibodies were directed against the same epitope,and neither had neutralizing activity,only the 932 strain had neutralizing activity.Therefore,932 monoclonal antibodies were prepared by ascites and purified by ammonium caprylate sulfate precipitation.The purified concentration was 3 mg / mL;the titer was up to 1: 256000 by indirect ELISA;identified by SDS-PAGE and Western blot.The purified monoclonal antibody has high purity and strong specificity with C.putrefium positive serum.Use the obtained monoclonal antibody to establish a blocking ELISA detection method,and use the square array titration experiment to determine the optimal antigen coating conditions,optimal blocking method,optimal antibody action conditions,enzyme label secondary antibody action time,chromogenic solution action time,etc.Screening and optimization were carried out separately to determine the best antigen coating condition is coating with 4?g / mL concentration at 4 ? for 12 h,and the best blocking method is 5% skim milk blocking at 37 ? for 2h,the best antibody action The conditions were as follows: the monoclonal antibody was diluted 1: 512000 and incubated at 37 ° C for 45 min.The enzyme-labeled secondary antibody was diluted 1: 15000 times and incubated at 37 ° C for 45 min.The coloring solution was at room temperature for 15 min in the dark.After determining the optimal reaction conditions,46 vaccine immune serum samples and negative serum samples stored in the laboratory were tested to determine the negative and positive cutoff values of the blocking ELISA method.In this study,a monoclonal antibody with high specificity,strong reactivity,and neutralizing activity was successfully prepared using the natural alpha toxin of C.putrefium as the immunogen.The method and its specificity and sensitivity have been verified,which can be used for the efficacy test of the inactivated sheep rapid-vaccine vaccine and the evaluation of the effect after vaccine immunization.