| Clostridium perfringens also known as Clostridium welchii is a widely distributed in nature and the animal digestive tract,which belongs to conditionally pathogenic bacteria.It can produce a variety of toxins and enzymes.The main toxins include alpha,beta and epsilon as well as iota toxin.Alpha toxin is the most important toxins which various types of Clostridium perfringens can produce.Presently,the main detection methods for the antibody of Clostridium perfringens are mice neutralization test and lecithin enzyme inhibition test.But it is complex and long time-consuming,is not suitable for detection of large quantities of clinical samples.A variety of testing kits for Clostridium perfringens disease have been developed in foreign,but the price is expensive.So in order to meet domestic blank and urgent need of the testing kits,establishing a fast,sensitive,specific the kits is essential and can be used to detect the diseases in large quantities.In this study,the hybridoma cell line F12 was used to develop monoclonal antibody,which is stored in our laboratory.The monoclonal antibody resistanced the alpha toxin was obtained through a series of processes,which contain expanding cultivation,injecting the mice,collecting ascites and purifying ascites.The titer of collected ascites was determined by indirect ELISA.The used alpha toxin was secreted by the standard strain NCTC528 of Clostridium perfringens stored in the laboratory.And the concentration of the purified alpha toxin was also detected.Selecting five healthy unvaccinated rabbits of 1.0-1.5 Kg,the rabbits were immunized with emulsion mixed by inactivated alpha toxin and Freund’s complete adjuvant in equal volume,according to the normal immunization procedure.Finally,hyper-immune serum was prepared in this study.The monoclonal antibody being as envelope antibody was coated in plastic plate.And the purified alpha toxin was as capture antigen.Through Hyper-immune serum being as positive serum of capture ELISA,method of capture ELISA for detection alpha toxin antibody was established.By the optimization of reaction conditions,we finally build the best working conditions: the dilution of monoclonal antibody was 1:6400 that was 0.33μg/ml,the dilutionof tested serum was 1:160 and antigen dilution was 1:1000 that was 2μg/ml.To get positive and negative cut-off values,33 negative sera were detected by the established ELISA.The calculated average of optical density(OD)was 0.243(X=0.243)and standard deviation(SD)was 0.015(SD=0.015).The calculated positive cut-off value was 0.288(0.288=X+3SD)and negative cut-off value 0.273(0.273=X+2SD).Under two values,detected serum was determined as positive when its tested value was ≥0.288,negative when the value was ≤ 0.273 and skeptical when the value was between 0.273 and 0.288.Specificity results showed that the method did not react with serum that obtained by rabbits immunized by Escherichia,Salmonella,Riemerella.Susceptibility test indicated that the sensitivity of this method was higher,with antibody dilution to 1:640 compared with classical mouse neutralized dilution to1:400.And the method had good repeatability.Repeated experiments showed that coefficient of variation in batch was 1.21% ~ 6.53%,and the coefficient of variation between batches was1.78% ~7.31%.Both of coefficients are less than 10%,which showed that the variation was little.The results showed that the capture ELISA was successfully established,with the specificity,sensitivity and reproducibility for the detection of alpha toxin antibody.The approach provides a new way for the determination of the immune effect,and lays a foundation for the clinical diagnosis of Clostridium perfringens disease.Meanwhile,it has a good application prospect in food safety. |
PDF Full Text Request