Establishment And Application Of Clostridium Perfringens NetB Toxin Blocking ELISA

Clostridium perfringens(CP) is an important zoonotic disease pathogen, which is widely distributed in nature. It could lead to a variety of animal disease, such as necrotic enteritis, intestinal toxemia, human gas gangrene and food poisoning. So far, it was found that these bacteria could produce 15 kinds of toxins. The first identification of necrotic enteritis B-like(NetB) toxin was produced in a bacterium isolate from chicken suffered from type A CP infection. This kind of toxin was reported to be critical for the development of the necrotic enteritis in chickens. Studying the biological functions and the diagnostic method of the NetB toxin has important significance for the comprehensive control and prevention of CP infection.In this paper, the purified and renatured recombinant expressed NetB protein was used to immunize rabbit and the polyclonal antibody was prepared. The ELISA titer of the polyclonal antibody was approximately 1:32 768. Western-blot analysis revealed that the antiserum had good reactivity with the recombinant expressed protein. After recovering and analyzing the biological activity of four hybridoma cell lines kept in the lab, F9G3 strain was selected to inject BALB/c mice intraperitoneally to prepare ascites of monoclonal antibodies(Mc Abs) against NetB toxin.The purified recombinant NetB protein was used as coating antigen, polyclonal antibody as binding antibody, and McAb F9G3 as detecting antibody to establish blocking ELISA. The results showed that the optimal antigen coating concentration was 0.5 μg/100μL, coated at 4℃ overnight; The optimal blocking solution was 1%BSA, incubated for 2 h; The optimal incubation time of sample was 90 min; The optimal working concentration of F9G3 McAb was 0.0625 μg/100μL, the reaction time of sample was 60 min; The optimal dilution of HRP-labeled goat-anti-mouse IgG was 1: 5000, the reaction time was 60 min; The optimal reaction time of substate was 10 min. Statistically analyzing the blocking ELISA test results with 90 negative serum samples of CP, It was concluded that the sample was judged positive as the blocking rate PI≥31.88%, PI≤22.08% for negative, and 22.08% <PI <31.88% for suspicious. The results of repeatability test and specificity test showed that the blocking ELISA method had good reproducibility and specificity, and has no cross reactions with other reference viral and bacterial positive serum. It was confirmed that the blocking ELISA was reliable and accurate after it was use to assay the dynamic changes of the serum antibody titers of chickens immunized with recombinant NetB expression protein. For the clinical samples, it was found 12 positive samples in total of 342 samples and this demonstrated that there appeared the infection of NetB toxin-producing CP in chicken farm of Heilongjiang Province...