Development And Application Of A Double-antibody Sandwich ELSA Method For Rabies Virus

Rabies, a devastating and preventable zoonosis, poses an serious threat to humans andanimals (especial canid species). No treatment is effective after the development of clinical signs,but pre-and post-exposure prophylaxis is necessary for prevention of rabies cases. Currently,inactivated vaccines are the most commonly used anti-rabies biologic for both humans anddomestic animals. In China, in the process of veterinary inactivated vaccine production, the titersof the cultured rabies virus are usually assayed by50%tissue culture infective dose (TCID50) withsome disadvantages such as an observation period of2-3days and biosafety.Enzyme-linked immunosorbent assay (ELISA) using a short time and low cost, especialsandwich ELISA, is a popular method for titration of antigen.Based on above problems, our studies are as follows:1. Development of the sandwich ELISA methodA sandwich ELISA for semi-quantification of rabies virus was developed in this study, usinganti-rabies sera as coated antibody, rabies virus strain SRV9assayed by TCID50method asstandards and HRP-labeled monoclonal antibody (McAb) against rabies viral nucleoprotein asindicator. The McAb was purified using Sepharose4B affinity chromatography and labeled withHRP by Guo’s method. The coating sera were prepared by mice immunization with inactivatedrabies virus. Standard curve was obtained by the OD (x-axis) and TCID50(y-axis) values of thecontrol samples. The optimizing the reaction conditions, sensitivity, specificity, fidelity andrepeatability of the ELISA method were determined.2. Application of the sandwich ELISA methodTo assess accuracy of the method, the virus titers of either half-finished or finished rabiesinactivated vaccine were assayed by the sandwich ELISA, and compared with the resultsdetermined by TCID50and NIH testThe results are as follows:The sandwich ELISA was established with a working range from1.0×106TCID50/ml to1.0×109TCID50/ml. The sensitivity of the ELISA described here was proved by assaying different rabiesvirus strains, and the results were all positive. The specificity was determined by assaying caninedistemper virus, canine parvovirus and canine adenovirus and the results are all negative. In the repeating test, the intra-assay and inter-assay standard deviation were respectively5.9%and11.7%.The results indicated that the semi-quantitation sandwich ELISA, with a detection range from106to109TCID50/ml, was established in this study, presenting high sensitivity and specificity dueto the usage of monoclonal antibody against rabies viral N protein. The results obtained by theELISA show a parallel relationship with those of TCID50assay and NIH test, which indicated thatit could be used for semi-quantitation of viral antigen in the half-finished products.In conclusion, the semi-quantitative sandwich ELISA for rabies virus is suitable for themeasurement of viral antigen in the process of inactivated vaccine.