Preparation And Preliminary Application Of Fluorescent Antibody To Rabies Virus

Rabies, caused by rabies virus(RV)is an acute and fatal zoonotic diseases. The fatality rate is almost 100%. All warm-blooded animals are susceptible to infection by the virus. It is considered to be a serious public health problem. The control and eradication of rabies depends on the development of safe, effective and economical vaccine that might be used in preexposure vaccination. However, checking virus dilution or effectiveness of rabies vaccines depends on the rapid,accurate and safe diagnostic method. Monoclonal antibody against rabies virus nucleoprotein was developed by means of the conventional protocol. The IgG was purified by affinity chromatography and conjugated with FITC. Virus titer and immunogenicity of rabies vaccines were detected by direct fluorescent antibody test (dFAT) and fluorescent antibody virus neutralization test (FAVN).After immunization of BALB/c mice with purified rabies virus(RV), three hybridoma cell strain against RV named C7,C4 and H3, respectively, were developed after fusion between SP2/0 myeloma cell and the stimulated splenocytes. The indirect ELISA results showed that the McAbs ascites titer were 1:1×106,1:5×105and 1:1×105; No cross reaction was found when McAbs reacted with CDV, CPV and CAV; Identification of subclass showed that C7 belonged to IgG2b, C4 and H3 belonged to IgG1 respectively; The result of western-blotting with purified RV is positive, which indicates thesemonoclonal antibodies all aimed directly at rabies virus nucleoprotein.The three McAbs were purified by protein G and labelled with FITC. The results of absorption test and blocking test showed that the fluorescent antibody (FA) has highly specificity and sensitivity , and has no interaction with CPV, CDV and CCV. Its optimal working concentration was 1:128.The FA to rabies nucleoprotein that was characterized correctly was used to detect virus titers of Rabies Vaccine with FAT and 30 serum samples from dogs with FAVN assay. The result of fluorescein stain showed that the TCID50 of RV ERA strain had been increased from 10 - 5.64 , 50μL before rejuvenescence to 10-8.16, 50μL after rejuvenescence; ED50 of the 30 examined serum samples were all > ED50 of the reference serum, then the titre was > 0.5 IU/mL, that is to say the titer of the antibody to RV had reached a protective level.FA diagnostic method that was established provided a convenient, fast and reliable technic to the antigen detection and epidemiologic survey of RV as well as immunologic surveillance and the detection of vaccine effectiveness. It has laid a foundation for prevention and cure of RV.