Establishment And Application Of Rabies Virus Fluorescent Antibody Virus Neutralization Test

Rabies is a kind of important zoonosis, and goes up year by year. Because lack of reasonable inoculated measures, rare species of vaccine and the low vaccinated effects. It is essencial to find a kind of effective, precise and suitable method to detect the neutralization antibody of serum in dogs. Meanwhile, to direct post exposure for precaution is the mainly reason. In China, the number of human rabies is on the rise in recent years. People and animals is usually infected rabies virus through a bite or a scratch from a rabid animal, so the prophylaxis of animal rabies is essential to the inhibition of transmitting to human. On the one hand, animals should be immunized with suited vaccine that is efficient to induce enough protective immunity. On the other hand, the titer of rabies-neutralizing antibody in animals should be assayed exactly in order to direct the inoculation and evaluate the efficacy of vaccine. Therefore, a new-type test is developed for the quantitation of rabies-neutralising antibody.The congress methods in detecting rabies virus including Mus Neutralization array (MNT), Rapid fluorescent focus inhibition test (RFFIT), Fluorescent antibody virus neutralization test (FAVN). The MNT is complex operation, long periodicity of assessment and not suitable for rapidly detection. The RFFIT is simple in operation, but is effect on subjective agent and needs the good experiences in detection and to apply on detecting antibody in clinical immunity of humans. FAVN based on 100 TCID50 rabies virus inhibited as assessment standard. The method is reliability and preferred way. Meanwhile, The specificity and sensitivity of FAVN is not significant with MNT and RFFIT. In my research, we have constructed a rapidly detecting way to determine neutralization antibody in serum on dogs and apply it on the clinical practice.Results of monoclonal antibody against rabies virus nucleoprotein was successfully produced by hybridoma 9G3. Monoclonal antibody against rabies virus nucleoprotein was developed by means of the conventional protocol in our lab. The subtype is IgG2a and prepare the abdominal dropsy. The IgG was purified by affinity chromatography and conjugated with FITC.The CVS-11 rabies virus was passaged and yielded on Baby Hamster Kidney (BHK-21)cells.The median tissue culture infectious dose (TCID50) was measured by direct immunofluorescent assay.The hyperimmune anti-rabies serum was prepared by immunizing dogs with commercial vaccine and monitored with the standard serum provided by WHO and OIE.A fluorescent antibody virus neutralization (FAVN) assay was established by referring to the protocol of WHO and OIE.51 serum samples from human and dogs were detected parallelly in this laboratory and the W eybridge Rabies Referrence Laboratory in UK.A simple software was programmed on the basis of Microsoft Excel and K?ber formula.In this trial, the sera of 500 dogs were collected from cities and villages in Guangdong, Hebei and Beijing, and assayed by FAVN for the presence and the level of neutralizing antibody against rabies. The results showed that the average neutralizing antibody level is significantly different in different districts of provinces or cities. Averagely, the rabies neutralizing antibody level in dogs from cities is higher than those from villages. The positive rate of neutralizing antibody is 46.3%. While the level of neutralizing antibody in village dogs is very low and the immunity coverage rate is 1.8%. However, the sera neutralization on dogs is lower the 0.5IU/mL regardless of city and village. From the previously datas, that reveals the immunizing level is not optimistic and especially in village.In addition, we apply on the FAVN method to detect the vaccinated serum of dog that inoculated with the recombinant live vector vaccine of CAV-2-G. In order to quantitative assessment on recombinant vaccine. Five hundreds 3-month-old unvaccinated dogs were split into two groups, 250 in each group. The dog sera were collected weekly before and after vaccination. Western blot analysis was performed to test the specific antibody. The result showed the neutralization antibody level arrive to 4.5±0.33IU, the antibody seroconversion is 87.5%, and lasted at least 54 weeks. The growth and decline regularity of antibody is not significant different both i.m. and orally vaccine group besides the difference of the induced immunoreactive time.To sum up the previously related, we sucessfully developed the FAVN method in our laboratory, and apply it on the clinical practice. Meanwhile, to identify the practicability and reliability and be not different with the international standard. It is active effect in preventing the rabies in our country.