| Rabies is an important zoonosis caused by rabies virus.It is the most acute infectious disease for its highest mortality so far.Once people and animals were unvaccinated,the highest mortality rate was almost 100%.Wild animal is a major reservoir of rabies virus.Dog play a key role in the course of human rabies distribution.For the past few years this disease was present an ascendant tendency year by year in our country.Because of the weakness of animal rabies prevention,human rabies has become seriously prevalent in our country and become an important public healthy problem now.As the requirement of animals international trade was increased,and to control the serious rabies epidemic situation day by day.Between country and country also need to take a severe quarantina and supervision to the dogs,cats and such animals which entry and exit.And animals must inoculate to the rabies vaccine.If the animals want to entry and exit,the neutralizing antibody titer must higher than 0.5 IU/ml.So WHO and OIE take the 0.5 IU/ml as the minimum standard of neutralizing antibody in immunizing serum.But because of the vaccine category,quality,technology,adjuvant and immunization times,immunization frequency was different in every,country.Although animals had immunized,the neutralizing antibody of them have discrepancy.So we need to construct a more quickly method to detect the neutralizing antibody level of immune animal.Due to rabies neutralization test is not only tedious and need much time,but also need a higher condition,so it is not suitable for quickly detection at the entry and exit port.We need to construct a quick,sensitive and characteristic method to instead of the rabies neutralization test.And even adapt to the inspection and quarantina of entry and exit.At this time,Enzyme linked immunosorbent assay was acquired an extensive application in animal plague inspection and quarantina of entry and exit.As this method has its advantage,such as sensitivity,characteristics and quickly.In domestic and overseas also have some literature report that enzyme linked immunosorbent assay can inspect the rabies virus antibody.But research of inspection method of rabies virus neutralizing antibody were no report.This research used purified rabies virus glycoprotein as the coating antigen.Through optimized the reaction condition to construct indirect enzyme linked immunosorbent assay. The standard procedure of indirect-enzyme linked immunosorbent assay test include the following steps, the concentration of coating antigen were 0.3μg/ml,by incubation at 37℃,2h and then in 4℃to stay overnight, the first antibody of dog serum was diluted 40 time.HRP-IgG of dog was diluted 1:2000.The best confining liquid is 1%ovalbumin and the blocking condition is 37℃,2h.Sample diluent and enzyme labelled antibody diluent were 1%ovalbumin.All the optimized incubated time of sample,the second antibody are 1h,the substrat solution is 10min.The values of P/N over 0.252 were considered as positive.Meanwhile,used fluorescent antibody virus neutralization test(FAVN) of OIE appointed to take a parallel and contrast test with ELISA.Use this two method to inspected different period dog serum of 376 which immunized.Then analyze the result.There were 299 positive and 77 negative by FAVN inspection.But there were 282 positive and 94 negative by indirect-ELISA inspection.Specificity of indirect-ELISA is 100%,sensitivity of indirect-ELISA is 94.31%.And the result concidence of this two method is 95.48%. Analysis by statistics method,the disparation of this two method is quiet.So we can confirm the advantage and practical value of the indirect-ELISA further.And also to establish the foundation for solving the difficult problem which perplexed the inspection of neutralizing antibody at the entry and exit port. |
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