Rabies Virus Antibody Enzyme-linked The Development Of Diagnostic Reagents

Kinds of rabies strains are normally used to produce rabies vaccine, such as PM, FLURY, AG, AV, CTN-1, etc. According to Literature, use one of those rabies strains to detect the immune serum, the positive rates are different. The purpose of this study was to compare the antigenic difference of different rabies vaccine strains used in domestic, optimize concentration ratio of the strains to coat EIA plate, and set up ELISA test system which can detection of antibody levels after immunization with different strains of rabies vaccine.NIH mice were intraperitoneally immunized with the he current uses of various strains of rabies vaccine in China. And we obtained all of the vaccine immune serum. Coat EIA plate with all of the vaccine antigen ,and titration each kind of the serum respectively; Then cross-react of every kind of vaccine strains and the immune serum ,and detect the amount of antigen by Sandwich ELISA before and after the reaction, using 50% inhibition rate as the end point to calculate the antigen ratio . Compare the antigen ratio between different pairs of vaccine antigen and evaluate the difference of the entire vaccine antigen. Then we use the antigen which has a much smaller difference with other strains to coat EIA plate and compare the results both in qualitative and quantitative to RFFIT test , AG: CTN-1=4∶1 group , which have a greater positive rate and a better correlation ship with RFFIT, was chosen as the coating antigen.We use high-titer anti-rabies virus antibody (CVS strains) to coat the EIA plate as capture antibody to react with the selected antigen, wash the plates and block it .Antibody against rabies virus CVS–HRP compete with the detecting antibody. And optimize the reaction conditions. Then we accomplished the capture competition ELISA. The specificity of the competition reaction, the best linear range, detection limits, precision and accuracy in a series of verification tests showed that the method has a good specificity. The best linearity of dose-response curves of the method were found in a range of 30 ~ 500m IU/ml, and quantification limit of 33mIU( r >0.99).The good precision and accuracy were obtained with the coefficient of variation(CV)of 6.68%～7.84%,and the recovery of 92%～99.75%. And comparison the kits through heat accelerate test, it shows a good stability .Parallel compare with the test results of RFFIT, through ROC curve analysis, set the cut-off value at 0.735IU/ml,which has a good specificity and sensitivity .Compare of the test results of competition ELISA with Ningbo Tianrun antibody test kits to 65 serums, the Kappa value is 0.844, which shows a good concordance. While compare to AutoBio Diagnostics kits, our kit have a greater relationship with RFFIT tests, either in qualitative agreement rate or Quantitative correlation ship .Parallel compare to RFFIT tests ,the Kappa value is 0.530;and through the regression analysis ,we got the equation ?=-0.475+3.246X;r=0.801,P<0.001.