The Optimizationof Indirect ELISA Method Of Rabies Virus Neutralizing Antibody

In China, the mortality caused by rabies is leaping into the front ranks of 37 statutory infections. Most of these cases were associated with the bites of dogs. So the prevention of dogs from rabies virus is the key way to inhibit them transmission to human. Up to date, the general way to quantify the ability of rabies virus defense of dogs is to measure the neutralizing antibody level in blood.In this research, we aimed to complete an ELISA kit that could exactly and conveniently detect the neutralizing antibody level of rabies virus. Firstly, select the best engineering bacteria that could express the advanced envelope antigen RV G3, and study the best way of inducing expression. Then get the RV G3 protein by purification which would be used for the ELISA to detect neutralizing antibody level of rabies virus.The results showed that, RV G3 was diluted to 163?g/mLin 0.1M carbonate/bicarbonate buffer?p H 9.6? and used to coat the wells of a Costar plate for 1.5h at 37?. After blocking by non-protein blocking fluid for 1h at room temperature, the serum samples diluted 1:10 in PBS/0.5% skim milk were added to the wells and the plates were incubated for 1.5h at 37?. Then peroxidase-labeled Ig G was diluted 1:1000 in PBS and added to the wells and the plates were incubated for 1h at 37 ?. After the finally wash, color reaction with 3,3',5,5'-Tetramethylbenzidine was lasted 15 min and stopped with 2M H₂SO₄.Repeated experiments showed that all the variable coefficients were below 10%. 22 serum samples of dogs were detected by this method and its computation compared with commercialization ELISA kit and FAVN method shared the same trends.In this research, we optimized the previous method and established the indirect ELISA method to quantify neutralizing antibody level of rabies virus, which contributes to the fast and general detection of neutralizing antibody level of rabies virus.