Preparation Of Monoclonal Antibodies Against RV N Protein And Establishment Of Immunological Diagnostic Methods For RV

Rabies is an acute, progressive encephalomyelitis, which causes almost 100% of fatality in animals and human. The human burden to rabies is estimated in excess of 55,000 deaths every year worldwide. Despite many efforts to control rabies, the disease continues to be a major problem in dogs and cats in developing countries. Therefore, antigen detection as well as evaluation of the effectiveness after vaccination for dogs and cats is the key to preventing and controlling the disease. Based on the immunological and genetic engineering techniques, we developed indirect ELISA to detect the antibodies against RV in the serum of dogs. Additionally, we preliminarily established a Double-Antibody Sandwich ELISA to detect RV.In our present study, the complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. We then transformed the recombinant plasmids into E. coli BL21 (DE3) strain and expressed them by adding 1 mM of IPTG. SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete RV-N, the partial protein (RV-N1) was expressed in a much higher level in E. coli BL21 (DE3). The antigenic specificity of RV-N1 protein was confirmed by western blot. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2μg/mL. The optimal concentration of serum samples and SPA-HRP were 1:100 and 1:4000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results showed that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.To generate monoclonal antibodies against RV-N, the purified pET-32a-RV-N protein was used as antigens for immunization of BALB/c mice. The spleen cells of the BALB/c mice were fused with the SP2/0 cells. Five hybridoma cell lines named 1D9, 2B6, 3C5, 6B5, 5F2, respectively, were screened and obtained by indirect ELISA with pGEX-6P-1-RV-N as an antigen. The subtype of 1D9, 2B6, 3C5 is IgG1 and the other two is IgM. The ascites from MAbs 2B6 reacted with purified pGEX-6P-1-RV-N protein in ELISA at a titer of 1:106. The results of IFA indicated that these MAbs were specific to rabies virus.We prepared a specific antiserum by immunizing rabbits with the purified pET-32a-RV-N protein. The titer of the PAbs is 1:105 detected by indirect ELISA. Purified by affinity chromatography, the PAbs were labeled by biotin. BAB-ELISA technique for detecting RV was established with MAbs, biotin-rabbit-anti-RVN-PAbs and HRP-avidin. By optimizing tHis method, the optimal concentration of MAbs 2B6 coating the ELISA plate was determined to be 1μg/mL. The optimal concentration of RV, PAbs and the HRP labled avidin were 1:2, 1:4000 and 1:104 respectively. Meanwhile, this method showed highly specificity and it can be preliminarily used to detect RV.