Preparation Of The Monoclonal Antibodies Against Canine Parainfluenza Virus And Establishment Of A Competitive ELISA For The Antibody Detection

Canine Parainfluenza Virus(CPIV),first isolated from dogs with respiratory tract infection, is one of several pathogens that cause kennel-cough. The main clinical symptoms of includ nose running, fever and cough. CPIV infection of dogs lead to death with bacteria and viruses. Serological survey shows that CPIV epidemics were common around the world. The survey data of epidemiological show a high positive rate of CPIV in serum samples of dogs and other wild animals. The disease hinders the healthy development of dog cultural industry, requires an accurate and convenient diagnostic method for diagnosis.In this study, NP gene of CPIV was cloned into the prokaryotic expression plasmid p ET-30 a vector and confirmed by gene sequencing and double enzyme digestion. After transforming the recombinant plasmids into BL21/DE3 competent cells, recombinant protein NP was expressed by inducing with IPTG. SDS-PAGE and Western blot showed that the purified recombinant protein NP can reacted with dog anti-CPIV serum specifically, and the molecular weight of recombinant protein was 64 ku as predicted.Purified NP recombinant proteins was used as antigen to immune BALB/c mice, then spleen cells from immunized BALB/c mice were fused with the SP2/0-Ag14 myeloma cells. The indirect ELISA with the purified CPIV as coating antigen was used toscreen the hybridoma cell culture supernatant.. All together,Three positive hybridomas were named as 2F1、3B9、4D11. Western bolt and indirect immimofluoresence assay showed all the three MAbs were positively reacted with CPIV. The ELISA titers of ascite of 4D11 can reach to 1:104.The competitive ELISA was established with the CPIV as coating antigen and monoclonal antibody 4D11 as detection antibody, and their optimal reactive conditions were determined. Detecting CPIV positive serum with competitive ELISA detection method, according to the positive statistical method: over 15.1% of inhibition rate was the positive judging standard. The ELISA method was only able to detect antibody against CPIV specifically, and no cross-reaction was shown with positive sera of canine parvovirus and canine distemper virus. The coefficients of variation for inter-assay was 1.30%~5.29% and intra-assay was 1.18%~8.55%. Moreover, 153 clinical sera samples were tested by indirect ELISA and hemagglutination inhibition. The coincidence rate of two methods was 92.16%.In summary, the highly sensitive and specifically competitive ELISA was established for detection of CPIV antibody with the CPIV as coating antigen. This method can be easily used for detection of a large number of clinical samples. The establishment of competitive ELISA lays the foundation for the rapid diagnosis, epidemiological investigation and antibody detection.