Preparation Of Monoclonal Antibody Against Canine Distemper Virus And Establishment And Evaluation Of Sandwich ELISA

Canine Distemper?CD?was a serious viral infectious disease caused by canine distemper virus?CDV?,and have been widely transmission all over the world.Its traditional host was carnivores,Its infection in Canidae,Catidae,Raccinidae,Mustelidae,Caniformia have been report many times.Until 2013,there were 1864 wild giant panda and 375 captivity giant panda in China.From December 2014 to April 2014,6 of 22 giant pandas from Shaanxi Research Center for Rescue and Feeding of Rare Wildlife were tested RNA positive for CDV,and 5 cases died.Until now,RT-QPCR and commercialization test strip were most common used detection methods of CDV infecting panda.However,RT-QPCR was high cost,complicated technology and easy pollution.For commercialization test strip,it was designed to detect the CDV infecting dog.It is necessary to establish a sandwich ELISA assay for detection of giant panda CDV.In this study,the extracellular region of F protein of CDV based on Louguantai strain was expessed in E.coli,the monoclonal antibody against CDV-F protein was preoared,and the double antibody sandwich ELISA for detecting CDV was established.2.The prokaryotic expression and purification of CDV F protein.To produce the CDV F protein to prepare monoclonal antibody,the recombinant CDV-F protein of panda CDV was successfully expressed in E.coli and purified by using a Ni-NTA resin column as an approximately 40 Kda His-tagged fusion protein.The purified CDV-F protein was used to Immune the mice.The antibody titer,tested by i-ELISA reach10^-5,suggesting the expected and immunogenicity.3.Preparation and purification of mAbs,and labling monoclonal antibody with HRPBALB/c mice were immuned with CDV-F protein.Three mAbs,including 1A5,1A5and 7D5 were prepared,and they were purified by using protein G column.The concentration of three purified mAbs,1A5,1A5 and 7D5 were determined to be 1.2mg/mL,1.2mg/mL and 1.8mg/mL,respectively.After that,these three mAbs were labled with HRP.3.The establishment of sandwich ELISAELISA showed that these three mAbs were against different epitopes.Using 1A5 as capture antibody and HRP-7D5 as detecting antibody,P/N value of sandwich ELISA was higher than other pairs of mAbs.checkerboard titration.The optimal concentrations of capture antibody was 800 mg/wells and HRP-7D5 was dilutions at 1?100,determined by checkerboard titration method.4.The sensitivity of sandwich ELISAThe CDV was detected by sandwich ELISA and test strip to deter mine the sensitivity of the established sandwich ELISA.The results showed that the detection limit was logTCID₅₀=1.8 by both sandwich ELISA and colloidal gold strip methods,suggesting similar sensitivity of sandwich ELISA with colloidal gold strip.5.The specificity of sandwich ELISAsupernatant to determined The specificity of developed sandwich ELISA was evaluated with CPV?Canine parvovirus virus?ICHV?Canine infectious hepatitis virus??CPIV?Canine parainfluenza virus?virus.Results showed that no cross-reaction was detected by this sandwich ELISA,suggesting good specificity.6.The coincidence of the sandwich ELISA with colloidal gold stripThirty panda serum and 30 dog serum were tested using sandwich ELISA and colloidal gold strip.No panda serum sample was positive,while six of dog serum samples were positive.The coincidence of sandwich ELISA with colloidal gold strip were 100%.