Preparation Of Monoclonal Antibody Against Caprine Parainfluenza Virus Type 3 And Establishment Of A Blocking ELISA Detection Method

Caprine parainfluenza virus type 3(CPIV3),a member of the genus Respirovirus in the family Paramyxoviridae as HPIV3 and BPIV3,is an enveloped virus with a non-segmented negative-sense RNA genome.A new PIV3 was first isolated in 2013 while the emerging disease with respiratory symptoms in some areas of Anhui and Jiangsu province.The serological detection method was desired for epidemiological investigation in goat herds.In this study,6~8 weeks-old Balb/c mice were immunized with purified CPIV3 with ultracentrifugation.After three normal and one enhanced immunization,the spleen cells of immunized mice were fused with the myeloma cells SP2/0.Two hybridomas named MAb2E6 and 4A4 were obtained after screening by indirect ELISA three times.The antibody titers of supernatant and ascites of these two MAbs were 29 and 2.56×105,29 and 1.28×105.The MAb2E6 and 4A4 showed highly specificity in Western Blot and IFA assy,indicated these two MAbs could react with CPIV3 specially.These two MAbs were used to detect positive serums by normal blocking ELISA method,the results showed that positive rate of MAb2E6 was 34/35 while the MAb4A4 was only 19/35.That indicated MAb2E6 provided a basic in building CPIV3 blocking ELISA method.The optimum conditions of blocking ELISA were confirmed by square titration test.A total 162 goat serums which had been confirmed by neutralization test were used as the reference serums.The cut-off point of blocking ELISA was ultimately confirmed as 35.45% by ROC curve method.BDV positive serum and PPRV positive serum were detected as negative by the blocking ELISA,which indicated that this method had a good specificity;The result of sensitivity test indicated this method could show a 100% positive detection when the serums had a neutralizing antibody titer more than 1:16;The CV of repeatability assay were lower than 10% indicated the blocking ELISA had a good repeatability.The consistency rate between blocking ELISA and virus neutralization test was 98% which was higher than the rate of 93%in the HI assay.It indicated that this ELISA method had a better consistency with VNT than HI assay.All of the serums collected during CPIV3 challenge experiment in goat were detected by blocking ELISA method,the result showed a disciplinary curve which presented antibody titers of diffrent days after challenging.These results indicated the blocking ELISA method had a highly specificity and sensitivity,corresponding with VNT,it will be replace VNT methord in detecting antibody titers of CPIV3 serums and make more contributions to monitoring and prevention of CPIV3 in the future.