Establishment And Application Of An Indirect ELISA For Detection Of Serum Antibodies Against Bovine Parainfluenza Virus Type 3

The genome of Bovine parainfluenza virus type 3(BPIV3)is a negative-strand RNA,the member of the respiratory virus of the Paramyxoviridae family,which is one of the major pathogens causing bovine respiratory disease complex.The bovine respiratory disease was caused by this virus after long-distance transportation,so BPIV3 is also called Shipping fever virus.The gene type C of BPIV3 was detected in China in 2008 for the first time,and then a few related research were carried out.At present,the detection method of BPIV3 serum antibodies in China is mainly virus neutralization test.This method is not suitable for large-scale detection of serum samples,and ELISA is a common method for detecting serum antibodies for animal infectious diseases.Therefore,this study developed the indirect ELISA methods for the detection of BPIV3 serum antibodies,so as to provide technical support for the prevention and control of BPIV3 in China.The nucleocapsid protein(NP)of BPIV3 is a structural protein with high content in virions,which can induce host to produce corresponding antibodies.In this respect,this study used prokaryotic expression system to express the nucleocapsid protein of BPIV3.The NP protein is expressed in partially soluble form by optimizing the induction of expression conditions.The recombinant protein NP was purified using a Ni column under non-denaturing conditions.The concentration of coated NP protein was determined to be 0.8 ?g/mL by square matrix titration,and the optimal dilution of serum sample was 1:200 times.Then the reaction conditions of ELISA were optimized to develop an indirect ELISA method for detecting BPIV3 serum antibody.Specific experiments showed that the recombinant protein NP did not cross-react with bovine infectious rhinotracheitis virus and bovine viral diarrhea virus positive sera.Repeatability experiments show that the method has good stability.BPIV3-positive bovine serum was still positive when it was diluted to 1:640,which showed a high sensitivity.The comparative test of 94 bovine serum samples showed that the coincidence rate of the method developed in this study with the virus neutralization test was 98%.At the same time,an indirect ELISA method for detecting BPIV3 serum antibody was also developed by using lysed BPIV3 virion as coating antigen.Firstly,BPIV3 was purified by sucrose density gradient centrifugation.The virus protein concentration was determined then the virus was fully lysed.The concentration of the coated antigen proteins was determined to be 0.2 ?g/mL and the optimal dilution of the serum was 1:50 times.Then the reaction conditions of the ELISA were further optimized.Specific experiments showed that the coated lysed BPIV3 did not cross-react with bovine infectious rhinotracheitis virus and bovine viral diarrhea virus positive sera.Repeatability experiments show that the method also has good stability.BPIV3-positive bovine serum was still positive at dilution to 1:1 280,and this method showed higher sensitivity.The comparative test of 94 bovine serum samples showed that the coincidence rate of the indirect ELISA method for lysing BPIV3 as the coating antigen with the virus neutralization test was 98.9%.A total of 394 bovine serum samples collected from cattle inoculated with BPIV3 inactivated vaccine were detected by the above two indirect ELISA methods.The results showed that the bovine sera detected by both methods were strongly positive.In addition,95 bovine serum samples collected from cattle not inoculated with BPIV3 vaccine were detected by the indirect ELISA using recombinant protein NP as coating antigen,with a positive rate of 80%.Subsequently,591 bovine serum samples collected from cattle not vaccinated with BPIV3 vaccine were detected by an indirect ELISA method using a split virus as coating antigen,and the positive rate was 91%.Comparing the results of the above two indirect ELISA methods with the virus neutralization test,the indirect ELISA method using the lysed virus as the coating antigen has a higher coincidence rate with the virus neutralization test,and is more suitable for the detection of BPIV3 serum antibody.The study provides technical support for the sero-epidemiological investigation and immunoassay of BPIV3 vaccine.