Preparation Of Monoclonal Antibody Against Bovine Infectious Rhinotracheitis Virus And Establishment Of Blocking ELISA

Bovine Infectious Rhinotracheitis Virus(IBRV)is an important viral pathogen causing bovine respiratory diseases.After infection,it mainly manifests clinical symptoms such as salivation,tearing,coughing,difficulty in breathing and elevated body temperature.In addition to causing respiratory inflammation,IBRV infection can also cause conjunctivitis,mastitis,pustular vulvovaginitis or glans foreskin,juvenile meningoencephalitis and abortion.In addition,IBRV can also cause immunosuppression of the body,which in turn causes bacterial or mycoplasma infections,leading to bovine respiratory disease complex(BRDC),which causes a large economic loss to the cattle industry.The serological epidemiological survey showed that the positive rate of IBRV in China was 46.03%(3933/8545).The samples investigated were all bovine serum with unimmunized IBRV vaccine.The high seroprevalence rate indicated that IBRV infection was very common in Chinese herds.At present,there is no diagnostic kit for commercial IBRV in China,and it is necessary to carry out research on related diagnostic techniques.In this study,IBRV Bartha nu/67 strain was purified by sucrose density gradient ultracentrifugation.The purified IBRV was inactivated and emulsified to immunize 6~8 weeks old BALB/c mice,The spleens of the mice were fused with sp2/0 cells after immunization,and 4 monoclonal antibodies were screened by indirect ELISA,named cp-1-1,cp-6-5,cp-8-1 and cp-8-2.The subclass identified 4 monoclonal antibodies with heavy chain of IgG1 and light chain of kappa.Ascites was prepared and the ascites titers were determined to be 10~4,10~4,10~5,and 10~4,respectively.By indirect ELISA,indirect immunofluorescence(IFA)and western blot,4 monoclonal antibodies were positively reacted with IBRV,showing good reactivity;IFA showed that 4 monoclonal antibodies only reacted positively with IBRV,It does not react with bovine viral diarrhea virus(BVDV)and bovine parainfluenza virus type 3(BPIV3)and shows good specificity.The relative affinity constants of the four monoclonal antibodies were determined by thiocyanate elution method to be 2.5 mol/L,3 mol/L,1.5 mol/L and 5 mol/L,respectively.Mass spectrometry and prokaryotic expression assays demonstrated that all 4 monoclonal antibodies were directed against IBRV VP8 protein.In this study,IBRV purified by sucrose density gradient centrifugation was used as a coating antigen,and monoclonal antibody cp-1-1 was used as a detection antibody to establish a blocking ELISA for detecting IBRV serum antibodies.50 IBRV antibodies were weakly positive(neutralizing antibody titer1:4~1:16)bovine serum as standard reference serum,the blocking rate of 52.06%was determined as the cut-off value of the method,When the blocking rate was higher than 52.06%,it was positive,and when it was lower than 52.06%,it was negative.Specificity test showed that only IBRV positive serum had a good blocking effect,while bovine viral diarrhea virus,bovine parainfluenza virus type 3,bovine adenovirus type 3 and bovine foot and mouth disease virus type O positive bovine serum all had poor blocking effect,indicating This method has good specificity.The diagnostic method can detect that the antibody induced by IBRV inactivated vaccine is the earliest time after the first immunization;in addition,the minimum neutralizing antibody titer detectable by this method is 1:4,which is basically consistent with the sensitivity of the neutralization test.Repeatability tests showed that the intra-and inter-assay coefficients of variation of the method were less than 10%,showing good repeatability.The coincidence rate test showed that the coincidence rate of the method with the neutralization experiment was 98.46%,and the coincidence rate with the IDEXX gB antibody diagnostic kit was 99.23%.The sera of immunization with IBRV inactivated vaccine were monitored by this method.The antibody positive rate was 99.51%(205/206),and 801 bovine serums were monitored in 8 provinces(municipalities and autonomous regions)in China.IBRV The positive rate was 41.57%(333/801).In summary,four monoclonal antibodies specific for IBRV VP8 protein were screened in this study,which laid a foundation for studying the structure and function of VP8 protein.A block ELISA method for the detection of IBRV bovine serum antibody was established with one of the monoclonal antibodies cp-1-1.The diagnostic method can be used for IBRV vaccine immunological monitoring and seroepidemiological investigation,provide technical support for the prevention and control of IBR in China.