Development And Application Of Blocking ELISA For Detection Of Neutralizing Antibodies Against Infectious Bovine Rhinotracheitis Virus

Infectious rhinotracheitis(IBR)is an infectious and latent infectious disease caused by IBRV.In recent years,the prevalence of IBRV serum antibody in China has increased year by year,and the individual positive rate has reached 46%.Diagnosis and vaccination are the main methods of prevention and control of IBRV.The current commercial diagnostic kits can not be used to evaluate the immune effect of vaccine.Therefore,there is an urgent need to establish a diagnostic method that can be used to evaluate the immune efficacy of IBRV vaccine.First of all,the sequence of IBRV gD gene was optimized based on the codon preference of baculovirus expression system and cloned it into pfast BacTM 1 to construct the recombinant transfer vector gD-pFast BacTM 1.The recombinant Bacmid-gD was prepared by transforming gD-pFast BacTM 1 into DH10BAC sensitive cells.The recombinant baculovirus rBac-gD was obtained by transferring the recombinant baculovirus into SF9 cells.The results of IFA showed that the anti-6× His tag antibody could specifically bind to SF9 of insect cells infected with recombinant baculovirus.the Western blot analysis showed that the bovine IBRV positive serum could specifically react with the recombinant gD protein,which indicated that the prepared protein had good reactivity and could be used as a detection antigen for the establishment of diagnosis method of IBRV.To prepare the monoclonal antibodies against gD protein of infectious bovine rhinotracheities virus(IBRV),Balb/C mice were immunized with purified IBRV.The hybridoma cell lines were screened by indirect ELISA coated with purified IBRV gD protein that was expressed from insect cells sf9.Three hybridoma cell lines secreting specific mAb against gD were obtained,designated 1B4,3E8 and 4F12.The sandwich ELISA for Ig subclass differentiated 1B4 and 3E8 mAbs as IgG2α,and 4F12 as IgG1.Western blot analysis and IFA assay revealed that three monoclonal antibodies could react with IBRV specifically.Micro-neutralization test demonstrated that 1B4 and 3E8 possessed neutralizing abilities to IBRV with titres of 1:320 individually.The blocking activity of McAb was analyzed by ELISA and the McAb 3E8 was selected as the detection antibody of blocking ELISA.The gD protein and the monoclonal antibody against IBRV gD was used to established a blocking ELISA for detecting neutralizing antibody of IBRV by optimizing the reaction conditions.There was no cross reaction between this method and B VDV,Brucella bovis and Paratuberculosis bovis,which indicated that this method had good specificity;the sensitivity test showed that the sensitivity of this method was lower than IDEXX IBRV gE antibody detection kit,but higher than neutralization test.The coefficient of variation within and between batches was less than 10%,which indicated that this method had good repeatability.The results of 15 clinical serum samples showed that the total coincidence rate between this method and IDEXX IBRV gE antibody detection kit was 93.85%(671/715),and the total coincidence rate with neutralization test was 99.58%(712/715).The blocking ELISA was used to detect 2352 bovine serum samples from 11 northern dairy farming provinces in China,including Beijing,Ningxia and Inner Mongolia.The results showed that 36 cattle farms surveyed were all positive for IBRV serum antibody,the average positive rate of individual was 50.08%(1178/2352).There were different degrees of IBRV infection in each province,among them,the lowest antibody positive rate was 12.5%(23/184)in Shandong Province and 97.14%(34/35)in Jilin Province.By monitoring the level of neutralizing antibody in the serum of immunized cattle,we established a functional relationship between the S/N value of blocking ELISA and the log2 value of neutralizing titer in the serum:y=9.3161e-2.335x(R2=0.9795),and determined that when the S/N value is not higher than 0.68,the vaccine can protect the cattle.In conclusion,this study established a blocking ELISA which can detect neutralizing antibody of IBRV by using IBRV gD protein as antigen and anti gD McAb with neutralizing activity as antibody.This method has the advantages of strong specificity,high sensitivity,strong sensitivity,good repeatability and high coincidence rate,which can be used to evaluate the immune effect of IBRV vaccine.It provides a good technical support for the prevention and control of IBRV in China.