Development Of An ELISA Antibody Detection Kit For Bovine Herpes Virus Type 1 GE Protein Blockade

Bovine herpesvirus 1(BoHV-1),also known as Infectious bovine rhinotracheitis virus(IBRV),is a DNA virus that can cause IBR in cattle.Generally,Bovine herpesvirus 1(BoHV-1)causes respiratory or reproductive tract infections,reduces beef cattle feed efficiency,lowers milk production in dairy cattle,miscarriages,infertility,endometritis in cows and significantly reducing production efficiency in cattle farms.Although the virus has a low mortality rate,its ability to remain latent makes it difficult to be eradicated,resulting in significant economic losses to the cattle industry.Therefore,purifying infectious bovine rhinotracheitis is of great significance for the improvement of Chinese cattle industry.The development of ELISA antibody detection kit for BoHV-1 g E protein,aided by the use of g E gene deletion marker vaccine,provides an effective solution for the prevention and control of bovine infectious rhinotracheitis.Currently,the BoHV-1 g E gene-deleted attenuated vaccine has been widely used abroad,and g E antibody detection kit has been developed,but the import cost is very expensive,which is not convenient for China to purify the disease in China.Based on the previous research on the BoHV-1g E~-/TK~-gene-deleted strain in our laboratory,this study purified BoHV-1 virus particles by sucrose density gradient centrifugation,which in turn was used as immunogens to get mouse-derived BoHV-1 g E protein monoclonal antibodies using an indirect ELISA screening method.Using BoHV-1 virion as a coated antigen,the BoHV-1 g E protein blocking ELISA method was established,and the assembly of the BoHV-1 g E protein blocking ELISA antibody assay kit was completed.The specific contents are as follows:1.Preparation and identification of BoHV-1 g E protein monoclonal antibodiesFirst,BoHV-1 virus particles were extracted and purified using sucrose density gradient centrifugation,and the purified virus particles were used as immunogens to immunize Balb/c mice with a rapid immunoadjuvant.After immunization,mice with the highest serum titer were selected,and their spleen cells were fused with myeloma cell.ELISA plates were coated with purified inactivated BoHV-1 virus particles and BoHV-1g E-/TK-virus particles,then positive hybridoma cells expressing g E protein antibodies were screened by indirect ELISA.Subsequently,three rounds of subcloning by limited dilution were performed,and seven hybridoma cell lines that could stably secrete g E protein monoclonal antibodies were obtained and named 1A1,1F5,1A7,1D7,1D2,2A4,and 2D7.Their biological characteristics were identified by additional experiments,indirect immunofluorescence,and Western blot,and the results showed that all seven obtained monoclonal antibodies had good specificity for g E protein.2.Establishment of BoHV-1 g E protein-blocking ELISA antibody detection methodPurified BoHV-1 virus particles by sucrose density gradient centrifugation were used as coating antigen,and g E protein monoclonal antibody labeled with horseradish peroxidase was used to establish a BoHV-1 g E protein-blocking ELISA antibody detection method.Through evaluation of the conditions for the blocking ELISA antibody detection method,the coating antigen concentration was determined to be 0.5μg/m L,and the dilution of the labeled monoclonal antibody was 1:15000.The suitable conditions for coating,blocking,and reaction steps were also determined.By drawing the ROC curve and analyzing the detection results of 120 clinical serum samples with known background,the criteria for determining the ELISA antibody detection results were established.The effectiveness of the developed BoHV-1 g E protein-blocking ELISA antibody detection kit was validated through sensitivity,specificity,repeatability,shelf-life,and compliance tests.The results showed that the kit had good sensitivity and specificity,high repeatability and compliance,and a long shelf-life.The kit was used to test 309 clinical serum samples originated from Tibet,Qinghai and other regions.3.Assembly of BoHV-1 g E protein-blocking ELISA antibody detection kitAfter establishing and optimizing the BoHV-1 g E protein-blocking ELISA antibody detection method,the antibody detection kit was assembled.The components of the kit included 2 antigen-coated plates,10μL labeled reagent,500μL positive control,500μL negative control,10 m L 10-fold concentrated diluent,50 m L 20-fold concentrated washing solution,25 m L substrate color solution,15 m L stop solution,2 serum dilution plates and1 instruction manual.The instruction manual clearly indicates the name,components,purpose,usage,specifications,precautions,and shelf-life of the kit.In conclusion,this study prepared g E protein,established and optimized the method for antibody detection of BoHV-1 g E protein-blocking ELISA,and completed the preliminary assembly of the kit.