Preparation of Monoclonal Antibody Against gB Protein of Porcine Pseudorabies Virus and Development of a Competitive ELISA Detection Method
[Objective]This study was aimed to prepare monoclonal antibodies against Porcine pseudorabies virus(PRV)gB protein and establish a competitive ELISA for detection of PRV antibody,provide reference for antibody detection of PRV and development of kits.[Method]In this study,BALB/c mice were immunized with PRV gB protein,and PEG1500 was used to facilitate cell fusion between mice spleen cells and SP2/0 cells.Positive hybridoma cells were screened by indirect ELISA.Through two rounds of sub-cloning using the limiting dilution method,positive hybridoma cells were induced in vivo to produce ascites,and monoclonal antibodies from the ascites were purified by ammonium sulfate precipitation.Furthermore,the monoclonal antibodies were used to develop a competitive ELISA.The chessboard method was used to optimize the reaction conditions and the negative sera were tested to calculate the cutoff value.The assay was evaluated and compared with commercial kits.[Result]Two strains of hybridoma cells,named as 2D6 and 2D8 were obtained.Both were IgG1 type with κ-chain light chain.The optimal dilution of monoclonal antibody of the established competitive ELISA method was 1∶400,the optimal coating concentration of protein was 1 μg/mL,the optimal dilution of serum to be examined was 1∶4.The optimal blocking solution was 1％BSA,and the optimal dilution of enzyme-labeled secondary antibody was 1∶4 000.It exhibited good specificity with no cross-reactivity with other porcine viruses.It remained positive when positive serum diluted to 1∶256,indicating that the method had high sensitivity.The coefficients of variation for both intra-assay and inter-assay were less than 10％,demonstrating that the established competitive ELISA method showed high repeatability.Compared with the commercialized kit,the consistency between the established ELISA and commercial test kit reached 93.3％.[Conclusion]In this study,monoclonal antibodies against the PRV gB protein were successfully prepared,and a PRV gB antibody competitive ELISA detection method with high specificity and sensitivity was established.
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