鸡传染性法氏囊病病毒VP2单克隆抗体的制备及其阻断ELISA方法的建立OA

This study aimed to prepare monoclonal antibodies(mAbs)against IBDV VP2 protein and to establish a highly sensitive blocking ELISA for supporting IBDV vaccine development and antibody de-tection.BALB/c mice were immunized with IBDV VP2 protein.Spleen cells from immunized mice were fused with SP2/0 myeloma cells to generate hybridomas.Selected hybridoma cell lines were screened and char-acterized using Western-blot,indirect immunofluorescence assay(IFA),and neutralization test.A block-ing ELISA was optimized using VP2 as the coating antigen and mAb 2F11 as the detection antibody.The as-say conditions were optimized,the cut-off value was determined by ROC curve analysis,and its perfor-mance was compared with a commercial ELISA kit.Two stable hybridoma cell lines,1D6(IgG2a)and 2F11(IgG1),secreting specific mAbs were obtained.The mAb 2F11 demonstrated neutralizing activity.Both mAbs specifically bound to the VP2 protein and to IBDV-infected chicken embryo fibroblast(CEF)cells,as confirmed by Western-blot and IFA.The optimal conditions for the blocking ELISA were:VP2 coating concentration at 0.015 μg/mL,2F11 concentration at 0.034 μg/mL,and blocking with 2%BSA.The estab-lished assay showed a sensitivity of 95.24%and a specificity of 97.61%(AUC=0.978).The total con-cordance rate with a commercial kit for clinical sample testing reached 97.5%.VP2-specific mAbs were successfully prepared,and the 2F11-based blocking ELISA provides a sensitive and reliable platform for IBDV antibody detection and vaccine evaluation.