小鼠细小病毒单克隆抗体的制备及双抗体夹心ELISA检测方法的建立OA

To prepare monoclonal antibodies(McAbs)against minute virus of mice(MVM)VP2 protein and establish an MVM antigen detection method based on double-antibody sandwich ELISA,the prokaryotic expression system was employed to express the VP2 protein.The purified protein was used to immunize female BALB/c mice for the preparation of McAbs.Using the obtained McAb 1C5 as the capture antibody and HRP-labeled McAb 8B9 as the detection antibody,conditions were optimized to establish a double-antibody sandwich ELISA detection method.The sensitivity,specificity,and repeatability of the method were systematically evaluated,and a comparative analysis was conducted with qPCR detection.The MVM VP2 protein was expressed and purified,and female BALB/c mice were immunized.Following the immunization protocol,two McAbs were screened and obtained,designated as 1C5 and 8B9.Western-blot and immunofluo-rescence assays confirmed that both antibodies specifically bound to MVM virus and recombinant protein.Using these two prepared antibodies,a double-antibody sandwich ELISA method was established with 1C5 as the capture antibody and HRP-labeled 8B9 as the detection antibody.The method demonstrated a minimum detection limit of 1034 TCID50/mL for MVM virus and 50 ng/mL for recombinant protein.It showed no cross-reactivity with four common murine viruses,exhibited an inter-assay coefficient of variation below 10％,and achieved 100％ overall concordance when compared with qPCR detection results.Two MVM McAbs were successfully prepared.The MVM double-antibody sandwich ELISA method established using these anti-bodies demonstrates favorable sensitivity and specificity,showing good concordance with qPCR detection.This study provides an effective detection approach for clinical diagnosis and high-throughput screening and monitoring of laboratory mice,thereby ensuring the SPF status of laboratory animals and providing healthy and reliable laboratory animals for life science research.