【摘要】 为建立一种非洲猪瘟病毒（ASFV）的快速检测方法，本研究利用真核系统表达后纯化与浓缩的ASFV重组CD2v蛋白（r CD2v）免疫BALB/c小鼠4次，采用杂交瘤技术制备ASFV CD2v蛋白单克隆抗体（MAb）。结果显示，共获得5株稳定分泌ASFV rCD2v MAb的杂交瘤细胞株。制备小鼠腹水，利用Protein A/G亲和层析柱纯化制备的小鼠MAb腹水，经SDS-PAGE电泳检测MAb的纯化效果，使用分光光度计测定纯化MAb的浓度，利用间接ELISA方法检测不同浓度MAb的效价。结果显示，5株纯化的MAb均分别在约55 ku（重链）和25 ku（轻链）处分别出现单一目的条带，且MAb的浓度均值为1.02±0.15 mg/mL。效价检测结果显示，除最低浓度外，其余浓度的5株MAb的OD_450nm值均大于0.2，其中MAb 11E2的OD_450nm值最高。采用过碘酸钠法将5株MAb标记HRP（MAb-HRP），经ELISA法检测标记效果及效价，将5株MAb与5株MAb-HRP两两组合后经双抗体夹心ELISA（DAS-ELISA）检测，以确定最佳的配对抗体。结果显示，5株纯化的MAb均标记上HRP，当以MAb 6D9作为捕获抗体，MAb 11E2-HRP作为检测抗体时的P/N值最高，因此采用这两种MAb配对进行后续实验。采用棋盘滴定法优化DAS-ELISA的反应条件，优化结果显示，MAb 6D9的最佳浓度为2μg/mL、r CD2v的最佳包被浓度为2.5μg/mL、MAb 11E2-HRP的最佳稀释度为1:400，初步建立了检测ASFV的DAS-ELISA方法。利用建立的DAS-ELISA检测猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪德尔塔冠状病毒、伪狂犬病病毒和ASFV，评估本研究建立方法的特异性；利用该DAS-ELISA方法检测2倍倍比稀释的ASFV rCD2v（2μg/mL～0.004μg/mL），评估该方法的敏感性；采用同一批次和不同批次rCD2v包被的酶标板，经DAS-ELISA方法分别检测3份ASFV猪阳性血清和1份ASFV猪阴性血清，评估该方法的重复性。结果显示，该方法仅能检测ASFV，其他常见的猪源病毒均为阴性结果，特异性强；该方法对rCD2v的检测限为0.0156μg/mL，敏感性高；批内和批间重复性试验的变异系数均在10%以下，重复性好。采用建立的DAS-ELISA与荧光定量PCR（q PCR）分别检测192份疑似ASFV感染的临床灭活猪血清样品。结果显示DAS-ELISA检出108份阳性样品，84份阴性样品；qPCR检出112份阳性样品，80份阴性样品。经计算两种方法的阳性符合率和总符合率分别为96.4%（108/112）与97.9%（188/192）。上述结果表明，本研究建立的DAS-ELISA方法适用于ASFV大规模临床猪血清样品的检测，为ASFV的检测及流行病学调查提供技术支撑。更多还原

【Abstract】 To establish a rapid detection method for African swine fever virus(ASFV), BALB/c mice were immunized four times with the recombinant CD2v protein(rCD2v) of ASFV, which was expressed in a eukaryotic system and purified. ASFV monoclonal antibodies(MAbs) were then prepared using hybridoma technology. The results showed that five hybridoma cell lines stably secreting MAbs against ASFV rCD2v were obtained. Ascites were prepared from mice and purified using Protein A/G affinity chromatography.The purification efficiency of the MAbs was assessed by SDS-PAGE electrophoresis, the concentration of the purified MAbs was measured using a spectrophotometer, and the reactivity of the MAbs was determined using an indirect ELISA method. The results showed that all five purified MAbs exhibited target bands at approximately 55 ku(heavy chain) and 25 ku(light chain), respectively.The average concentration of the purified MAbs was 1.02 ± 0.15 mg/mL. The five MAbs were labeled with horseradish peroxidase(HRP) using the sodium periodate method. A pairing test was performed using double-antibody sandwich ELISA(DAS-ELISA). The results indicated that the combination of MAb 6D9 as the capture antibody and MAb 11E2-HRP as the detection antibody yielded the highest P/N value and was selected for subsequent experiments. A checkerboard titration method was employed to optimize the reaction conditions for DAS-ELISA. The results showed that the optimal concentration of MAb 6D9 was 2μg/mL, the optimal coating concentration of rCD2v was 2.5μg/mL, and the optimal dilution for MAb 11E2-HRP was 1:400.Specificity tests showed that the established DAS-ELISA specifically detected only ASFV, with no cross-reactivity with PEDV, TGEV, PDCoV, and PRV. The limit of detection(LOD) for rCD2v was 0.0156??g/mL. The coefficients of variation for intra-and inter-batch repeatability test were both below 10%. A total of 192 inactivated clinical serum samples suspected of ASFV infection were tested by both DAS-ELISA and qPCR. The DAS-ELISA detected 108 positive and 84 negative samples, while q PCR detected 112 positive and 80 negative samples.The positive agreement rates and total agreement rates of the two methods were 96.4%(108/112) and 97.9%(188/192), respectively.All these results suggest that the DAS-ELISA established in this study is suitable for the detection of large-scale and inactivated clinical serum samples suspected, providing technical support for the clinical diagnosis and epidemiological investigation of ASFV.更多还原