【摘要】 为建立一种快速检测A型塞内卡病毒（SVA）的方法，本研究利用氯化铯密度梯度离心法纯化的SVA粒子免疫BALB/c小鼠3次，采用杂交瘤技术制备SVA单克隆抗体（MAb）。结果显示，共获得7株能够稳定分泌SVA MAb的杂交瘤细胞株。采用过碘酸钠法将其中5株MAb标记HRP（MAb-HRP），并采用ELISA法检测标记效果。将7株MAb与5株MAb-HRP两两组合进行抗体配对试验。结果显示，5株MAb均标记上HRP，且其中各稀释度标记的MAb 2C8-HRP的反应性均最强；当MAb 2G7作为捕获抗体，MAb 2C8-HRP作为检测抗体时，P/N值最大，表明这两种MAb的配对效果最好。在此基础上，通过优化各反应条件初步建立检测SVA的双抗体夹心ELISA（DAS-ELISA）方法。利用建立的DAS-ELISA方法检测猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪轮状病毒、猪瘟病毒、O型和A型口蹄疫病毒和SVA，结果显示，该方法仅能检测SVA，其他常见的猪源病毒均为阴性结果，表明该方法特异性强。利用本研究建立的DAS-ELISA方法分别检测系列稀释的SVA上清液(1～1:80 000，对应的病毒滴度分别为10^7.5TCID₅₀/mL～1.25×10^2.5TCID₅₀/mL)和2倍倍比稀释的SVA猪阳性猪血清（1:2～1:256），同时采用猪塞内卡病毒抗原（SVA-Ag）ELISA试剂盒分别检测上述SVA上清液和SVA猪阳性血清，评估本研究建立DAS-ELISA方法的敏感性。结果显示SVA稀释40 000倍后，其OD_450nm值仍大于临界值0.115，即该方法对SVA上清液的检测限为2.5×10^2.5TCID₅₀/mL，且1:64倍稀释的SVA猪阳性血清样品仍为阳性；（SVA-Ag）ELISA结果显示，该方法检测的SVA最大稀释度为1:20000，即对SVA上清液的检测限为5×10^2.5TCID₅₀/mL，且仅能检测到稀释至32倍的SVA猪阳性血清，均低于本实验建立的DAS-ELISA方法，表明该方法敏感性高。对4份SVA猪阳性血清和1份SVA猪阴性血清采用DAS-ELISA方法进行批内和批间重复性试验，结果显示批间和批内重复性试验的变异系数均在10%以下，重复性较好。对采集的205份疑似SVA感染的猪扁桃体组织、鼻咽拭子及血清样品分别采用DAS-ELISA及RT-PCR方法检测。DAS-ELISA的检测结果显示，36份样品为阳性，169份样品为阴性；RT-PCR检测结果显示，50份样品为阳性，155份样品为阴性。经计算两种方法的阳性符合率为72%（36/50），总符合率为93%（191/205）。以上结果表明，本研究建立的DAS-ELISA适用于大规模多种临床样品的检测，为SVA的检测及流行病学调查提供技术支撑。更多还原

【Abstract】 To establish a method for rapid detection of Seneca virus type A （SVA）,BALB/c mice were immunized three times with SVA particles purified by cesium chloride density gradient centrifugation,and SVA monoclonal antibodies （MAbs） were prepared by hybridoma technology.The results showed seven hybridoma cell lines that could stably secrete anti-SVA MAb were obtained.Five of these MAbs were labled with horseradish peroxidase （HRP） using the sodium periodate method,and the conjugation efficiency was assessed by enzyme-linked immunosorbent assay （ELISA）.Seven strains of MAb and five strains of MAb-HRP were matched respectively.The results showed that all five MAbs were successfully conjugated with HRP,and the 2C8 MAb conjugated HRP had the strongest reactivity at each dilutions.The results of the pairing test showed that when MAb 2G7 was used as the capture antibody and2C8-HRP was used as the detection antibody,the P/N value was the largest,indicating that the pairing effect of the two MAbs was the best.Based on these findings,we established a double antibody sandwich ELISA （DAS-ELISA） method for SVA detection by optimizing various reaction conditions.Using the DAS-ELISA method to detect PRRSV,PRV,PPPV,PCV2,PEDV,TGEV,RV,CSFV,type O/A FMDV and SVA,the results showed that the method had no cross-reactivity with other common swine viruses except for the specific reaction with SVA,indicating that the method had highly specificity.The sensitivity of the DAS-ELISA method were evaluated by detecting a range of SVA supernatant dilutions (10^7.5TCID₅₀/m L-1.25×10^2.5TCID₅₀/m L) and 2-fold serially diluted SVA-positive pig serum（1:2-1:256）.Concurrently,the porcine Senecavirus A antigen （SVA-Ag） ELISA kit was utilized for comparative detection of the same samples.The DAS-ELISA method showed the OD_450nmvalue was above the cut-off when the SVA virus culture medium was diluted40000-fold,with a detection limit at 2.5×10^2.5TCID₅₀/m L and the SVA pig positive serum sample diluted 1:64 times was still positive;In contrast,the SVA-Ag ELISA kit had a detection limit of 1:20000 for SVA supernatant and could detect SVA-positive pig serum up to32-fold dilutions,which were lower than those detected by the DAS-ELISA method,indicating the high sensitivity of the established method.DAS-ELISA was used to test the intra-and inter-batch repeatability of 4 SVA pig positive sera and 1 SVA pig negative serum.The results showed that the coefficient of variation of the inter-and intra-batch repeatability tests were all below 10%,indicating good repeatability of this method.The 205 samples of tonsillar tissue,nasopharyngeal swabs and serum samples collected from pigs suspected to be infected with SVA were tested by DAS-ELISA and RT-PCR methods,respectively.The DAS-ELISA showed that 36 samples were positive and 169 samples were negative;RT-PCR showed that 50 samples were positive and 155 samples were negative.The positive coincidence rate and the overall coincidence rate of the DAS-ELISA test with RT-PCR were 72%（36/50） and 93%（191/205）,respectively.All these results suggest that the DAS-ELISA established in this study is suitable for the detection of large-scale and multi-species clinical samples,providing technical support for the clinical differential diagnosis and epidemiological investigation of SVA.更多还原