检测牛冠状病毒抗体间接ELISA方法的建立与应用

背景]牛冠状病毒(Bovine coronavirus,BCoV)是引起新生犊牛死亡的主要病原之一,有效的检测手段是防治该病的前提。目前BCoV ELISA检测方法存在敏感性低、不稳定等缺陷。目的]对原有BCoV ELISA方法进行改进,建立间接ELISA检测方法。方法]应用我国BCoV流行毒株CD株n基因为模板,预测N蛋白抗原表位,通过原核表达制备可溶性的重组N蛋白作为抗原,建立间接ELISA方法,应用该方法对黑龙江省2010-2017年的BCoV感染进行血清流行病学调查。结果]该ELISA方法最佳工作条件为:用50 mmol/LpH 9.6碳酸盐作为包被液,抗原包被浓度2.5μg/mL;用PBST作为样本稀释液,稀释浓度1:50,37℃孵育1.5 h;HRP-羊抗牛IgG稀释浓度1:7 500,37℃孵育1.0 h;用1%明胶37℃封闭30 min。阴阳性临界值为0.225。该方法与BRV、BRSV、BVDV、IBRV、BPIV3和E.coli阳性血清均无交叉反应。批内和批间变异系数均小于10%,与病毒中和试验的符合率高达93.5%。对黑龙江省部分地区共603份奶牛血清样品检测结果显示,BCoV抗体阳性率为98.84%。结论]建立的ELISA方法特异性强、敏感性高、稳定性好,为进一步研发ELISA试剂盒提供了技术基础。

Establishment and application of indirect ELISA for detection of bovine coronavirus antibody

Background] Bovine coronavirus(BCoV) is one of the main causes of neonatal calf death, and effective detection is the prerequisite to prevent and control the disease. Objective] At present, BCoV ELISA detection method has some defects, such as low sensitivity, instability and so on. This study aims to improve these defections to establish indirect ELISA detection method. Methods] The method of indirect ELISA was established by using the soluble recombinant N protein of the epidemic BCoV-CD strain as an antigen. The epitope of the N protein was predicted by DNAStar soft, and was prepared by prokaryotic expression in the non-denatured condition. The seroepidemiological investigation of BCoV infection in Heilongjiang province in recent 5 years was carried out by using this method. Results] The optimum working conditions of the ELISA method were as follows: the coating solution was 50 mmol/L pH 9.6 carbonate, and the antigen coating concentration was 2.5 μg/mL;The sample diluent was PBST, the dilution concentration was 1 μg/mL, and incubated at 37 °C for 1.5 h;The dilution concentration of HRP-labeled secondary antibody was 1:7 500, and incubated at 37 °C for 1.0 h;The blocked condition was 1% gelatin at 37 °C for 30 minutes. The negative-positive cut off value was 0.225. The method had no cross-reaction with positive serum of bovine rotavirus, bovine viral diarrhea virus, bovine respiratory syncytial body, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 and Escherichia coli. The intra-and inter-assay coefficient of variation was less than 10%, and the coincident rate with virus neutralization test was 93.5%. The results showed that the positive rate of BCoV antibody was 98.84% in 603 serum samples of cows in some areas of Heilongjiang Province. Conclusion] The ELISA method established in this study has strong specificity, high sensitivity and good stability, which provides a technical basis for the further development of ELISA kit.