南非Ⅱ型口蹄疫间接ELISA检测方法的建立

The Development of Indirect ELISA for Detection of FMDV SAT Ⅱ

为满足口岸对南非II型口蹄疫检测、监测的需求，在对南非II型口蹄疫抗原表位分析及相关基因合成的基础上，进行融合蛋白pGEX—VP1-VP3的体外表达，采用柱上酶切方法制备VP1-VP3蛋白。将纯化VP1-VP3抗原包被96微孔板，分别优化抗原包被浓度、血清稀释度、酶标二抗稀释度，确定阴性临界值。经反复优化，建立的南非II型间接ELISA检测方法的最佳抗原包被浓度为6．4ug／ml，血清最佳稀释度为1：200，酶标二抗最佳稀释度为1：4000，阴性临界值（0D450）为0．562。检测样品判定标准为：OD450〉0．622为阳性，OD450〈0．502为阴性，0．502≤OD450≤0．622为可疑。特异性试验结果表明，所建立方法与其它血清型口蹄疫间无交叉反应。南非II型口蹄疫间接ELISA检测方法的建立为我国口岸口蹄疫检疫提供了新的查验方法。

In order to meet the requirements for detecting and monitoring SAT II foot-and-mouth disease virus （FMDV）, the related nucleotide of the specific antigen epitopes was synthesized and expressed in vitro. The expressed fusion protein pGEX-VP1-VP3 was digested on column to prepare the VP1-VP3 protein which was used to coat 96 well plates. The concentration of coating antigen, serum dilution and HRP-anti-eattle rabbit IgG dilution were optimized respectively. The negative threshold was determined using the indirect ELISA assay. Results showed that the optimized antigen coating concentration was 6.4ug/ml, dilution of serum was 1：200, dilution of HRP-anti-cattle rabbit IgG was 1：4000 and the negative critical value （OD450 was 0.562. The established standard of ELISA assay was defined as.- OD450〉0.622 was positive, OD450〈0.502 was negative, 0.502≤OD450≤0.622 as suspicious. Specificity tests showed that the developed method has no cross reaction with other sero-types of FMDV. In conclusion, the established Indirect ELISA assay could provide a new method for the prevention and control of FMDV in port .