基于PEDV Nsp7蛋白间接ELISA抗体检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2017.11.028

摘要/Abstract

摘要：

本研究旨在建立检测猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）不同毒株抗体的间接ELISA方法。试验以纯化的非结构蛋白7（nonstructural protein 7，Nsp7）作为包被抗原，通过优化ELISA反应条件建立了PEDV不同毒株抗体检测的间接ELISA方法。结果显示，其最佳反应条件为：抗原包被量为0.2 μg/孔，包被条件为37℃孵育1 h后4℃过夜；血清稀释度为1：300，作用时间为2 h；酶标二抗最适稀释度为1：10 000，作用时间为1.5 h；TMB显色液作用时间为15 min。在优化条件下，临界值的判定标准为：样品S/P值>0.1694判为阳性，S/P值<0.1398判为阴性。所建立的ELISA方法特异性、重复性及敏感性均良好。用所建立的ELISA方法对40份来自于疑似猪PEDV血清样品进行检测，该方法与商品化试剂盒检测之间的符合率为95%。本研究建立的ELISA方法在临床上可用于PEDV不同毒株抗体水平的检测，也有用于PEDV早期诊断的潜质，从而为制定有效防控PEDV的措施提供一定的参考依据。

关键词: 猪流行性腹泻病毒(PEDV); 非结构蛋白7(Nsp7); 间接ELISA

Abstract:

This study was aimed to establish an indirect ELISA to detect antibodies against different strains of porcine epidemic diarrhea virus (PEDV). Puried nonstructural protein 7(Nsp7) was used as coating antigen, and the indirect ELISA was established by optimizing the ELISA reaction conditions. The results showed that the optimal reaction conditions were as follow:The amount of coating antigen was 0.20 μg/well, and the coating condition was at 37℃ incubation for 1 h then 4℃ overnight; The working dilution of serum samples and HRP-labelled secondary antibody were 1:300 and 1:10 000, and the incubation time were 2 and 1.5 h, respectively; TMB substrate incubation time was 15 min. Serum sample was determined as positive when its S/P>0.1694 and negative when its S/P<0.1398. The ELISA was specific, reproducible and sensitive. Forty samples of suspected PEDV serum samples were tested by the established ELISA, and the coincidence rate between the ELISA and the commercial kit was 95%. The ELISA established in this study could be used clinically to detect the antibody level of different strains of PEDV, and it also had the potential for early diagnosis of PEDV, providing a basis for the development of effective measures to control PEDV.

Key words: porcine epidemic diarrhea virus (PEDV); nonstructural protein 7(Nsp7); indirect ELISA

中图分类号:

S852.65⁺9.6

李红杰, 任豪杰, 刘延珂, 高冬生, 赵军. 基于PEDV Nsp7蛋白间接ELISA抗体检测方法的建立与应用[J]. 《中国畜牧兽医》, 2017, 44(11): 3306-3312.

LI Hong-jie, REN Hao-jie, LIU Yan-ke, GAO Dong-sheng, ZHAO Jun. Development and Application of a PEDV Nsp7-based Indirect ELISA for Antibody Detection[J]. , 2017, 44(11): 3306-3312.

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