猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立

王晨燕; 王隆柏; 吴学敏; 陈如敬; 车勇良; 周伦江

doi:10.19303/j.issn.1008-0384.2017.08.003

猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立

doi: 10.19303/j.issn.1008-0384.2017.08.003

福建省农业科学院畜牧兽医研究所/福建省畜禽疫病疾病防治工程技术研究中心, 福建福州 350013

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2017R1023-8

福建省农业科学院青年创新基金项目 MYQJ2015-

福建省科技创新平台建设项目——福建省畜禽疫病防控技术重大研发平台 2014N2003-4

详细信息

作者简介:
王晨燕(1988-), 女, 硕士, 助理研究员, 主要从事动物传染病研究(E-mail:329177624@qq.com)

通讯作者:
周伦江(1973-)男, 博士, 研究员, 主要从事动物病毒学与分子生物学(E-mail:lunjiang@163.com)

中图分类号: S767.5
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出版历程
- 收稿日期: 2017-05-16
- 修回日期: 2017-07-07
- 刊出日期: 2017-08-28

Double Antibody Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences/Fujian Animal Diseases Control Technology Development Center, Fuzhou, Fujian 350013, China

摘要

摘要: 采用特异性好的鼠源抗猪流行性腹泻病毒（PEDV）单克隆抗体为捕获抗体，兔源多克隆抗体为检测抗体，建立PEDV双抗体夹心ELISA检测方法。结果显示，该方法的最佳反应条件为：抗PEDV单克隆抗体E1包被质量浓度4.40 μg·mL^-1，37℃包被2 h，采用5% BSA封闭液封闭1 h，兔抗PEDV抗体工作质量浓度为5.91 μg·mL^-1，酶标二抗稀释度为1：2000，以OD_450nm ≥ 0.381作为阳性判定标准。该ELISA方法对猪轮状病毒和猪传染性胃肠炎病毒无交叉反应。敏感度可达30 μg·mL^-1（5×10^3.12）；重复性变异系数小于10%。采用该方法和RT-PCR方法同时检测临床样品42份，阳性样品符合率为92.30%，表明建立的PEDV双抗体夹心ELISA检测方法具有特异性好、敏感性高和方便快捷等优点，可用于PEDV快速检测。
- 猪流行性腹泻病毒 /
- 单克隆抗体 /
- 双抗体夹心ELISA
Abstract: A double antibody sandwich ELISA (DAS-ELISA) was developed using the high specificity, mouse-derived monoclonal antibody (Mab) as the capture antibody and the rabbit-derived polyclonal antibody against porcine epidemic diarrhea virus (PEDV) as the detecting antibody. The optimal reaction conditions for DAS-ELISA was determined to include a coating concentration of 4.40 g·mL^-1 for PEDV MAb E1 with 1 h incubation at 37℃, the use of 5% BSA solution for blocking for 1 h, an application of 5.91 μg·mL^-1 in concentration of rabbit polyclonal antibodies against PEDV, a 2 000×dilution of HRP, and the positive OD equal or greater than 0.381 at 450 nm wave length on the spectrophotometer measurement. The developed method showed no cross-reaction between porcine rotavirus and transmissible gastroenteritis virus. The detection sensitivity of the method was 30 g·mL^-1(5×10^3.12); and, the coefficient variation of repetition, less than 10%. Furthermore, a total of 42 clinical samples were positively detected by the method in conjunction with RT-PCR at a rate of 92.30%. Consequently, it was concluded that the newly developed DAS-ELISA methodology was highly specific, sensitive, rapid, and hence, applicable for PEDV detection.
- porcine epidemic diarrhea virus /
- monoclonal antibody /
- double antibody sandwich ELISA

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图 1 双抗体夹心ELISA特异性试验

Figure 1. Specificity of DAS-ELISA

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图 2 双抗体夹心ELISA敏感性试验

Figure 2. Sensitivity of DAS-ELISA

下载: 全尺寸图片幻灯片

表 1 批内和批间重复试验结果(n=5)

Table 1. Intra-and inter-batch reproducibility test results on DAS-ELISA

样品	批内重复试验			批间重复试验
样品	平均数	方差	变异系数 /%	平均数	方差	变异系数 /%
1	1.050	0.021	2.003	0.778	0.023	2.998
2	0.655	0.020	3.128	0.897	0.036	4.056
3	0.774	0.031	4.007	0.920	0.033	3.588
4	0.589	0.018	3.122	0.691	0.020	2.975
5	1.255	0.058	4.589	1.320	0.049	3.745

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参考文献(14)

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