Double Antibody Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus
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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OD450nm ≥ 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（5×103.12）；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。
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关键词:
- 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(5×103.12); 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. -
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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 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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