猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立

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4.40μg·mL~(-1),37℃包被2h,采用5%BSA封闭液封闭1h,兔抗PEDV抗体工作质量浓度为5.91μg·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30μg·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。

Double Antibody Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus

A double antibody sandwich ELISA (DAS-ELISA) was developed using the high specificity,mousederived 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× 10312);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.