PEDV双抗体夹心ELISA检测方法的建立

Establishment of a double antibody sandwich ELISA for the detection of PEDV

为了快速检测猪流行性腹泻病毒,本研究以纯化的家兔抗猪流行性腹泻病毒多隆抗体为捕获抗体,以小鼠抗猪流行性腹泻单克隆抗体为检测抗体,并对单克隆抗体进行HRP标记,由此建立了双抗体夹心ELISA(DAS-ELISA)检测法,检测抗体的最佳工作浓度为浓度为0.75ug/m L。当D450≥0.233且P/N≥2时判定为阳性。该方法特异性强,与猪传染性胃肠炎、猪轮状病毒均无交叉反应,最低检测量为3.125×103TCID50/m L。该方法重复性较好:批间和批内重复率均小于10%,临床样品检测结果与RT-PCR方法比较,符合率为90.5%。上述结果表明,本研究建立的检测猪流行性腹泻病毒的双抗体夹心ELISA特异性强、灵敏度高,可用于对猪粪样中PEDV的检测。

In order to effeciently detect porcine epidemic diarrhea virus(PEDV),a double antibody sandwich ELISA(DAS-ELISA)was established by using the purified rabbit polyclonal antibody against PEDV as capture antibody and HRP-labelled mouse monoclonal antibody agsinst PEDV as detection antibody.The results showed that the optimal incubation concentration of detection antibody was 0.75 ug/m L,and it could be considered to be positive when D450≥0.233 and P/N≥2.The minimum detectable limit was3.125×103 TCID50/m L,with no cross reaction with transmissible gastroenteritis virus(TGEV)or porcine rotavirus(Po RV).Meanwhile,the inter-batch and intra-batch repetition rates were both lower than 10%,indicating good repeatability.Also,the consistency rate of this method with RT-PCR in detecting clinial samples could reach 90.5%.In conclusion,this study result provides a useful tool for detecting PEDV in clinical samples.