猪流行性腹泻病毒感染小鼠血清抗体间接ELISA方法的建立

Establishment of indirect ELISA for detecting PEDV antibody in mouse serum

为建立检测感染小鼠血清中猪流行性腹泻病毒(PEDV)抗体的方法,本研究应用PEDV受体结合区(receptor binding domain, RBD)重组蛋白作为抗原,通过探索各反应的最佳条件,并进行了该方法灵敏度、重复性、特异性分析。结果显示,最佳蛋白包被浓度为5μg/mL,待检血清稀释浓度为1∶400,孵育时间为37℃30 min,羊抗鼠IgG-HRP作为二抗,孵育条件为37℃30 min,底物显色时间为15 min。对建立的ELISA检测方法进行验证,阳性对照血清的稀释度为1∶204 800倍时OD450值仍大于临界值,批内和批间的变异系数均小于10%,对猪传染性胃肠炎病毒和猪δ冠状病毒抗体小鼠阳性血清检测均不发生交叉反应。结果表明本研究建立的小鼠血清PEDV抗体间接ELISA方法具有很好的灵敏度、重复性、特异性。为PEDV候选疫苗的小鼠血清特异性抗体检测提供了一种精准、高效的方法。

In order to establish a method for detecting porcine epidemic diarrhea virus(PEDV)antibody in mouse serum;the recombinant protein of a PEDV receptor binding domain(RBD)was used as an antigen;the optimal reaction conditions were explored;and the sensitivi-ty,repeatability and specificity of the method were analyzed.The results showed as follows:The optimal protein coating concentration was 5μg/mL.The diluted concentration of the serum to be tested was 1:400.The incubation temperature and time were 37℃ for 30 min.The goat anti-mouse IgG-HRP was used as the secondary antibody.The incubation condition was 37 C for 30 min,and the substrate color de-velopment time was 15 min.Then,the established ELISA method was verified.When the dilution of the positive control serum was 1:204 at 800 times,the OD4so value was still greater than the critical value,and the coefficient of variation within and between the batches was less than 10%.There was no cross reaction in the positive serum of the mice with the TCEV or PDCoV antibody.These results indicated that the indirect ELISA method of mouse serum PEDV antibody established here possessed good sensitivity,repeatability and specificity,which pro-vided an accurate and efficient method for detection of specific antibodies in mouse serum against PEDV candidate vaccines.