猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法的建立及应用

An Indirect ELISA Assay for Detecting Specific SIgA Antibody of Porcine Epidemic Diarrhea Virus

【目的】快速了解猪群感染猪流行性腹泻病毒或免疫猪流行性腹泻病毒相关疫苗后猪群特异性SIgA抗体水平。【方法】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)ZJ08株进行纯化,采用纯化的病毒作为包被抗原,以辣根过氧化物酶标记的SC(Secretory component,SC)多克隆抗体为标记抗体,通过优化检测条件,建立乳汁中猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法。【结果】成功建立了猪流行性腹泻病毒SIgA抗体检测方法。PEDV抗原的最佳包倍浓度为0.6μg·孔−1,样本最佳稀释比例为1∶5,作用时间1 h;SC标记抗体最适稀释比例为1∶100,作用时间1.5 h。建立的ELISA方法能够特异性检测样本中抗PEDV SIgA抗体,与PoRV(Porcine rotavirus,PoRV)、TGEV(Transmissible gastroenteritis virus,TGEV)的阳性乳汁均无交叉反应;批内、批间重复试验的变异系数小于10%,具有良好的特异性和重复性。通过临床采集的乳汁样品测定发现,样本中PEDV IgA抗体阳性率达到98.1%,而采用针对特异性SIgA的抗体方法检测,其阳性率仅为69.2%,样本中IgA和SIgA抗体符合率仅为70.6%,提示仅仅依据PEDV IgA抗体水平并不能很好地反映猪群免疫该疫苗后特异性黏膜免疫的真实水平。【结论】建立的PEDV特异性SIgA抗体检测方法可为分析、评估猪群免疫PEDV疫苗后乳汁中特异SIgA抗体产生及猪群的保护情况提供可靠依据。

【Objective】A method for detecting SIgA antibodies of porcine epidemic diarrhea virus(PEDV)was established.【Methods】Using a purified PEDV ZJ08 strain as coating antigen and the antibody for secretory component(SC)labeled by horseradish peroxidase(HRP)as secondary antibody,an indirect ELISA assay with optimized conditions was developed to detect the specific SIgA antibody of PEDV.【Result】The optimized assay applied a working antigen concentration of 0.6μg per well,a 1∶5 sample dilution,a coating at 37℃for 1 h,and an 100×dilution of the labeled antibody for 1.5 h incubation at 37℃.Other than the target detection,the assay did not react with porcine rotavirus or transmissible gastroenteritis virus.The inter-or intra-batch variations of the assay on test results were less than 10%.On the milk from vaccinated pigs,a 98.1%positive detection on IgA,which was significantly higher than 69.2%on SIgA and merely 70.6%on combined IgA and SIgA,was achieved indicating the deficiency of a single IgA test for an accurate determination on the mucosal immunity of the animal.【Conclusion】The established assay appeared to appropriately reveal the specific SIgA antibody produced in pigs for protection of the livestock from PEDV by the vaccination.