间接法Dot-ELISA检测猪流行性腹泻抗体的研究

Indirect DOT ELISA for the Detection of Antibodies Against Porcine Epidemic Diarrhea Virus

使用非洲绿猴肾（Ｖｅｒｏ）细胞增殖适应传代细胞培养的猪流行性腹泻病毒（ＰＥＤＶ），并经聚乙二醇（ＰＥＧ）沉淀法分离纯化ＰＥＤＶ抗原，建立斑点酶联免疫吸附试验（Ｄｏｔ－ＥＬＩＳＡ）检测猪流行性腹泻（ＰＥＤ）抗体。在最适工作条件下，进行了敏感性和特异性试验，结果表明，该法检测ＰＥＤＶ抗体，敏感、特异、重复性好，且方便、快捷，适用于大批量试样的检测，可作为一种诊断ＰＥＤ的比较理想的方法。采用此方法分别对来自加拿大、台湾省进口的种猪和海南省和广东省内种猪群的血清样共８３４份进行了检测，检测得ＰＥＤＶ抗体阳性率达２１％。

Viral antigens of porcine epidemic diarrhea virus (PEDV) PEDV G1 strain,which was adapted to Vero,PK 15 and ST cells without the existing of trypsin,were extracted from infected Vero cell cultures by polyethylene glycol precipitation.A Dot-ELISA based on the antigens was developed to detect antibodies against PEDV.The antibody titres of PED hyperimmune sera and sera of natural in fected pigs were 1∶200￣1∶6400,whereas sera of 50 pigs from PED free farms gave negative results.The minimum amount of pruified PEDV IgG giving a positive result was 12.5ng.Repeatitive tests of 22 PED positive sera and 16 PED negative sera for 4 times showed that the coincident rate was 97.7% and 100% respectively.The antigen membrane could gave the same detection result after being stored for at least 60 days at 4℃and 30 days at room temperature.Both cross reaction test and block test showed that the assay could specifically detect antibodies against PEDV.A detection of 594 sera from Canada and Taiwan,Guangdong and Hainan provinces in China showed that the positive rate was 21.04%.The Dot ELISA had the advantages of simplicity,sensitivity,specificity and rapidity,thus provided a new method for rapid diagnosis of PEDV.