单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

畜牧兽医学报 ›› 2006, Vol. 37 ›› Issue (5): 474-479.doi:

单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

刘建文，余兴龙，张丽颖，涂长春*

军事医学科学院军事兽医研究所，长春 130062

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-05-25 发布日期:2006-05-25

Establishment of Antigen-captured ELISA Using Monoclonal Antibodies to Detect Classical Swine Fever Virus

LIU Jianwen， YU Xinglong， ZHANG Liying， TU Changchun*

The Institute of Veterinary Sciences, Academy of Military Medicine Sciences, Changchun 130062, China

Received:1900-01-01 Revised:1900-01-01 Online:2006-05-25 Published:2006-05-25

摘要/Abstract

摘要： 分别用原核表达的猪瘟病毒(CSFV)主要抗原 E2蛋白和猪瘟基因疫苗免疫BALB/c小鼠，通过细胞融合与克隆筛选出5株稳定分泌CSFV抗体的杂交瘤细胞株1E2、1G7、3A2、3B7和4B6。间接免疫荧光和Western-blotting试验结果表明，5株单抗均与CSFV E2蛋白和全病毒抗原反应。将筛选的CSFV特异性单抗1E2、3B7和4B6纯化后等量混合后包被酶标板（捕捉抗体），与兔抗CSFV IgG（检测抗体）联合应用，建立起CSFV抗原捕捉ELISA(AC-ELISA)方法。随后采用方阵滴定法确定了单抗与多抗的最适工作浓度及判定检测结果的OD450临界值。最后以建立的AC-ELISA检测CSFV细胞培养物、CSFV攻毒死亡猪的病料和临床猪瘟组织样品，结果表明，该方法敏感、特异、重复性好，与病毒分离和RT-PCR方法符合率分别为86.2%和90.3%。

Abstract: The five monoclonal antibodies (mAbs) against classical swine fever virus were generated by immunization of BALB/c mice respectively with CSFV DNA vaccine plasmid (expressing glycoprotein E2) and E.coli-expressed E2 protein. These mAbs could react with CSFV growing in PK-15 cells in indirect immunofluorescent assay (IFA), as well as with expressed E2 protein in western-blotting. Of them, the mAbs 1E2, 3B7 and 4B6 are CSFV specific antibody without cross reaction with E2 protein of BVDV, therefore have been used, along with rabbit anti-CSFV IgG, to develop the antigen-captured ELISA(AC-ELISA) for detecting CSFV antigen directly from clinical tissue samples. The cut-off value was determined by testing negative tissue samples of 24 healthy pigs. The method was testified by comparison with virus isolation and RT-PCR of 31 positive tissue samples from piglets experimentally infected with CSFV shimen strain,11 clinically CSF cases and 12 healthy samples. The results showed that the AC-ELISA is sensitive, specific and has high consistency with virus isolation and RT-PCR.

刘建文;余兴龙;张丽颖;涂长春. 单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立[J]. 畜牧兽医学报, 2006, 37(5): 474-479.

LIU Jianwen;YU Xinglong;ZHANG Liying;TU Changchun. Establishment of Antigen-captured ELISA Using Monoclonal Antibodies to Detect Classical Swine Fever Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2006, 37(5): 474-479.

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