兔出血症病毒双夹心ELISA检测方法的建立  

Establishment of double sandwich enzyme-linked immunosorbent assay to detect rabbit hemorrhagic disease virus

作　　者：董婷[1,2] 王芳[1] 熊富强[2] 王先炜[2] 范志宇[1] 胡波[1] 王敏敏[1] 李洪广[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部动物疫病免疫与诊断重点歼放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2011年第3期571-576,共6页Jiangsu Journal of Agricultural Sciences

基　　金：现代农业产业技术体系建设专项基金项目(nycytx-44);江苏省农业科技自主创新基金项目[CX(10)214];江苏省“六大人才高峰”第六批农业行业省直项目(5311002)

摘　　要：为建立特异性好、敏感度高、稳定可靠的检测兔出血症病毒的双夹心ELISA方法。该试验制备了兔出血症病毒多抗和小鼠腹水单抗,并将他们提纯。用方阵法筛选最佳反应浓度,用特异性试验、敏感度试验和重复性试验对该方法进行鉴定,并同时用双夹心ELISA和血凝试验(HA)对52份待测兔肝脏样品进行检测,比较这两种方法的符合率。结果显示:兔出血症病毒多抗最佳稀释度为1∶400,浓度为19.50 mg/L;单抗的最佳稀释度为1∶800,浓度为2.86 mg/L。特异性试验和敏感性试验结果表明,该方法特异性强、敏感度高。对52份待检兔肝脏样品的检测结果显示:双夹心ELISA和HA试验的阳性检出率分别为82.7%(43/52)和75.0%(39/52),双夹心ELISA与HA试验的符合率为90.7%。用不同批次包被的酶标板重复检测已知样品显示出良好的重复性。以上试验结果表明该双夹心ELISA方法是检测RHDV的一种特异、敏感、快速、经济的免疫学检测技术。To establish a stable double sandwich enzyme-linked immunorbent assay（ELISA） method with good specificity and sensitivity for detection of rabbit hemorrhagic disease virus（RHDV）,rabbit polyclonal antibody and monoclonal antibody in mice′s ascites were prepared and purified.Chessboard titration crossing test was used to determine the optimal working concentrations of these two antibodies.Specificity,sensitivity and repeatability of the constructed ELISA were determined,meanwhile both the double sandwich ELISA method and hemagglutination test（HA） were used to test 52 rabbit liver samples and the coincidence rates of the two methods were compared.The results show that the best working concentration of the purified polyclonal antibody（19.50 mg/L） and monoclonal antibody（2.86 mg/L） were 1∶400 and 1∶800,respectively.The double sandwich ELISA method developed in this study possessed high specificity and sensitivity.The coincidence of double sandwich ELISA and HA method was 90.7%.Forty-three of fifty-two（82.7%） rabbit liver samples were positively detected by the double sandwich ELISA.Thirty-nine of fifty-two（75.0%） were positively detected by HA test.The results tested by different batches of coated microtiter plates showed good repeatability.The results suggest that the double sandwich ELISA is a rapid,sensitive,economic and specific method for detection of RHDV.

关 键 词：兔出血症病毒 双夹心ELISA 多抗 单抗 

分 类 号：S852.52[农业科学—基础兽医学]