摘要
目的制备牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)gB蛋白单克隆抗体,并建立双抗体夹心ELISA方法。方法应用重组IBRV gB蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,利用IBRV全病毒免疫家兔制备多克隆抗体,采用方阵滴定法确定兔抗IBRV IgG多抗与单抗的最适工作浓度,建立双抗体夹心ELISA检测方法,并对该方法进行灵敏度和特异性验证。结果获得了1株稳定分泌抗IBRV gB单抗的杂交瘤细胞株,命名为2C4;兔抗IBRVIgG多抗和单抗的最适工作浓度分别为2.620 9和2.634 1μg∕ml,酶标二抗的最适稀释度为1∶30 000;应用建立的双抗体夹心ELISA法对100份IBRV阳性血清样品进行检测,结果均呈阳性,符合率为100%;检测牛口蹄疫病毒、牛流行热病毒、牛副流感3型病毒的结果均呈阴性。结论成功制备了IBRV gB蛋白单克隆抗体,并建立了IBRV双抗体夹心ELISA检测方法,可用于牛传染性鼻气管炎的诊断和流行病学调查。
Objective To prepare the monoclonal antibody against gB protein of infections bovine rhinotracheitis virus （IBRV） and develop a double antibody sandwich ELISA method. Methods BALB/c mice were immunized with recombinant IBRV gB protein to prepare monoclonal antibody by hybridoma technique, and rabbits were immunized with whole IBRV to prepare polyclonal antibody. The working concentrations of prepared monoclonal and polyclonal antibodies were optimized by block titration, based on which a double antibody sandwich ELISA method was developed and verified for sensitivity and specificity. Results One hybridoma cell strains stably secreting monoclonal antibody against IBRV gB, named as 2C4, was obtained. The optimal working concentrations of polyclonal and monoclonal antibodies were 2. 620 9 and 2. 634 1 μg / ml respectively, and the optimal dilution of the secondary antibody was 1 ： 30 000. All the determination results of 100 IBRV-positive samples by the developed double antibody sandwich ELISA method were positive, indicating a coincidence rate of 100%. However, all the determination results of bovine foot- and-mouth disease virus, bovine epidemic fever virus and bovine parainfluenza virus type 3 were negative. Conclusion The monoclonal antibody against IBRV gB was successfully prepared, and a double antibody sandwich ELISA method was developed, which might be used for the diagnosis and epidemic investigation of bovine rhinotracheitis.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第2期206-208,共3页
Chinese Journal of Biologicals
关键词
疱疹病毒Ⅰ型
gB蛋白
抗体
单克隆
酶联免疫吸附测定
Herpes virus type I
gB protein
Antibody, monoclonal
Enzyme-linked immunosorbent assay
