三株抗人巨细胞病毒pp65蛋白单克隆抗体的鉴定和应用  

Identification and application of three anti-HCMVpp65 McAbs

作　　者：钱春艳[1] 王升年[2] 张玥[1] 陈盈盈[2] 施艳[2] 何妍[2] 陆皓[2] 季育华[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院检验科,200025 [2]生物芯片上海国家工程研究中心

出　　处：《中华检验医学杂志》2007年第2期196-198,共3页Chinese Journal of Laboratory Medicine

基　　金：上海市科学技术委员会2004年重大攻关项目(04D219114)

摘　　要：目的 制备抗HCMVpp65蛋白的单克隆抗体（简称单抗），并对其进行相关研究。方法 以重组表达的HCMVpp65蛋白为抗原，运用杂交瘤技术制备单抗，对获得的单抗进行Ig亚型、特异性、效价和亲和常数的鉴定与测定，并以免疫荧光检测法与进口同类产品进行比较。结果 获得3株针对HCMVpp65蛋白的单抗，即B6、G12、2812；Ig亚型依次为IgG1κ型、IgG1κ型、IgMg型；亲和常数依次为3．6×10^7L／mol、3．8×10^7L／mol、3．1×10^7L／mol；免疫印迹试验结果表明，3株单抗都与HCMVpp65蛋白特异性的结合，除2B12外，B6、G12与EB病毒（EBV）、单纯疱疹病毒（HSV）Ⅰ、HSVⅡ均无交叉反应；与进口单抗平行检测140份临床标本，结果一致（r=1．00，P〉0．05）。结论 成功获得3株稳定分泌抗HCMVpp65蛋白的杂交瘤细胞株，所产生的抗体特异性和亲和力较理想，为HCMVpp65蛋白抗原血症检测国产化试剂盒的研制奠定了基础。Objective To generate and analyze monoclonal antibodies against human cytomegalovirus- pbosphoprotein 65 （HCMVpp65）. Methods The protein which was obtained previously was immunized the Balb/c mice. Hybridoma technique was used to prepare and screen the hybridoma cell lines that could secret the target antibodies. After antibodies were purified, their sub-classes, isotypes, titers, affinity constants and specificities were identified respectively. In addition, immunofluorescence method was used to compare our McAb with other similar imported products. Results Three hybridoma cell lines was obtained which named 136 ,G12,2B12 respectively. The sub-classes and isotypes were IgG1κ ,IgG1κ ,IgMK, their affinity constants were 3.6 x 107 L/mol,3.8 × 10^7 L/mol,3.1×10^8 L/mol,respectively. All of these three antibodies could be recognized by HCMVpp65 protein,the antibodies did not show cross-reactions with EBV,HSV Ⅰ and HSV Ⅱ except 2B12. When the McAbs were compared with imported products in 140 clinical cases, the agreement was very good （ r = 1.00, P 〉 0. 05 ）. Conclusion Three Hybridoma cell lines which secrete the target antibodies with satisfied affinities and specificities have been successfully raised, which provides a basis to produce a domestic-made HCMVpp65 antigen diagnosis kit.

关 键 词：巨细胞病毒 PP65蛋白 抗体 单克隆 

分 类 号：R446.6[医药卫生—诊断学]