摘要
目的 :为了获得抗人巨细胞病毒 (HCMV)单克隆抗体 (McAb) ,并将其用于人HCMV感染后的抗体检测。方法 :采用杂交瘤技术获得抗重组HCMV(rhCMV)gp 5 2蛋白的McAb。并用抗人 μ链抗体包被、rhCMVgp5 2蛋白作抗原和HRP -McAb作标记抗体 ,建立了检测抗HCMV抗体的捕获ELISA。用该法检测献血员、病毒性肝炎患者及疑似CMV感染的肝炎综合征患者血清共计 939份。结果 :最终获得 4株 (3E9,5A6 ,4G1 2 ,2H2 )能稳定分泌高效价抗rhCMVMcAb的杂交瘤细胞。它们分别识别gp5 2蛋白上的 2个不同抗原表位 ,2H2识别gp5 2蛋白上的一个表位 ,3E9,5A6和 4G1 2则共同识别另一个表位。用其建立的捕获ELISA ,gp5 2蛋白抗原最佳浓度为1 μg·mL- 1,酶标单抗的工作浓度为 1 :1 6 0 0 ,批内平均变异系数 3.7% ,批间平均变异系数 7.9%。6 34例献血员IgM抗体阳性 1 7例 (2 .7% ) ;2 88例病毒性肝炎患者 ,阳性 1 0例 (3.5 % ) ;1 7例肝炎综合征患者 ,阳性 1 1例 (6 4.7% )。均经免疫印迹法证实。结论 :用gp5 2蛋白及其单抗建立的捕获ELISA ,特异性强 ,灵敏度高 ,不受类风湿因子的影响 ,结果可靠 ,优于全病毒抗原构成的检测试剂 。
Objective:To obtain monoclonal antibody (McAb) against recombinant human cytomegalovirus (rhCMV) for the detection of human cytomegalovirus (HCMV) IgM antibody following infection. Methods:McAbs against rhCMV gp52 protein were obtained by hybridoma technique.Antibody-capture ELISA (AC-ELISA) were composed of coated antibody (anti-human μ chain),antigen (rhCMV gp52)and marker antibody (specific HRP-McAbs). 939 sera were examined for CMV-IgM by AC-ELISA. Results: Four hybridoma cell lines (3E9,5A6,4G12 and 2H2) secreting high specific McAbs against rhHCMV were obtained, which recognized 2 different epitopes on rhCMV gp52 protein. 2H2 recognized one epitope while 3E9,5A6, and 4G12 recognized together the other one.The proper content of gp52 was 1μg·mL -1 and working dilution of HRP-McAbs was 1:1 600. Within-run CV was 3.7%, between-run CV was 7.9%. 17 of 643 (2.7%) blood donors, 10 of 288 (3.5%) patients with virus hepatitis and 11 of 17(64.7%) patients with hepatitis syndrome developed anti-HCMV IgM. Conclusion:The AC-ELISA using recombinant gp52 antigen and McAbs is specific, sensitive, not interfered by rheumatoid factor and reliable for the detection of serum HCMV-IgM.It is better than CMV antigen and rabbit antibody ELISA and is suitable for clinical diagnosis and epidemiological investigation.
出处
《解放军预防医学杂志》
CAS
北大核心
2003年第2期82-85,共4页
Journal of Preventive Medicine of Chinese People's Liberation Army
基金
南京军区资金赞助项目 (No .0 2 0 1 37)
