抗巨细胞病毒pp65抗原单克隆抗体的制备及临床初步比对检测

The preparation of monoclonal antibodies against the CMV pp65 antigen and preliminary clinical comparison test

目的制备抗巨细胞病毒pp65单克隆抗体并建立外周血巨细胞病毒抗原血症免疫荧光检测方法。方法 CMV AD169感染人胚胎肺成纤维细胞MRC-5后24、48、72h,超声波破碎和甲醛灭活制备CMV抗原,以及CMV pp65重组抗原免疫BALB/C小鼠,标准方法制备抗巨细胞病毒pp65单克隆抗体。利用所得单克隆抗体建立检测外周血巨细胞病毒抗原血症检测的实验方法,并与进口试剂盒CMV BriteTM Kit进行比对。结果共有两株杂交瘤细胞产生特异性抗体,克隆名称为13D1-3和29F1-1,免疫球蛋白亚型分别是IgG1型和IgG2a型,制备的腹水抗体纯化后仍具有良好的免疫学特性。单克隆抗体13D1-3与CMV BriteTM Kit试剂盒比对,阳性符合率为88.8%,阴性符合率为98.9%,总符合率为97.3%,Kappa值为0.895,吻合度较高。结论成功制备抗巨细胞病毒pp65单克隆抗体,建立以13D1-3单克隆抗体为基础的检测外周血巨细胞病毒抗原血症方法,与CMV BriteTM Kit试剂盒比对具有良好的符合性。

Objective To prepare monoclonal antibodies （McAbs） against the CMV pp65 antigen ,and estab‐lish immumofluorescence method for detection of CM V antigenemia in peripheral blood leukocytes .Methods 24 ,48 , 72 h after CMV AD169 infected human embryonic lung fibroblast （MRC‐5） ,CMV antigen was prepared through ul‐trasonic fragmentation and formalin inactivation ,BALB/C mice were immunonized by the CMV pp65 antigens ,and the standard method was used to produce monoclonal antibodies against the CMV pp65 antigen .The prepared mono‐clonal antibodies were used to establish immumofluorescence method for detection of CMV antigenemia in peripheral blood leukocytes ,and the established method was compared to CMV BriteTM kit .Results There was two hybridoma cells producing specific monoclonal antibodies ,and named 13D1‐3 and 29F1‐1 ,respectively .The immunoglobulin sub‐types were IgG1 and IgG2a .After purification ,monoclonal antibodies of ascites fluid also had good immunological characteristics .Compared monoclonal antibody 13D1‐3 with CMV BriteTM kit ,the positive coincidence rate was 88 .8% ,negative coincidence rate was 98 .9% and the total coincidence rate was 97 .3% （kappa= 0 .895） ,predicted high consistency .Conclusion This study might have successfully produced monoclonal antibodies against the CMV pp65 antigen and established immumofluorescence method for detection of CMV antigenemia in peripheral blood leu‐kocytes ,which could indicate high consistency compared with CMV BriteTM kit .