CAJ | 학술논문

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 CM V AD169感染人胚胎肺成纤维细胞M RC‐5后24、48、72h ，超声波破碎和甲醛灭活制备CM V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CM V 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 CM V 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CM V BriteTM Kit试剂盒比对具有良好的符合性。
목적：제비항거세포병독pp65단극륭항체병건립외주혈거세포병독항원혈증면역형광검측방법。방법 CM V AD169감염인배태폐성섬유세포M RC‐5후24、48、72h ，초성파파쇄화갑철멸활제비CM V항원，이급CMV pp65중조항원면역BALB／C소서，표준방법제비항거세포병독pp65단극륭항체。이용소득단극륭항체건립검측외주혈거세포병독항원혈증검측적실험방법，병여진구시제합CM V BriteTM Kit진행비대。결과공유량주잡교류세포산생특이성항체，극륭명칭위13D1‐3화29F1‐1，면역구단백아형분별시IgG1형화IgG2a형，제비적복수항체순화후잉구유량호적면역학특성。단극륭항체13D1‐3여 CM V BriteTM Kit시제합비대，양성부합솔위88．8％，음성부합솔위98．9％，총부합솔위97．3％，Kappa치위0．895，문합도교고。결론성공제비항거세포병독pp65단극륭항체，건립이13D1‐3단극륭항체위기출적검측외주혈거세포병독항원혈증방법，여CM V BriteTM Kit시제합비대구유량호적부합성。
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 .