外周血中nf-κb检测方法的建立及应用.docVIP

人外周血中NF-κB p65定量检测方法的建立及临床应用 林青1 郑健2 陈宇新1 艾斯2 王芳1 林礼兴1 （1.福建中医学院附属人民医院检验科，2. 福建中医学院附属人民医院儿科,福建 福州 350004） 建立一种能定量测定人外周血中核转录因子-κB(Nuclear transcription factor- kappa B,NF-κB) p65活化量的检测方法，为临床监测NF-κB 活化情况及预后判断提供参考。方法 用分离外周血单个核细胞，提取核蛋白，应用Bradford法进行核蛋白定量，以识别NF-κB p65不同抗原表位的2种抗NF-κB 单克隆抗体为基础，建立可定量检测人外周血NF-κB p65活化量水平的双抗体夹心酶联免疫吸附试验法。 本研究所建立的方法批内、批间变异系数分别为9.78 % ± 3.99 %、12.51 % ± 4.53％灵敏度为0.030 mg/L回收率为%。与NF-κB突变型寡核苷酸不发生交叉反应。微小病变型肾病综合征患者（68例）治疗前外周血中NF-κB p65活化量较健康人（20名）显著增高（P 0.01），治疗后（68例）与治疗前NF-κB p65活化量差异有统计学意义（P 0.01），但与健康人NF-κB p65活化量差异无统计学意义（P 0.05）。结论 本研究建立的人外周血中NF-κB p65活化量定量检测方法、特异且可靠，为临床监测NF-κB p65活化情况提供了条件。 NF-κB ，p65亚基；单个核细胞微小病变型肾病综合征Establishment of quantitative analysis of NF-κB p65 levels in human peripheral blood and clinic application LIN Qing1, ZHENG Jian2, CHEN Yu-xin1, AI Si2, WANG Fang1, LIN Li-xin1 Department of Clinical Laboratory, 2.Department of Paediatrics, The Affiliated People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou 350004,China) 基金项目: 福建省自然科学基金计划资助项目(No. C0740002，C0440015); 福建中医学院B类课题(No. XB2006011)林青女，医学硕士，副主任技师实验诊断新方法的开发、疾病早期诊断等研究。 Abstract Objective To establish a quantitative method to measure the activated level of Nuclear transcription factor- kappa B (NF-κB) p65 in human peripheral blood for clinical monitoring activation of NF-κB and predicting prognosis in patients. Methods To separate mononuclear cell in human peripheral blood with density gradient centrifugation, the nuclear proteins were extracted from nuclear compartments of the cell, the quantification of nucleoprotein was assayed with Bradford method, anti-NF-κB monoclonal antibodies recognizing two different epitopes on NF-κB p65 were used as capture antibody and detecting antibody respectively to establish a sandwich enzyme-linked immunosorbent assay to quantify NF-κB p65 activation in human peripheral blood. Results The intra-and inter-run imprecision (CV) of the developed method were 9.78 % ± 3.99 % and 12.51 % ± 4.53%, respect