夹心ELISA检测血清结缔组织生长因子及其初步临床的应用.docVIP

夹心ELISA检测血清结缔组织生长因子及其初步临床的应用 　　【摘要】 目的 应用抗结缔组织生长因子（CTGF）单克隆抗体和抗CTGF多克隆抗体，建立CTGF免疫分析方法，用其方法检测血清中CTGF含量初步验证其临床应用价值。方法 使用抗CTGF的单克隆抗体包被反应板，辣根过氧化物酶标记兔抗人CTGF多克隆抗体(二抗)建立双抗体夹心方法，通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度；以纯化的CTGF抗原为标准品建立标准曲线；以重复性、灵敏性和回收率实验评价ELISA方法。使用该检测系统和进口试剂盒对照，检测其准确性和可比性。利用该系统检测临床有肝纤维化症状的患者血清标本，初步评价其临床应用价值。结果 该方法有较高的灵敏度、特异性及稳定性，最低检测限批间和批内变异系数分别为0.98 ng/ml、9.8%、4.9%。与进口试剂盒有很强的可比性，两者的相关系数r=0.988。初步临床标本检测证实，肝硬化、慢性肝炎急性肝炎患者的血清CTGF含量明显高于建康对照者(?P?0.01)，肝硬化患者的血清CTGF水平明显高于慢性肝炎和急性肝炎患者（?P?0.01）。结论 成功地建立了一种测定血清CTGF的双抗体夹心方法，该反应系统有较高的灵敏度和特异性，和进口试剂盒有很强的可比性，为临床广泛使用打下了基础。? 　　【关键词】 乙型肝炎； 结缔组织生长因子； ELISA?? 　　The establishment of sandwich ELISA measuring serum connective tissue growth factor and its clinical value ?SHI ?Yu-?ling.The First People?s Hospital of Xiangcheng in Henan Province，Xiangcheng 466200，China?? 　　【Abstract】 Objective ?To establish immunologic assay detecting serum connective tissue growth factor(CTGF)using anti-CTGF monoclone antibodies and anti-CTGF polyclone antibodies.Validated the clinical value of the ELISA system.Methods Plates were coated by anti-CTGF monoclonal antibodies，HRP conjugated anti-CTGF polyclonal antibody was used as detecting antibodies.The optimization reactivity conditions were analyzed by chessboard titrimetric method.Compare the ELISA system and imported ELISA kit detecting CTGF for validating the accurate and comparability of the ELISA system.Results The ELISA system had perfect sensitivity，specificity and stability，the minimum determinable dose，intra-assay and inter-assay coefficients of variation(CV)was 0.18 ng/ml，9.8% and 4.5%，respectively.Correlation coefficient was 0.988 between the ELISA system and imported ELISA kit.Serum level of CTGF in patients with liver cirrhosis was significantly higher than that in chronic hepatitis and control group.Conclusion The sandwich ELISA detecting serum CTGF was successfully established.The ELISA had a perfect sensitivity，specificity and comparability with imported ELISA kit，which lay the foundation fo