【作者】 沈丹丹；

【导师】 何国平；

【作者基本信息】 江苏大学 ， 内科学， 2009， 硕士

【摘要】 背景:C-反应蛋白(CRP)是一种高度保守的血浆蛋白,在脊椎动物和多种非脊椎动物中存在同系物,参与全身炎症反应。机体处于炎症状态时,其血浆CRP浓度升高,因此CRP作为炎症标记物被广泛应用于临床。CRP是一种模式识别分子,通常与死亡细胞或病原体表面的特殊分子构型结合。在组织损伤或感染后数小时内,其合成迅速增加,提示CRP参与机体的宿主防御和固有免疫反应。最近,血浆CRP水平的轻微升高和未来心血管事件的相关性被广泛证实。因此,建立高灵敏度的CRP测定方法是一项重要的实用性研究领域,引起研究工作者和临床医生的兴趣和关注。目的:初步建立一种可用于检测人超敏C-反应蛋白(hs-CRP)的双抗体夹心ELISA方法,并初步探讨其临床应用价值。方法:1.采用Protein A亲和层析柱纯化本室制备的抗CRP单克隆抗体(mAb),并行聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western-blot)对纯化抗体特性进行鉴定;利用简易过碘酸钠法对抗CRP mAb进行辣根过氧化物酶(HRP)标记。2.采用dp-Aminophenyl phosphoryl Cholnie Gel亲和层析柱从肿瘤患者胸水中分离、纯化CRP,并通过SDS-PAGE与Western-blot检测其纯度与特异性。3.通过抗体配对实验确定最佳配对抗体,行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CRP抗原为标准品建立标准曲线;以重复性、灵敏性、回收性实验评价ELISA方法。4.对冠状动脉无狭窄者(冠脉正常组)68例、狭窄直径＜50%者(冠状动脉硬化症组)59例和狭窄直径≥50%者(冠心病组)67例采用双抗体夹心ELISA方法测定血浆hs-CRP水平。5.统计学方法:采用SPSS16.0软件包进行统计分析。Hs-CRP数据呈非正态分布,以中位数(M)和四分位数间距(QR)表示。非正态分布资料多组间比较用Kruskal-Wallis检验,两两比较用Mann-Whitney检验。结果:1.采用protein A亲和层析柱从小鼠腹水中纯化出55KD、25KD各一条带,SDS-PAGE检测纯度超过95%的蛋白,行western-blot检测未见杂带。并行HRP酶标,酶标抗体行western-blot检测未见杂带。2.采用dp-Aminophenyl phosphoryl Cholnie Gel亲和层析法从肿瘤患者胸水中成功纯化出大小为24KD蛋白,SDS-PAGE检测纯度超过95%,用抗CRP mAb进行western-blot检测未见杂带。3.最佳配对组合为抗CRP mAb 1C10和HRP标记抗CRP mAb 2C11,最适工作浓度分别为10ug/ml和1∶2000,该方法的批内、批间变异系数分别为3.1%～9.7%和3.6～13.6%,灵敏度达8.3ng/ml,回收率为90%～109%。4.用ELISA法测定的血浆hs-CRP水平:冠状动脉硬化症组明显高于冠脉正常组[(1.15±3.65mg/L)vs(0.74±1.75mg/L)](P＜0.05),冠心病组明显高于冠状动脉硬化症组[(3.29±8.93mg/L)vs(1.15±3.65mg/L)](P＜0.05)。结论:纯化、鉴定和标记了抗CRP mAb,纯化和鉴定了CRP抗原,初步建立了一种可用于临床检测CRP的双抗体夹心ELISA方法。更多还原

【Abstract】 Background:C-reactive protein(CRP) is a phylogenetically highly conserved plasma protein,with homologs in vertebrates and many invertebrates,that participates in the systemic response to inflammation.Its plasma concentration increases during inflammatory states,a characteristic that has long been employed for clinical purposes.CRP is a pattern recognition, binding to specific molecular configuration that are typically exposed during cell death or found on the surfaces of pathogens.Its rapid increase in synthesis within hours after tissue injury or infection suggests that it contributes to host defense and that it is part of the innate immune response. Recently,an association between minor CRP elevation and future major cardiovascular events has been recognized.Therefor,it is important to establish a high sensitive method for measurement of CRP and it is also a practical work to explore a method with high sensibility.Objective:To establish a sandwich ELISA for quantitative measurement of hs-CRP, and to explore its clinical application.Methods:1.Anti-CRP mAbs prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western-blot to test their characteristics.All the mAbs were labeled with horseradish peroxidase by sodium oxidation method.2.Human CRP was isolated from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel.The malignant ascites fluid was obtained from cancer patients and the investigation conforms to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.Purified human CRP was assayed by SDS-PAGE and western-blot analysis.3.Antibody pairs were performed using anti-CRP mAb as coating antibody and HRP labeled anti-CRP mAb as labeled antibody,which optimal concentrations were defined by titration.Standard curve was performed using purified CRP and was judged by sensitivity,reproducibility and recovery rate.4.According to the result of coronary angiography,plasma hs-CRP level was detected in 68 normal patients(without coronary artery stenosis),59 coronary atherosclerotic patients(the severity of coronary artery stenosis＜50%) and 67 coronary artery disease patients(the severity of coronary artery stenosis≥50%).5.Statistics:The data are presented as M±QR.The Kruskal-Wallis and Mann-Whitney tests were performed with the use of SPSS 16.0 statistical software.Statistical significance was accepted at P＜0.05.Results:1.Anti-CRP purified from mice ascites fluid using protein A was in the dipolymer form(55KD,25KD) with no detection of other protein by SDS-PAGE(purity up to 95%) and western-blot.2.CRP purified from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel was in the monomeric form (24KD) with no detection of other proteins by SDS-PAGE(purity up to 95%) and western-blot.3.The optimal paired antibodies were anti-CRP mAb 1C10 and HRP labeled anti-CRP mAb 2C11 which optimal concentrations were 10ug/ml and 1:2000,respectively.The sensitivity of this assay was 8.3ng/ml.The coefficients of variation were 3.1%to 9.7%within assay and 3.6%to 13.6%between assays.The recovery rate was 90%to 109%.4.The results showed that the plasma hs-CRP level in coronary atherosclerotic patients was significantly higher than normol patients[(1.15±3.65mg/L) vs(0.74±1.75mg/L)](P＜0.05),the plasma hs-CRP level in coronary artery disease patients was also significantly higher than coronary atherosclerotic patients[(3.29±8.93mg/L) vs(1.15±3.65mg/L)](P＜0.05).Conclusion:A sandwich ELISA assay for detecting hs-CRP was obtained.更多还原