定量检测人超敏C-反应蛋白双抗体夹心ELISA方法的建立及初步临床应用
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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。
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.
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。
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.
引文
1.Kinlay S,Schwartz GG,Olsson AG,et al.High-dose atorvastatin enhances the decline in inflammatory markers in patients with acute coronary syndromes in the MIRACL study.Circulation,2003,108(13):1560-1566.
2.Gabay C,Kushner I.Acute-phase proteins and other systemic responses to inflammation.N Engl J Med,1999,340(17):448-454.
3.Yudkin JS,Kumari M,Humphries SE,et al.Inflammation,obesity,stress and coronary heart disease:is interleukin-6 the link? Atherosclerosis,2000,148(2):209-214.
4.Chang MK,Binder C J,Torzewski M,et al.C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand:phosphorylcholine of oxidized phospholipids.Proc Acad Sci U S A,2002,99(20):13043-13048.
5.Ridker PM..C-reactive protein and the predition of cardiovascular events among those at intermediate risk:moving an inflammatory hypothesis toward consensus.J Am Coll Cardiol,2007,49(21):2129-2138.
6.Bansal S,Ridker PM.Comparison of characteristics of future myocardial infarction in women with baseline high versus baseline low levels of hs-C-reactive protein.Am J Cardiol,2007,99(11):1500-1503.
7.周玉,李岩松,潘风光,等.小鼠腹水IgG类单克隆抗体纯化方法的研究.黑龙江畜牧兽医,2006,10(6):41-61.
8.Bouvet JP.A modified gel filtration tenique predicting an unusual exclusion volume of IgM:a simple way of preparing monoclonal IgM.J Immumol Meth,1984,66(2):229-230.
9.Clegardin P.Trandem purification of mouse IgM monoclonal antibodies produced in vitro using anion-exchange and gel fest protein liquid chromatography.J Chromatogar,1986,358(1):209-218.
10.Jiskoot W.Two-step purification of a murine monoclonal anti-bodies intended for therapeutic application in man.Optimisation of purification condition and scaling up.J Immunol Meth,1989,124(1):143-156.
11.Lamoyi E.Preparation of F(ab)'fragrnents from mouse IgG of various subclasses.J Immunol Meth,1983,56(2):235-243.
12.Parham P.Monoclonal antibodies:Purification tragrnentation and application to structural and functional studies of class I MHC antigens.J Immunol Meth,1982,53(2):133-173.
13.Barton DE,Yang Feng TL,Mason A J,et al.Mapping of genes for inhibin submits alpha,teta A,and beta B on human and mouse chromosomes and studies ofjad mice.Genomics,1989,5(1):91-99.
14.Roberts V J,Barth SL.Expression of messenger ribonucleic acid encoding the inhibin lactivin system during mid- and late-gestation rat embogogenesis.Endocrinol,1994,134(2):914-923.
15.李红乐,王宏卫,夏辉明,等.应用Protein A-Sepharose CL-4B亲和层析法纯化单克隆抗体.河南医科大学学报,1999,34(4):11-12.
16.林清华.免疫学实验.武汉:武汉大学出版社,1999:124-128.
17.Steven B,Irving K,David S.C-reactive protein.J Biol Chem,2004,279(47):48487-48490.
18.Schwick HG,Haupt H.Properties of acute phase proteins of human plasma.Behring Institute Mitteilungen 1986:1-10.
19.Hutchinson WL,Kahan MC,et al.Transgenic human CRP is not pro-atherogenic,pro-atherothrombotic or pro-inflammatory in apoE-/-mice.Atherosclerosis,2008,196(1):248-255.
20.Volanakis JE,Narkates AJ.Interaction of C-reactive protein with artificial phosphatidylcholine bilayers and complement.J Immunol,1981,126(5):1820-1825.
21.Volanakis JE,Kaplan MH.Interaction of C-reactive protein complexes with the complement system.Ⅱ.Consumption of guinea pig complement by CRP complexes:requirement for human Clq.J Immunol,1974,113(6):2135-2147.
22.Ikuta T,Okubo H,Yukimura H,et al.Purification of C-reactive protein.Biochemistry international,1983,6(2):275-282.
23.Volanakis JE,Wirtz KW.Interaction of C-reactive protein with artificial phosphatidylcholine bilayers.Nature,1979,281(1):155-157.
24.Li JJ,Fang CH.C-reactive protein is not only an inflammatory marker but also a direct cause of cardiovascular disease.Med Hypothese,2004,62 (6):499-506.
25.Fujita Y,Kakino A,Nishimichi N,et al.Oxidized LDL Receptor LOX-1 Binds to C-Reactive Protein and Mediates Its Vascular Effects,linical Chemistry,2009,55 (2):285-294.
26.Zwaka TP,Hombach V,Torzewski J.C-Reactive Protein-Mediated Low Density Lipoprotein Uptake by Macrophages.Circulation,2001,103 (9):1194-1197.
27.Ridker PM.Clinical application of C-reactive protein for cardiovascular disease detection and prevention.Circulation,2003,107 (3):363-369.
28.Ridker PM,Hennekens CH,Buring JE,et al.C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women.N Engl J Med,2000,342 (7):836-843.
29.Ridker PM.High-sensitivity C-reactive protein:potential adjunct for global risk assessment in the primary prevention of cardiovascular disease.Circulation,2001,103 (13):1813-1818.
30.Pischon T,Hu FB,Rexrode KM,et al.Inflammation,the metabolic syndrome,and risk of coronary heart disease in women and men.Atherosclerosis,2008,197 (1):392-399.
31.Bassuk SS,Rifai N,Ridker PM.High-sensitivity C-reactive protein:clinical importance.Curr Probl Cardiol,2004,29 (8):439-493.
32.Biasucci LM,Liuzzo G,Bona RD,et al.Different Apparent Prognostic Value of hsCRP in Type 2 Diabetic and Nondiabetic Patients with Acute Coronary Syndromes.Clinical Chemistry,2009,55 (10):365-368.
1.Steven B,Irving K,David S.C-reactive protein.J Biol Chem,2004,279(47):48487-48490.
2.Pearson T,Ballantyne C,.Veltri E,et al.Pooled Analyses of Effects on C-Reactive Protein and Low Density Lipoprotein Cholesterol in Placebo-Controlled Trials of Ezetimibe Monotherapy or Ezetimibe Added to Baseline Statin Therapy.The American Joumal of Cardiology,2009,103(3):369-374.
3.Du Clos TW.Function of C-reactive protein.Ann Med,2000,32(4):274-278.
4.Thompson D,Pepys MB,Wood SP.The physiological structure of human C-reactive protein and its complex with phosphocholine.Structure,1999,7(2):169-177.
5.Whitehead AS,Bruns GAP,Colten HR,et al.Isolation of human C-reactive protein complementary DNA and localization of the gene to chromosome 1.Science,1983,221(1):69-71.
6.Chang MK,Binder C J,Torzewski M,et al.C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand:Phosphorylcholine of oxidized phospholipids.Proc.Natl.Acad.Sci.U.S.A,2002,99(20):13043-13048.
7.Zwaka TP,Hombach V,Torzewski J.C-Reactive Protein-Mediated Low Density Lipoprotein Uptake by Macrophages.Circulation,2001,103(9):1194-1197.
8.Griselli M,Herbert J,Hutchinson WL,et al.C-reactive Protein and Complement Are Important Mediators of Tissue Damage in Acute Myocardial Infarction.Journal of Experimental Medicine,1999,190 (12):1733-1740.
9.Pischon T,Hu F,Rexrode K,et al.Inflammation,the metabolic syndrome,and risk of coronary heart disease in women and men.Atherosclerosis,2008,197 (1):392-399.
10.Mora S,Musunuru K,Blumenthal RS.The Clinical Utility of High-Sensitivity C-Reactive Protein in Cardiovascular Disease and the Potential Implication of Jupiter on Current Practice Guidelines.Clinical Chemistry,2009,5 (10):219-228.
11.Ridker PM.Clinical Application of C-Reactive Protein for Cardiovascular Disease Detection and Prevention.Circulation,2003,107 (3):363-369.
12.Wang TJ,Gona P,Larson MG,et al.Multiple Biomarkers for the Prediction of First Major Cardiovascular Events and Death.The New England journal of medicine,2006,355 (25):2631-2639.
13.Pasceri V,Willerson JT,Yeh E T.Direct proinflammatory effect of C-reactive protein on human endothelial cells.Circulation,2000,102 (18):2165-2168.
14.Ridker PM,Cannon CP,Morrow D,et al.C-Reactive Protein Levels and Outcomes after Statin Therapy.New England Journal of Medicine,2005,352 (1):20-28.
15.Blake GJ,Ridker PM.Novel clinical markers of vascular wall inflammation.Circ Res,2001,89(9):763-771.
16.Lowe GDO,Pepys MB.C-reactive protein and cardiovascular disease:Weighing the evidence.Current Cardiovascular Risk Reports,2006,8 (5):421-428.
17.Ridker PM,Rifai N,Rose L.Comparison of C-Reactive Protein and Low-Density Lipoprotein Cholesterol Levels in the Prediction of First Cardiovascular Events.New England Journal of Medicine,2002,347 (14):1557-1565.
18.Medina-Urrutia A,Juarez-Rojas J,Martinez-Alvarado R,et al.High-density lipoprotein subclasses distribution and composition in Mexican adolescents with low HDL cholesterol and/or high triglyceride concentrations,and its association with insulin and c-reactive protein.Atherosclerosis,2008,201 (2):392-397.
19.Hübner-Wo(?)niak E,Malara M,Ok(?)cka-Szyma(?)ska J,et al.Cholesterol fractions,C-reactive protein and creatine kinase activity in plasma of female athletes.Physical Education and Sport,2008,52 (26):88-91.
20.Li J J,Fang C H,Chen MZ,et al.Rapid effects on lipid profile and C-reactive protein by simvastatin in patients with hypercholesterolemia.Clin Cardiol,2003,26(3):472-476.
21.Rifai N,Ridker PM.High-sensitivity C-reactive protein:a novel and promising marker of coronary heart disease.Clin Chem,2001,47 (3):403-411.
22.Paul A,Ko KWS,Li L,et al.C-Reactive Protein Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice.Circulation,2004,109(5):647-655.
23.Pepys MB,Hirschfield GM,Tennent GA,et al.Targeting C-reactive protein for the treatment of cardiovascular disease.Nature,2006,440 (7088):1217-1221.
1.Gretarsdottir S,Sveinbjomsdottir S,Jonsson HH,et al.The gene encoding phosphodiesterase 4D confers risk of ischemic stroke.Nat Genet,2003,35 (1):131-138.
2.吴丽娥,刘鸣,张月辉,等.缺血性卒中TOAST病因分型和预后.中华神经科杂志.2004,37(4):292-295.
3.Houslay,MD,Adams,DR.PDE4 cAMP phosphodiesterase:modular enzymes that orchestrate signaling cross-talk,desensitization and compartmentalization.Biochem.J,2003,370(1):1-18.
4.Qing Song,John W.Cole,Jeffrey R.O'Connell,et al.Phosphodiesterase 4D polymorphisms and the risk of cerebral Infarction in a biracial population:the Stroke Prevention in Young Women Study.Hum Mol Genet,2006,15(16):2468-2478.
5.杨晓军.磷酸二酯酶4D基因与缺血性卒中.国外医学脑学管疾病分册,2004,12(4):310-312.
6.Meschia JF,Brott TG,Brown RD,et al.Phosphodiesterase 4D and 5-lipoxygenase activating protein in ischemic stroke.Ann Neurol,.2005,58(3):351-361.
7.Woo D,Kaushal R,Kissela B,et al.Association of phosphodiesterase 4D with ischemic stroke:A population,based case-control study.Stroke,2006,37(2):371-376.
8.Saleheen D,Bukhari S,Haider SR,et al.Association of Phosphodiesterase 4D gene with ischemic stroke in a Pakistani population.Stroke,2005,36(10):2275-2277.
9.Nakayama T,Asais S,Sato N,et al.Genotype and haplotype association study of STRK 1 region on 5q12 among Japanese:a case-control study.Stroke,2006,37(1):69-76.
10.Xue H,Wang H,Song X,et al.Phosphodiesterase 4D gene polymorphism is associated with ischaemic and haemorrhagic stroke.Clin Sci(Lond),2009,116(4):335-340.
11.Lohmussaar E,Gschwendtner A,Mueller JC,et al.ALOX5AP Gene and the PDE4D gene in a central European population of stroke patients.Stroke,2005,36(10):731-736.
12.Bevan S,Porteous L,Sitzer M,et al.Phosphodiesterase 4D gene,Ischemic stroke,and asymptomatic carotid atherosclerosis.Stroke,2005,36(5):949-953.
13.Nilsson-Ardnor S,Wiklund PG,Lindgren P,et al.Linkage of ischemic stroke to the PDE4D region on 5q in a Swedish population.Stroke,2005,36(8):1666-1671.
14.李明才,张成,张鸿炼,等.磷酸二酯酶4D基因与缺血性卒中的遗传易感性研究.中华神经科杂志,2006,39(3):180-182.
15.徐波,邓春艳,张彩英,等.磷酸二酯酶4D基因三个多态性与缺血性卒中的关系.医学世界,2007,2(3):55-57.
16.Hsieh MS,Yu SC,Chung WT,et al.Phosphodiesterase 4D(PDE4D) Gene Variants and Risk of Ischemic Stroke in the Taiwanese Population.Lab Medicine,2009,40(6):87-90.
2.Gabay C,Kushner I.Acute-phase proteins and other systemic responses to inflammation.N Engl J Med,1999,340(17):448-454.
3.Yudkin JS,Kumari M,Humphries SE,et al.Inflammation,obesity,stress and coronary heart disease:is interleukin-6 the link? Atherosclerosis,2000,148(2):209-214.
4.Chang MK,Binder C J,Torzewski M,et al.C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand:phosphorylcholine of oxidized phospholipids.Proc Acad Sci U S A,2002,99(20):13043-13048.
5.Ridker PM..C-reactive protein and the predition of cardiovascular events among those at intermediate risk:moving an inflammatory hypothesis toward consensus.J Am Coll Cardiol,2007,49(21):2129-2138.
6.Bansal S,Ridker PM.Comparison of characteristics of future myocardial infarction in women with baseline high versus baseline low levels of hs-C-reactive protein.Am J Cardiol,2007,99(11):1500-1503.
7.周玉,李岩松,潘风光,等.小鼠腹水IgG类单克隆抗体纯化方法的研究.黑龙江畜牧兽医,2006,10(6):41-61.
8.Bouvet JP.A modified gel filtration tenique predicting an unusual exclusion volume of IgM:a simple way of preparing monoclonal IgM.J Immumol Meth,1984,66(2):229-230.
9.Clegardin P.Trandem purification of mouse IgM monoclonal antibodies produced in vitro using anion-exchange and gel fest protein liquid chromatography.J Chromatogar,1986,358(1):209-218.
10.Jiskoot W.Two-step purification of a murine monoclonal anti-bodies intended for therapeutic application in man.Optimisation of purification condition and scaling up.J Immunol Meth,1989,124(1):143-156.
11.Lamoyi E.Preparation of F(ab)'fragrnents from mouse IgG of various subclasses.J Immunol Meth,1983,56(2):235-243.
12.Parham P.Monoclonal antibodies:Purification tragrnentation and application to structural and functional studies of class I MHC antigens.J Immunol Meth,1982,53(2):133-173.
13.Barton DE,Yang Feng TL,Mason A J,et al.Mapping of genes for inhibin submits alpha,teta A,and beta B on human and mouse chromosomes and studies ofjad mice.Genomics,1989,5(1):91-99.
14.Roberts V J,Barth SL.Expression of messenger ribonucleic acid encoding the inhibin lactivin system during mid- and late-gestation rat embogogenesis.Endocrinol,1994,134(2):914-923.
15.李红乐,王宏卫,夏辉明,等.应用Protein A-Sepharose CL-4B亲和层析法纯化单克隆抗体.河南医科大学学报,1999,34(4):11-12.
16.林清华.免疫学实验.武汉:武汉大学出版社,1999:124-128.
17.Steven B,Irving K,David S.C-reactive protein.J Biol Chem,2004,279(47):48487-48490.
18.Schwick HG,Haupt H.Properties of acute phase proteins of human plasma.Behring Institute Mitteilungen 1986:1-10.
19.Hutchinson WL,Kahan MC,et al.Transgenic human CRP is not pro-atherogenic,pro-atherothrombotic or pro-inflammatory in apoE-/-mice.Atherosclerosis,2008,196(1):248-255.
20.Volanakis JE,Narkates AJ.Interaction of C-reactive protein with artificial phosphatidylcholine bilayers and complement.J Immunol,1981,126(5):1820-1825.
21.Volanakis JE,Kaplan MH.Interaction of C-reactive protein complexes with the complement system.Ⅱ.Consumption of guinea pig complement by CRP complexes:requirement for human Clq.J Immunol,1974,113(6):2135-2147.
22.Ikuta T,Okubo H,Yukimura H,et al.Purification of C-reactive protein.Biochemistry international,1983,6(2):275-282.
23.Volanakis JE,Wirtz KW.Interaction of C-reactive protein with artificial phosphatidylcholine bilayers.Nature,1979,281(1):155-157.
24.Li JJ,Fang CH.C-reactive protein is not only an inflammatory marker but also a direct cause of cardiovascular disease.Med Hypothese,2004,62 (6):499-506.
25.Fujita Y,Kakino A,Nishimichi N,et al.Oxidized LDL Receptor LOX-1 Binds to C-Reactive Protein and Mediates Its Vascular Effects,linical Chemistry,2009,55 (2):285-294.
26.Zwaka TP,Hombach V,Torzewski J.C-Reactive Protein-Mediated Low Density Lipoprotein Uptake by Macrophages.Circulation,2001,103 (9):1194-1197.
27.Ridker PM.Clinical application of C-reactive protein for cardiovascular disease detection and prevention.Circulation,2003,107 (3):363-369.
28.Ridker PM,Hennekens CH,Buring JE,et al.C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women.N Engl J Med,2000,342 (7):836-843.
29.Ridker PM.High-sensitivity C-reactive protein:potential adjunct for global risk assessment in the primary prevention of cardiovascular disease.Circulation,2001,103 (13):1813-1818.
30.Pischon T,Hu FB,Rexrode KM,et al.Inflammation,the metabolic syndrome,and risk of coronary heart disease in women and men.Atherosclerosis,2008,197 (1):392-399.
31.Bassuk SS,Rifai N,Ridker PM.High-sensitivity C-reactive protein:clinical importance.Curr Probl Cardiol,2004,29 (8):439-493.
32.Biasucci LM,Liuzzo G,Bona RD,et al.Different Apparent Prognostic Value of hsCRP in Type 2 Diabetic and Nondiabetic Patients with Acute Coronary Syndromes.Clinical Chemistry,2009,55 (10):365-368.
1.Steven B,Irving K,David S.C-reactive protein.J Biol Chem,2004,279(47):48487-48490.
2.Pearson T,Ballantyne C,.Veltri E,et al.Pooled Analyses of Effects on C-Reactive Protein and Low Density Lipoprotein Cholesterol in Placebo-Controlled Trials of Ezetimibe Monotherapy or Ezetimibe Added to Baseline Statin Therapy.The American Joumal of Cardiology,2009,103(3):369-374.
3.Du Clos TW.Function of C-reactive protein.Ann Med,2000,32(4):274-278.
4.Thompson D,Pepys MB,Wood SP.The physiological structure of human C-reactive protein and its complex with phosphocholine.Structure,1999,7(2):169-177.
5.Whitehead AS,Bruns GAP,Colten HR,et al.Isolation of human C-reactive protein complementary DNA and localization of the gene to chromosome 1.Science,1983,221(1):69-71.
6.Chang MK,Binder C J,Torzewski M,et al.C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand:Phosphorylcholine of oxidized phospholipids.Proc.Natl.Acad.Sci.U.S.A,2002,99(20):13043-13048.
7.Zwaka TP,Hombach V,Torzewski J.C-Reactive Protein-Mediated Low Density Lipoprotein Uptake by Macrophages.Circulation,2001,103(9):1194-1197.
8.Griselli M,Herbert J,Hutchinson WL,et al.C-reactive Protein and Complement Are Important Mediators of Tissue Damage in Acute Myocardial Infarction.Journal of Experimental Medicine,1999,190 (12):1733-1740.
9.Pischon T,Hu F,Rexrode K,et al.Inflammation,the metabolic syndrome,and risk of coronary heart disease in women and men.Atherosclerosis,2008,197 (1):392-399.
10.Mora S,Musunuru K,Blumenthal RS.The Clinical Utility of High-Sensitivity C-Reactive Protein in Cardiovascular Disease and the Potential Implication of Jupiter on Current Practice Guidelines.Clinical Chemistry,2009,5 (10):219-228.
11.Ridker PM.Clinical Application of C-Reactive Protein for Cardiovascular Disease Detection and Prevention.Circulation,2003,107 (3):363-369.
12.Wang TJ,Gona P,Larson MG,et al.Multiple Biomarkers for the Prediction of First Major Cardiovascular Events and Death.The New England journal of medicine,2006,355 (25):2631-2639.
13.Pasceri V,Willerson JT,Yeh E T.Direct proinflammatory effect of C-reactive protein on human endothelial cells.Circulation,2000,102 (18):2165-2168.
14.Ridker PM,Cannon CP,Morrow D,et al.C-Reactive Protein Levels and Outcomes after Statin Therapy.New England Journal of Medicine,2005,352 (1):20-28.
15.Blake GJ,Ridker PM.Novel clinical markers of vascular wall inflammation.Circ Res,2001,89(9):763-771.
16.Lowe GDO,Pepys MB.C-reactive protein and cardiovascular disease:Weighing the evidence.Current Cardiovascular Risk Reports,2006,8 (5):421-428.
17.Ridker PM,Rifai N,Rose L.Comparison of C-Reactive Protein and Low-Density Lipoprotein Cholesterol Levels in the Prediction of First Cardiovascular Events.New England Journal of Medicine,2002,347 (14):1557-1565.
18.Medina-Urrutia A,Juarez-Rojas J,Martinez-Alvarado R,et al.High-density lipoprotein subclasses distribution and composition in Mexican adolescents with low HDL cholesterol and/or high triglyceride concentrations,and its association with insulin and c-reactive protein.Atherosclerosis,2008,201 (2):392-397.
19.Hübner-Wo(?)niak E,Malara M,Ok(?)cka-Szyma(?)ska J,et al.Cholesterol fractions,C-reactive protein and creatine kinase activity in plasma of female athletes.Physical Education and Sport,2008,52 (26):88-91.
20.Li J J,Fang C H,Chen MZ,et al.Rapid effects on lipid profile and C-reactive protein by simvastatin in patients with hypercholesterolemia.Clin Cardiol,2003,26(3):472-476.
21.Rifai N,Ridker PM.High-sensitivity C-reactive protein:a novel and promising marker of coronary heart disease.Clin Chem,2001,47 (3):403-411.
22.Paul A,Ko KWS,Li L,et al.C-Reactive Protein Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice.Circulation,2004,109(5):647-655.
23.Pepys MB,Hirschfield GM,Tennent GA,et al.Targeting C-reactive protein for the treatment of cardiovascular disease.Nature,2006,440 (7088):1217-1221.
1.Gretarsdottir S,Sveinbjomsdottir S,Jonsson HH,et al.The gene encoding phosphodiesterase 4D confers risk of ischemic stroke.Nat Genet,2003,35 (1):131-138.
2.吴丽娥,刘鸣,张月辉,等.缺血性卒中TOAST病因分型和预后.中华神经科杂志.2004,37(4):292-295.
3.Houslay,MD,Adams,DR.PDE4 cAMP phosphodiesterase:modular enzymes that orchestrate signaling cross-talk,desensitization and compartmentalization.Biochem.J,2003,370(1):1-18.
4.Qing Song,John W.Cole,Jeffrey R.O'Connell,et al.Phosphodiesterase 4D polymorphisms and the risk of cerebral Infarction in a biracial population:the Stroke Prevention in Young Women Study.Hum Mol Genet,2006,15(16):2468-2478.
5.杨晓军.磷酸二酯酶4D基因与缺血性卒中.国外医学脑学管疾病分册,2004,12(4):310-312.
6.Meschia JF,Brott TG,Brown RD,et al.Phosphodiesterase 4D and 5-lipoxygenase activating protein in ischemic stroke.Ann Neurol,.2005,58(3):351-361.
7.Woo D,Kaushal R,Kissela B,et al.Association of phosphodiesterase 4D with ischemic stroke:A population,based case-control study.Stroke,2006,37(2):371-376.
8.Saleheen D,Bukhari S,Haider SR,et al.Association of Phosphodiesterase 4D gene with ischemic stroke in a Pakistani population.Stroke,2005,36(10):2275-2277.
9.Nakayama T,Asais S,Sato N,et al.Genotype and haplotype association study of STRK 1 region on 5q12 among Japanese:a case-control study.Stroke,2006,37(1):69-76.
10.Xue H,Wang H,Song X,et al.Phosphodiesterase 4D gene polymorphism is associated with ischaemic and haemorrhagic stroke.Clin Sci(Lond),2009,116(4):335-340.
11.Lohmussaar E,Gschwendtner A,Mueller JC,et al.ALOX5AP Gene and the PDE4D gene in a central European population of stroke patients.Stroke,2005,36(10):731-736.
12.Bevan S,Porteous L,Sitzer M,et al.Phosphodiesterase 4D gene,Ischemic stroke,and asymptomatic carotid atherosclerosis.Stroke,2005,36(5):949-953.
13.Nilsson-Ardnor S,Wiklund PG,Lindgren P,et al.Linkage of ischemic stroke to the PDE4D region on 5q in a Swedish population.Stroke,2005,36(8):1666-1671.
14.李明才,张成,张鸿炼,等.磷酸二酯酶4D基因与缺血性卒中的遗传易感性研究.中华神经科杂志,2006,39(3):180-182.
15.徐波,邓春艳,张彩英,等.磷酸二酯酶4D基因三个多态性与缺血性卒中的关系.医学世界,2007,2(3):55-57.
16.Hsieh MS,Yu SC,Chung WT,et al.Phosphodiesterase 4D(PDE4D) Gene Variants and Risk of Ischemic Stroke in the Taiwanese Population.Lab Medicine,2009,40(6):87-90.