蛋白质芯片技术定量检测血清铁蛋白和可溶性转铁蛋白受体方法的研究
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摘要
目的建立一个利用蛋白质芯片技术同时检测血样铁蛋白、可溶性转铁蛋白受体的检测方法,并对此检测方法的应用进行评价。
方法以Western Blot印迹技术对所选抗原SF、sTfR的抗原性、抗原与抗体特异性结合能力进行验证。以酶联免疫技术对所选抗体定量检测目标蛋白进行抗体浓度确定、灵敏度确定、线性范围评价、标准曲线建立、及精密度准确度评价。利用所选抗体,进行蛋白质芯片夹心技术定量检测目标蛋白的固定探针选择、点样方式选择、点样一致性比较、抗体特异性交叉反应、固定探针与检测抗体浓度优化、封闭时间比较、封闭剂与二抗优化、灵敏度分析、线性范围评价与标准曲线建立、可报告范围确定、精密度与准确度确定及干扰性评价、方法稳定性评价、检测时效评价等研究。
结果Western Blot实验结果证明所选抗原具有稳定的抗原性,所选抗体具有与目标抗原特异性结合的能力,能够用于进一步的方法建立。酶联免疫实验初步建立了对两种目标蛋白进行定量检测的技术方法,为完成蛋白芯片检测方法奠定了基础。蛋白质芯片检测SF、sTfR的检测方法最终选用鼠抗做为固定探针、以接触式点样为点样方式、选取前40点为预点样区段、40—200点为点样一致性区段、交叉反应实验结果显示不同抗体与不同抗原之间不存在交叉反应；SF固定探针与检测抗体的浓度确定为0.5 mg/ml与5μg/ml, sTfR固定探针与检测抗体的浓度确定为0.5 mg/ml与0.36μg/ml;不同封闭时间对检测结果不产生有统计学差异的影响；选择以3%BSA为封闭剂、以GE公司的Cy3标记的羊抗兔IgG为二抗；SF检测低限为0.788 ng/ml、生物检测下限为2 ng/ml, sTfR的检测低限为0.446ng/ml,生物检测限为1.56 ng/m；分别选择以一项式回归方程为SF检测回归方程、以二项式回归方程为sTfR检测回归方程；方法所确定的二者的可报告范围分别是：SF可报告上限为4100 ng/ml、下限为2 ng/ml, sTfR可报告上限为5000 ng/ml、下限为1.56 ng/ml;SF批内变异系数最大时不超过8.69%,批间变异系数最大时不超过18.38%,sTfR批内变异系数最大时不超过8.88%,批间变异系数最大时不超过12.8%；SF稀释回收率最小时为104%,加标回收率的系统比例误差最小时不超过5%,sTfR稀释回收率最小时为107.5%,加标回收率最小时不超过5%；方法学比较的配对t检验结果显示,蛋白芯片检测SF与sTfR的结果与比浊法间的差异无统计学意义,蛋白芯片法检出铁缺乏者的能力与比浊法间的差异经X2检验无统计学意义；人血清白蛋白与甘油三油酸酯对蛋白芯片检测方法不存在干扰；蛋白芯片在点样后38天之内检测浓度结果间的差异无统计学意义；在完成二抗杂交后22天之内,蛋白芯片的浓度结果间的差异无统计学意义。
结论建立了利用蛋白质芯片技术同时检测SF与sTfR的检测方法,此方法具有较好的精准度与实用性,能够满足实验室检测需要。
Object The objective of this research is to establish a protein microarray measurement method for combined measurement of serum ferritin (SF) and soluble transferrin receptor (sTfR) and to estimate the method.
Method To validate the activity of the antigen and the specificity of the antibody by Western Blot. To obtain the concentration of antibody, sensitivity, standard curve, the range of report of SF and sTfR by Enzyme-Linked Immunoassay (ELISA) detection. To choose the probe, printing method, homogeneity of the spots of SF and sTfR probe, the specificity cross-reaction, the optimized concentration of probe and detection antibody, the blocking time,the optimized blocking reagents and second antibodies, the sensitivity, the standard curve, range of reportable, theprecision and accuracy, and to estimate the interference, stability, detection time limitation and the detection cost.
Result The result of Western Blot and ELISA showed that the antibody and antigen could be used in the further study of protein mciroarray measurement. In the established protein chip method, the mouse antibody was selected as the probe, the contact printing as the printing method and the pre-printing in 40 spots The homogeneity range of the spots of SF and sTfR probe was from the 40th spot to 200th and the cross-reaction experiment result suggested that there is no cross-reaction between different antibody and antigen. The concentrations of SF probe and detection antibody were 0.5mg/ml and 5μg/ml, and 0.5 mg/ml and 0.36μg/ml for sTfR. The 3% BSA and goat anti-rabbit IgG antibody of GE were chosen as the blocking reagent and second antibody. Differences among different blocking time didn't affect on the results of dection. The lower limit of detection and biologic limit of detection of SF was 0.788ng/ml and 2 ng/ml and 0.446 ng/ml and 1.56 ng/ml for sTfR. The linear equation was used to detect the SF and the quadratic equation was usedto detect the sTfR. The range of reportable was from 2 ng/ml to 4100 ng/ml for SF and from 1.56 ng/ml to 5000 ng/ml for sTfR. The interior coefficient of variation (CV) and the intermediate CV for SF were less than 8.69% and 18.38% respectively and for sTfR were less than 8.88% and 12.8% respectively. The dilution recovery ratios for SF and sTfR were 104% and 107.5% respectively. The minimal sysytem ratio error was less than 5% for both of SF and sTfR. There was no statistics difference between protein chip detection method and Immunoturbidimetry for SF and sTfR detection by paired design and the ability to finding iron deficiency between them by Chi-square test. There were no interference observed in SF and sTfR measurement in one protein microarray chip. The protein chip could keep stable in 38 days in this study and it can be scan in 22days after finished the experiment.
Conclusions A protein microarray quantitative analysis method was established for simultaneous measurement of SF and the method could be applied in the measurement of SF and sTfR.
方法以Western Blot印迹技术对所选抗原SF、sTfR的抗原性、抗原与抗体特异性结合能力进行验证。以酶联免疫技术对所选抗体定量检测目标蛋白进行抗体浓度确定、灵敏度确定、线性范围评价、标准曲线建立、及精密度准确度评价。利用所选抗体,进行蛋白质芯片夹心技术定量检测目标蛋白的固定探针选择、点样方式选择、点样一致性比较、抗体特异性交叉反应、固定探针与检测抗体浓度优化、封闭时间比较、封闭剂与二抗优化、灵敏度分析、线性范围评价与标准曲线建立、可报告范围确定、精密度与准确度确定及干扰性评价、方法稳定性评价、检测时效评价等研究。
结果Western Blot实验结果证明所选抗原具有稳定的抗原性,所选抗体具有与目标抗原特异性结合的能力,能够用于进一步的方法建立。酶联免疫实验初步建立了对两种目标蛋白进行定量检测的技术方法,为完成蛋白芯片检测方法奠定了基础。蛋白质芯片检测SF、sTfR的检测方法最终选用鼠抗做为固定探针、以接触式点样为点样方式、选取前40点为预点样区段、40—200点为点样一致性区段、交叉反应实验结果显示不同抗体与不同抗原之间不存在交叉反应；SF固定探针与检测抗体的浓度确定为0.5 mg/ml与5μg/ml, sTfR固定探针与检测抗体的浓度确定为0.5 mg/ml与0.36μg/ml;不同封闭时间对检测结果不产生有统计学差异的影响；选择以3%BSA为封闭剂、以GE公司的Cy3标记的羊抗兔IgG为二抗；SF检测低限为0.788 ng/ml、生物检测下限为2 ng/ml, sTfR的检测低限为0.446ng/ml,生物检测限为1.56 ng/m；分别选择以一项式回归方程为SF检测回归方程、以二项式回归方程为sTfR检测回归方程；方法所确定的二者的可报告范围分别是：SF可报告上限为4100 ng/ml、下限为2 ng/ml, sTfR可报告上限为5000 ng/ml、下限为1.56 ng/ml;SF批内变异系数最大时不超过8.69%,批间变异系数最大时不超过18.38%,sTfR批内变异系数最大时不超过8.88%,批间变异系数最大时不超过12.8%；SF稀释回收率最小时为104%,加标回收率的系统比例误差最小时不超过5%,sTfR稀释回收率最小时为107.5%,加标回收率最小时不超过5%；方法学比较的配对t检验结果显示,蛋白芯片检测SF与sTfR的结果与比浊法间的差异无统计学意义,蛋白芯片法检出铁缺乏者的能力与比浊法间的差异经X2检验无统计学意义；人血清白蛋白与甘油三油酸酯对蛋白芯片检测方法不存在干扰；蛋白芯片在点样后38天之内检测浓度结果间的差异无统计学意义；在完成二抗杂交后22天之内,蛋白芯片的浓度结果间的差异无统计学意义。
结论建立了利用蛋白质芯片技术同时检测SF与sTfR的检测方法,此方法具有较好的精准度与实用性,能够满足实验室检测需要。
Object The objective of this research is to establish a protein microarray measurement method for combined measurement of serum ferritin (SF) and soluble transferrin receptor (sTfR) and to estimate the method.
Method To validate the activity of the antigen and the specificity of the antibody by Western Blot. To obtain the concentration of antibody, sensitivity, standard curve, the range of report of SF and sTfR by Enzyme-Linked Immunoassay (ELISA) detection. To choose the probe, printing method, homogeneity of the spots of SF and sTfR probe, the specificity cross-reaction, the optimized concentration of probe and detection antibody, the blocking time,the optimized blocking reagents and second antibodies, the sensitivity, the standard curve, range of reportable, theprecision and accuracy, and to estimate the interference, stability, detection time limitation and the detection cost.
Result The result of Western Blot and ELISA showed that the antibody and antigen could be used in the further study of protein mciroarray measurement. In the established protein chip method, the mouse antibody was selected as the probe, the contact printing as the printing method and the pre-printing in 40 spots The homogeneity range of the spots of SF and sTfR probe was from the 40th spot to 200th and the cross-reaction experiment result suggested that there is no cross-reaction between different antibody and antigen. The concentrations of SF probe and detection antibody were 0.5mg/ml and 5μg/ml, and 0.5 mg/ml and 0.36μg/ml for sTfR. The 3% BSA and goat anti-rabbit IgG antibody of GE were chosen as the blocking reagent and second antibody. Differences among different blocking time didn't affect on the results of dection. The lower limit of detection and biologic limit of detection of SF was 0.788ng/ml and 2 ng/ml and 0.446 ng/ml and 1.56 ng/ml for sTfR. The linear equation was used to detect the SF and the quadratic equation was usedto detect the sTfR. The range of reportable was from 2 ng/ml to 4100 ng/ml for SF and from 1.56 ng/ml to 5000 ng/ml for sTfR. The interior coefficient of variation (CV) and the intermediate CV for SF were less than 8.69% and 18.38% respectively and for sTfR were less than 8.88% and 12.8% respectively. The dilution recovery ratios for SF and sTfR were 104% and 107.5% respectively. The minimal sysytem ratio error was less than 5% for both of SF and sTfR. There was no statistics difference between protein chip detection method and Immunoturbidimetry for SF and sTfR detection by paired design and the ability to finding iron deficiency between them by Chi-square test. There were no interference observed in SF and sTfR measurement in one protein microarray chip. The protein chip could keep stable in 38 days in this study and it can be scan in 22days after finished the experiment.
Conclusions A protein microarray quantitative analysis method was established for simultaneous measurement of SF and the method could be applied in the measurement of SF and sTfR.
引文
[1]WHO/UNICEF/UNU Iron Deficiency Anemia:Prevention Assessment and Control, Report of a Joint WHO/UNICEF/UNU Consultation. World Health Organization, Geneva, Switzerland.1998.
[2]KONHN A M, HERSHKO C. Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release[J]. BrJ Haem,1977,37:7-16.
[3]BRUGNARA C, ZURAKOWSKI D, DICANZIO J, et al. Reticulocyte hemoglobin content to diagnoseiron deficiency in children[J]. JAMA,1998,281:2225-2230.
[4]LABBE R F, DEWANJ I A. Iron assessment tests:transferrin receptor vis-avis zinc protoporphyrin[J]. Clin Biochem,2004,37:165-174.
[5]MARY E COGSWELL, ANNE C LOOKER, CHRISTINE M PFEIFFER,et al. Assessment of iron deficiency in US preschool children and nonpregnant females of childbearing age:National Health and Nutrition Examination Survey 2003-2006[J]. Am J Clin Nutr,2009,89 (5):1334-1342.
[6]FRANK T WIERINGA, J ACQUES BERGER, MARJOLEINE A DIJKHUIZEN, et al. Combined Iron and Zinc supplementation in infants improved iron and zinc status, but interactions reduced efficacy in a multicountry trial in southeast asia[J]. Nutr,2007,137(2):466-471.
[7]JUERGEN G.E., JOHN E., CHRESTINE M.P, et al.Combined measurement of ferritin, soluble transferrin receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J]. J. Nutr,2004,134(11):3172-3132.
[8]ANITA J. FUGLESTAD, ASHTON E. LEHMANN,et al.Iron deficiency in international adoptees from eastern europe[J]. J. Ped,2008,153(8):272-277
[9]KUNIHIKO HASHIMOTO,SANAINOGUCHI, YASUHIKO MORIMOTO, et al. HbA1C, but not serum glycated albumin, is elevated in late pregnancy due to iron deficiency [J]. DIABETES CARE,2008,7.
[10]S ARNON, T DOLFIN, S BAUER, et al. Iron supplementation for preterm infants receiving restrictive red blood cell transfusions:reassessment of practice safety[J]. J PERINATOL,2010,3
[11]PAULI SUOMINEN, KARIPUNNONEN, ALLAN RAJAMAKI, et al. Automated immunoturbidimetric method for measuring serum transferrin receptor[J]. Clin Chem,1999,45(8):1302-1305.
[12]RIIKKA VIKSTEDT, PIIA VON LODE, TIMO TAKALA, et al.Rapid one-step immunofluorometric assay for measuring soluble transferrin receptor in whole blood[J].Clin Chem,2004,50(10):1831-1833.
[13]CARRIAGA M T, SKIKNE B S, FINLEY B, et al. Serum transferrin receptor for the detection of iron deficiency in prgnancy[J]. Am J Clin Nutr,1991,54:1077-1081.
[14]SKIKNE B S.Circulating Transferrin Receptor Assay-Coming of Age[J].Clin Chem,1998,44(1):7-9
[15]BAYNES R D. Refining the assessment of body iron status[J]. Am J Clin Nutr.1996,64:793-794.
[16]梁建功,何治柯.蛋白质芯片及其分析应用新进展.分析化学,2004,32(2)：244-247.
[17]LuekingA,HornM,EickhoffH,etal.Proteinmicroarrays for Gene Expression and Antibody Screening[J].Anal Biochem,1999,270(1):103-111.
[18]Haab BB,Dunham MJ,Brown PO.Proteinmicroarrays for highly parallel detection and quantitation of specific proteins and antibodies in complexsolutions[J].GenomeBiol,2001,2(2):RESEARCH004.
[19]彭聪,李桂源.蛋白质组与蛋白质芯片[J].国外医学.2003,23(3)：123
[20]Li Min,Zhou Zongcan. Proteinchip [J].Chemistry of Life,2001,21(2):156-157
[21]Zhu H,Snyder M. Protein arrays and microarrays.Curr.Opin.Chem.Biol.,2001 Feb,5(1):40-45
[22]Yuan Quan, Zhao Fukun.New Frontiers in the Proteome Research:Quantitative Proteomics[J].Acta Bioph,Sin.,2001,33(5):477-482.
[23]Kodadek T. Protein microarrays:prospects and problems[J].Chemistry &Biology,2001Feb,8(2):105-115.
[24]陈忠斌.生物芯片技术.化学工业出版社,2005：126-127.
[25]MACBEATH G, SCHREIBER S L. Printing proteins as microarrays for high throughput functior determination[J].Science,2000,289(5485):1760-1763.
[26]ZHOU H, ROY S, SCHULMAN H, et al. Solution and chip arrays in protein profiling[J].Trends Biotechnol,2001,19(10 Supply):534-539.
[27]LEE YS, MRKSICH M. Protein chips:from concept to practice[J]. Trends Biotechnol,2002,20(12 Suppl):S14-18.
[28]KODADEK T. Development of protein-detecting microarrays and related devices[J]. Trends Biochem Sci,2002,27(6):295-300.
[29]高志贤,王艳,房彦军.小分子阿特拉津和罂粟碱检测的免疫芯片技术研究[J].分析化学,2005,33(4)：455-458.
[30]Grow AE, Wood LL, Claycomb JL,et al. New biochip technology for label-free detection of pathogens and their toxins [J].J Microbiol Methods,2003,53(2):221-233.
[31]Stokes DL, Griffin GD, Vo-Dinh T, et al. coli using a microfluidics-based antibody biochip detectior system [J].Fresenius J Anal Chem,2001,369(3-4):295-301.
[32]林果为.铁蛋白测定的临床意义[J].临床内科杂志,1989,6(3)：11-12
[33]Matthias Kaup, Katrin Dassler, Christoph Weise et al. Shedding of the Transferrin Receptor Is Mediatec Constitutively by an Integral Membrane Metalloprotease Sensitive to Tumor Necrosis Factor a Protease Inhebitor-2.The Journal of Biological Chemistry[J].2002,277(41):38494-38502.
[34]冯丰仁.分析灵敏度(检测限)[J].上海医学检验杂志,2002,17(3)：133-136.
[35]张海晨,宋云霄.血清可溶性转铁蛋白受体参考范围的建立及其在缺铁性贫血诊断中的意义[J].诊断学理论与实践,2008,7(3)：339-340.
[36]黄颖.标记免疫分析的质量控制和标准化(二)[J].标记免疫分析与临床,2006,13(4)：240-243.
[37]黄颖.标记免疫分析的质量控制和标准化(一)[J].标记免疫分析与临床,2006,13(3)：171-174.
[38]钟春英,彭蓉,彭建新.蛋白质芯片技术[J].生物技术通报,2004,2：34-37.
[39]STEARS RL, MARTINSKY T, SCHENA M. Trends in microarray analysis[J].Techno(?) Trends,2003,9:140-145.
[40]NEWMAN J D, TURNER A P F. Ink-jet printing for the fabrication of amperometric glucose biosensors[J]. Anal Chim Acta,1992,262:13-17.
[41]BLANCHARD A P, KAISER R J, HOOD L E. High-density oligonucleotide arrays[J]. Biosens Bic electr,1996,11:687-690.
[42]EMMO A V, FISHER J T. GEYSEN H M et al. Characterization of an inkjet chemical microdispense(?) for combinatorial library synthesis[J]. Anal Chem,1997,69:543-551.
[43]Mark Schena. Protein Microarrays.Jones and Bartlett Publishers,2005:175.
[44]江凌晓,郭兆彪,陈泽良,等.蛋白质芯片制作条件的优化[J].第一军医大学学报,2004,24(11)：1230-1232.
[45]陈保民,陈维娜.酶联免疫吸附实验全白动检测的控制[J].职业与健康,2002,18(5)：46-47.
[46]蒋理,徐建,杨瑞霞.散射比浊法测定血清铁蛋白的方法学评价[J].放射免疫学杂志,200821(2)：182-183.
[47]COOK J D, FLOWERS C H, SKIKNE B S. The quantitative assessment of body iron[J].Blood 2003,101:3359-3364.
[48]JUERGEN G E, JOHN E, CHRESTINE M P, et al. Combined measurement of ferritin, soluble transferrin receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J].J Nutr,2004,134:3127-3132.
[49]齐斌,谷文英,谢岩黎.酶联免疫吸附法检测黄精皂苷[J].吉林农业大学学报,2006,28(3)：292-295.
[50]苏敏,沙玲,台虹STAGO-STA型全自动血凝仪APTT测定总精密度评价[J].中国医学检验杂志,2001,2(5)：348-350.
[51]胡瑜,成立科.血清转铁蛋白受体与血清铁鉴别缺铁性贫血和慢性病贫血的意义[J].江苏大学学报(医学版),2002,12(1)：44-46
[52]李剑虹,朴建华,杨丽琛等.血红蛋白判断育龄妇女缺铁性贫血阈值研究[J].中国公共卫生,2008,24(2)：157-158.
1葛可佑.中国营养科学全书(上、下册)[M].第一版.北京：人民卫生山版社,2004：129.
2 WHO/UNICEF/UNU Iron Deficiency Anemia:Prevention Assessment and Control, Report of a Join WHO/UNICEF/UNU Consultation. World Health Organization, Geneva, Switzerland.1998.
3 KONIIN AM, HERSHKO C. Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release[J] BrJ Haem,1977,37:7-16.
4 BRUGNARA C, ZURAKOWSKI D, DICANZIO J, et al. Reticulocyte hemoglobin content to diagnoseiror deficiency in children[J]. J AMA,1998,281:2225-2230.
5 LABBE R F, DEWANJ I A. Iron assessment tests:transferrin receptor vis-avis zinc protoporphyrin[J]. Clir Biochem,2004,37:165-174.
6 DAVID A L, BARRY A SKIKNE, CAROLYN THOMPSON. Precision and accuracy of serum ferritir measurements[J]. Am J Clin Nutr,1981,34:951.
7刘绣云.放射免疫法与酶联免疫法测定血清铁蛋白的比较[J].营养学报,1985,7(2)：130-136.
8 DUPUY AM, DEBARGE L, POULAIN M et al. Determination of serum ferritin using immunoturbidimetry or chemiluminescent detection in comparison with radioimmunoassay a compendium o(?) a methodological juxtaposition[J]. Clin Lab,2009,55(5-6):207-15.
9董毅,刘蕾,朱太岗,等.血.清铁蛋白检测在贫血.性疾病中的临床价值[J].中华全科医学2010,8(4)：437-438.
10张宏,李强,李友生.乙肝肝硬化患者血清铁蛋白、转铁蛋白水平检测及意义[J].标记免疫分析与临床,2009,16(2)：75-76.
11孙件绣.血清铁蛋白放免测定在肿瘤疾病的临床应用[J].中国医疗前沿,2009,4(18)：61.
12 M PAGE, L THERTAULT, M NILSSON. Solid-phase ELISA for serum ferritin[J]. Clin Lab Invest 1980,40 (11):641-645.
13梁国栋,邓贤碧,江希蓉等.血清铁蛋白酶联免疫测定法[J].第四军医大学学报,1982(2)：107-110.
14 MARY E COGSWELL, ANNE C LOOKER, CHRISTINE M PFEIFFER,et al. Assessment of iror deficiency in US preschool children and nonpregnant females of childbearing age:National Health and Nutrition Examination Survey 2003-2006[J]. Am J Clin Nutr,2009,89 (5):1334-1342.
15 FRANK T WIERINGA, J ACQUES BERGER, MARJOLEINE A DIJKHUIZEN, et al. Combined Iror and Zinc supplementation in infants improved iron and zinc status, but interactions reduced efficacy in a multicountry trial in southeast asia[J]. Nutr,2007,137(2):466-471.
16 JUERGEN G.E., JOHN E., CHRESTINE M.P, et al.Combined measurement of ferritin, soluble transferrir receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J]. J.Nutr,2004,134(11):3172-3132.
17杨秀岑,伍莉萍,陈达进等.用化学发光法测定辣根过氧化物酶几种体系的研究[J].华西医大学报1990,21(3)：293-297.
18 ANITA J. FUGLESTAD, ASHTON E. LEHMANN,et al.Iron deficiency in international adoptees from eastern europe[J]. J. Ped,2008,153(8):272-277
19 KUNIHIKO HASHIMOTO,SANAI NOGUCHI, YASUHIKO MORIMOTO, et al. HbA1C, but not serurr glycated albumin, is elevated in late pregnancy due to iron deficiency [J]. DIABETES CARE,2008,7.
20 S ARNON, T DOLFIN, S BAUER, et al. Iron supplementation for preterm infants receiving restrictive rec blood cell transfusions:reassessment of practice safety[J]. J PERINATOL,2010,3
21常玉荣,葛庆峰,刘培光.血清铁、铁蛋白和铁染色对缺铁性贫血的诊断价值[J].华北煤炭医学院学报,2007,9(4)：500-501.
22安镜如,林金明,陈曦.电化学发光研究及其在分析化学上的应用[J].分析化学,1991,19(11)： 1340-1346.
23李桂彩,陈德平,肖静坤.电化学发光免疫法与放射免疫法测定血清铁蛋白的评价[J].实用医技杂志,2006,5(6)：853-855.
24蒋理,徐建.散射比浊法测定血清铁蛋白的方法学评价[J].放射免疫学杂志,2008,21(2)：182-183.
25 KOHGO Y, NISHISATO T, KONDO H, et al. Circulating transferrin receptor in human serum[J]. Br J Haematol,1986,64:277.
26 HUEBERS HA, BEGUIN Y, POOTRAKUL P, et al. Intact transferrin receptors in human plasma and their relation to erythropoiesis[J]. Blood,1990,75(1):102-107.
27 CH FLOWERS, B S SKIKNE, A M COVELL,et al. The clinical measurement of serum transferrin receptor[J]. J Lab Clin Med,1989,114(4):368-377.
28 Cook J D, Flowers C H, Skikne B S. The quantitative assessment of body iron[J].Blood,2003,101:3359-3367.
29李强,苟清,廖清奎,等.单克隆及多克隆双抗体夹心ELISA法测定血清转铁蛋白受体[J].华西医大学报,1995,26(4)：398-402.
30吴鹏,王学文,庄昱洲,等.可溶性转铁蛋白受体及其复合参数在缺铁性贫血及慢性病贫血鉴别诊断中的意义[J].医学研究生学报,2003,16(6)：438-441.
31 PAULI SUOMINEN, KARI PUNNONEN, ALLAN RAJAMAKI, et al. Automated immunoturbidimetric method for measuring serum transferrin receptor[J]. Clin Chem,1999,45(8):1302-1305.
32卢忠,沈俊娅sTfR对慢性炎症患者并发缺铁性贫血的诊断价值[J].放射免疫学杂志,2007,20(1)：66-68.
33 RIIKKA VIKSTEDT, PIIA VON LODE, TIMO TAKALA, et al.Rapid one-step immunofluorometric assay for measuring soluble transferrin receptor in whole blood[J].Clin Chem,2004,50(10):1831-1833.
34 WHO/UNICEF/UNU Iron Deficiency Anemia:Prevention Assessment and Control, A guide for programme managers. World Health Organization, Geneva, Switzerland.2001.
35 CLYNEB,JONATHAN S OLSHAKER.The C-reactive protein[J]. J Emer Med,1999,17(6):1019-1025.
36陈杭,杨洁.C反应蛋白——天然防御蛋白研究进展[J].细胞生物学杂志,2003,25(5)：264-268.
37 S EDA, J KAUFMANN, M MOLWITZ, et al. A new method of measuring C-reactive protein, with a low limit of detection, suitable for risk assessment of coronary heart disease[J]. Scand J Clin Lab Invest Suppl, 1999,230:32-35.
38 M E WARIS, N J MELTOLA, J T SOINI, et al. Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein[J]. Anal Biochem,2002,309(1):67-74.
39 ALLEN J, BACKSTROM K R, COOPER J A, et al. Measurement of soluble transferrin receptor in serum of healthy adults[J]. Clin Chem,1998,44(l):35-39
40 SUOMINEN P, PUNNONEN K, RAJAMAKI A, et al. Evaluation of new immunoenzymometric assay for measuring soluble transferrin receptor to detection deficiency in anemic patients[J]. Clin Chem,1997, 43(9):1641-1646
41 BARRY S SKIKNE.Circulating transferrin receptor assay-coming of age[J].Clin Chem,1998,44(1):7-9
42庞鹏宇,揭玉丽,刘秀川等.血清可溶性转铁蛋白受体在缺铁性贫血诊断中的应用[J].中国热带医学,2007,7(8)：1483-1487.
43李剑虹,朴建华,杨晓光.转铁蛋白受体评估铁过量状况最佳临界值的研究[J].营养学报,2007,29(5)：431-434.
44张海晨,宋云霄.血清可溶性转铁蛋白受体参考范围的建立及其在缺铁性贫血诊断中的意义[J].诊断学理论与实践,2008,7(3)：339-340.
45杨振修.C-反应蛋白的检测[J].上海医学检验杂志,1999,14(5)：261-263.
46 ROBERTS WL, MOULTON L, LAW TC,et al. Evaluation of nine automated high-sentivity C-reactive protein method:implications for clinical and epidemiological applications Part 2. Clin Chem,2001 47(3):418-425.
47简序,王金和,程佩兰.C反应蛋白的临床研究进展[J].国外医学临床生物化学与检验学分册,200425(5)：471-473.
48鞠晓红,方德洋,马爱新等.C-反应蛋白水平的性别和年龄差异[J].广东医学,2007,28(6)：923-924.
[2]KONHN A M, HERSHKO C. Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release[J]. BrJ Haem,1977,37:7-16.
[3]BRUGNARA C, ZURAKOWSKI D, DICANZIO J, et al. Reticulocyte hemoglobin content to diagnoseiron deficiency in children[J]. JAMA,1998,281:2225-2230.
[4]LABBE R F, DEWANJ I A. Iron assessment tests:transferrin receptor vis-avis zinc protoporphyrin[J]. Clin Biochem,2004,37:165-174.
[5]MARY E COGSWELL, ANNE C LOOKER, CHRISTINE M PFEIFFER,et al. Assessment of iron deficiency in US preschool children and nonpregnant females of childbearing age:National Health and Nutrition Examination Survey 2003-2006[J]. Am J Clin Nutr,2009,89 (5):1334-1342.
[6]FRANK T WIERINGA, J ACQUES BERGER, MARJOLEINE A DIJKHUIZEN, et al. Combined Iron and Zinc supplementation in infants improved iron and zinc status, but interactions reduced efficacy in a multicountry trial in southeast asia[J]. Nutr,2007,137(2):466-471.
[7]JUERGEN G.E., JOHN E., CHRESTINE M.P, et al.Combined measurement of ferritin, soluble transferrin receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J]. J. Nutr,2004,134(11):3172-3132.
[8]ANITA J. FUGLESTAD, ASHTON E. LEHMANN,et al.Iron deficiency in international adoptees from eastern europe[J]. J. Ped,2008,153(8):272-277
[9]KUNIHIKO HASHIMOTO,SANAINOGUCHI, YASUHIKO MORIMOTO, et al. HbA1C, but not serum glycated albumin, is elevated in late pregnancy due to iron deficiency [J]. DIABETES CARE,2008,7.
[10]S ARNON, T DOLFIN, S BAUER, et al. Iron supplementation for preterm infants receiving restrictive red blood cell transfusions:reassessment of practice safety[J]. J PERINATOL,2010,3
[11]PAULI SUOMINEN, KARIPUNNONEN, ALLAN RAJAMAKI, et al. Automated immunoturbidimetric method for measuring serum transferrin receptor[J]. Clin Chem,1999,45(8):1302-1305.
[12]RIIKKA VIKSTEDT, PIIA VON LODE, TIMO TAKALA, et al.Rapid one-step immunofluorometric assay for measuring soluble transferrin receptor in whole blood[J].Clin Chem,2004,50(10):1831-1833.
[13]CARRIAGA M T, SKIKNE B S, FINLEY B, et al. Serum transferrin receptor for the detection of iron deficiency in prgnancy[J]. Am J Clin Nutr,1991,54:1077-1081.
[14]SKIKNE B S.Circulating Transferrin Receptor Assay-Coming of Age[J].Clin Chem,1998,44(1):7-9
[15]BAYNES R D. Refining the assessment of body iron status[J]. Am J Clin Nutr.1996,64:793-794.
[16]梁建功,何治柯.蛋白质芯片及其分析应用新进展.分析化学,2004,32(2)：244-247.
[17]LuekingA,HornM,EickhoffH,etal.Proteinmicroarrays for Gene Expression and Antibody Screening[J].Anal Biochem,1999,270(1):103-111.
[18]Haab BB,Dunham MJ,Brown PO.Proteinmicroarrays for highly parallel detection and quantitation of specific proteins and antibodies in complexsolutions[J].GenomeBiol,2001,2(2):RESEARCH004.
[19]彭聪,李桂源.蛋白质组与蛋白质芯片[J].国外医学.2003,23(3)：123
[20]Li Min,Zhou Zongcan. Proteinchip [J].Chemistry of Life,2001,21(2):156-157
[21]Zhu H,Snyder M. Protein arrays and microarrays.Curr.Opin.Chem.Biol.,2001 Feb,5(1):40-45
[22]Yuan Quan, Zhao Fukun.New Frontiers in the Proteome Research:Quantitative Proteomics[J].Acta Bioph,Sin.,2001,33(5):477-482.
[23]Kodadek T. Protein microarrays:prospects and problems[J].Chemistry &Biology,2001Feb,8(2):105-115.
[24]陈忠斌.生物芯片技术.化学工业出版社,2005：126-127.
[25]MACBEATH G, SCHREIBER S L. Printing proteins as microarrays for high throughput functior determination[J].Science,2000,289(5485):1760-1763.
[26]ZHOU H, ROY S, SCHULMAN H, et al. Solution and chip arrays in protein profiling[J].Trends Biotechnol,2001,19(10 Supply):534-539.
[27]LEE YS, MRKSICH M. Protein chips:from concept to practice[J]. Trends Biotechnol,2002,20(12 Suppl):S14-18.
[28]KODADEK T. Development of protein-detecting microarrays and related devices[J]. Trends Biochem Sci,2002,27(6):295-300.
[29]高志贤,王艳,房彦军.小分子阿特拉津和罂粟碱检测的免疫芯片技术研究[J].分析化学,2005,33(4)：455-458.
[30]Grow AE, Wood LL, Claycomb JL,et al. New biochip technology for label-free detection of pathogens and their toxins [J].J Microbiol Methods,2003,53(2):221-233.
[31]Stokes DL, Griffin GD, Vo-Dinh T, et al. coli using a microfluidics-based antibody biochip detectior system [J].Fresenius J Anal Chem,2001,369(3-4):295-301.
[32]林果为.铁蛋白测定的临床意义[J].临床内科杂志,1989,6(3)：11-12
[33]Matthias Kaup, Katrin Dassler, Christoph Weise et al. Shedding of the Transferrin Receptor Is Mediatec Constitutively by an Integral Membrane Metalloprotease Sensitive to Tumor Necrosis Factor a Protease Inhebitor-2.The Journal of Biological Chemistry[J].2002,277(41):38494-38502.
[34]冯丰仁.分析灵敏度(检测限)[J].上海医学检验杂志,2002,17(3)：133-136.
[35]张海晨,宋云霄.血清可溶性转铁蛋白受体参考范围的建立及其在缺铁性贫血诊断中的意义[J].诊断学理论与实践,2008,7(3)：339-340.
[36]黄颖.标记免疫分析的质量控制和标准化(二)[J].标记免疫分析与临床,2006,13(4)：240-243.
[37]黄颖.标记免疫分析的质量控制和标准化(一)[J].标记免疫分析与临床,2006,13(3)：171-174.
[38]钟春英,彭蓉,彭建新.蛋白质芯片技术[J].生物技术通报,2004,2：34-37.
[39]STEARS RL, MARTINSKY T, SCHENA M. Trends in microarray analysis[J].Techno(?) Trends,2003,9:140-145.
[40]NEWMAN J D, TURNER A P F. Ink-jet printing for the fabrication of amperometric glucose biosensors[J]. Anal Chim Acta,1992,262:13-17.
[41]BLANCHARD A P, KAISER R J, HOOD L E. High-density oligonucleotide arrays[J]. Biosens Bic electr,1996,11:687-690.
[42]EMMO A V, FISHER J T. GEYSEN H M et al. Characterization of an inkjet chemical microdispense(?) for combinatorial library synthesis[J]. Anal Chem,1997,69:543-551.
[43]Mark Schena. Protein Microarrays.Jones and Bartlett Publishers,2005:175.
[44]江凌晓,郭兆彪,陈泽良,等.蛋白质芯片制作条件的优化[J].第一军医大学学报,2004,24(11)：1230-1232.
[45]陈保民,陈维娜.酶联免疫吸附实验全白动检测的控制[J].职业与健康,2002,18(5)：46-47.
[46]蒋理,徐建,杨瑞霞.散射比浊法测定血清铁蛋白的方法学评价[J].放射免疫学杂志,200821(2)：182-183.
[47]COOK J D, FLOWERS C H, SKIKNE B S. The quantitative assessment of body iron[J].Blood 2003,101:3359-3364.
[48]JUERGEN G E, JOHN E, CHRESTINE M P, et al. Combined measurement of ferritin, soluble transferrin receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J].J Nutr,2004,134:3127-3132.
[49]齐斌,谷文英,谢岩黎.酶联免疫吸附法检测黄精皂苷[J].吉林农业大学学报,2006,28(3)：292-295.
[50]苏敏,沙玲,台虹STAGO-STA型全自动血凝仪APTT测定总精密度评价[J].中国医学检验杂志,2001,2(5)：348-350.
[51]胡瑜,成立科.血清转铁蛋白受体与血清铁鉴别缺铁性贫血和慢性病贫血的意义[J].江苏大学学报(医学版),2002,12(1)：44-46
[52]李剑虹,朴建华,杨丽琛等.血红蛋白判断育龄妇女缺铁性贫血阈值研究[J].中国公共卫生,2008,24(2)：157-158.
1葛可佑.中国营养科学全书(上、下册)[M].第一版.北京：人民卫生山版社,2004：129.
2 WHO/UNICEF/UNU Iron Deficiency Anemia:Prevention Assessment and Control, Report of a Join WHO/UNICEF/UNU Consultation. World Health Organization, Geneva, Switzerland.1998.
3 KONIIN AM, HERSHKO C. Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release[J] BrJ Haem,1977,37:7-16.
4 BRUGNARA C, ZURAKOWSKI D, DICANZIO J, et al. Reticulocyte hemoglobin content to diagnoseiror deficiency in children[J]. J AMA,1998,281:2225-2230.
5 LABBE R F, DEWANJ I A. Iron assessment tests:transferrin receptor vis-avis zinc protoporphyrin[J]. Clir Biochem,2004,37:165-174.
6 DAVID A L, BARRY A SKIKNE, CAROLYN THOMPSON. Precision and accuracy of serum ferritir measurements[J]. Am J Clin Nutr,1981,34:951.
7刘绣云.放射免疫法与酶联免疫法测定血清铁蛋白的比较[J].营养学报,1985,7(2)：130-136.
8 DUPUY AM, DEBARGE L, POULAIN M et al. Determination of serum ferritin using immunoturbidimetry or chemiluminescent detection in comparison with radioimmunoassay a compendium o(?) a methodological juxtaposition[J]. Clin Lab,2009,55(5-6):207-15.
9董毅,刘蕾,朱太岗,等.血.清铁蛋白检测在贫血.性疾病中的临床价值[J].中华全科医学2010,8(4)：437-438.
10张宏,李强,李友生.乙肝肝硬化患者血清铁蛋白、转铁蛋白水平检测及意义[J].标记免疫分析与临床,2009,16(2)：75-76.
11孙件绣.血清铁蛋白放免测定在肿瘤疾病的临床应用[J].中国医疗前沿,2009,4(18)：61.
12 M PAGE, L THERTAULT, M NILSSON. Solid-phase ELISA for serum ferritin[J]. Clin Lab Invest 1980,40 (11):641-645.
13梁国栋,邓贤碧,江希蓉等.血清铁蛋白酶联免疫测定法[J].第四军医大学学报,1982(2)：107-110.
14 MARY E COGSWELL, ANNE C LOOKER, CHRISTINE M PFEIFFER,et al. Assessment of iror deficiency in US preschool children and nonpregnant females of childbearing age:National Health and Nutrition Examination Survey 2003-2006[J]. Am J Clin Nutr,2009,89 (5):1334-1342.
15 FRANK T WIERINGA, J ACQUES BERGER, MARJOLEINE A DIJKHUIZEN, et al. Combined Iror and Zinc supplementation in infants improved iron and zinc status, but interactions reduced efficacy in a multicountry trial in southeast asia[J]. Nutr,2007,137(2):466-471.
16 JUERGEN G.E., JOHN E., CHRESTINE M.P, et al.Combined measurement of ferritin, soluble transferrir receptor,retinol binding protein, and C-reactive protein by an inexpensive,sensitive, and simple sandwich Enzyme-linked immunosorbent assay technique[J]. J.Nutr,2004,134(11):3172-3132.
17杨秀岑,伍莉萍,陈达进等.用化学发光法测定辣根过氧化物酶几种体系的研究[J].华西医大学报1990,21(3)：293-297.
18 ANITA J. FUGLESTAD, ASHTON E. LEHMANN,et al.Iron deficiency in international adoptees from eastern europe[J]. J. Ped,2008,153(8):272-277
19 KUNIHIKO HASHIMOTO,SANAI NOGUCHI, YASUHIKO MORIMOTO, et al. HbA1C, but not serurr glycated albumin, is elevated in late pregnancy due to iron deficiency [J]. DIABETES CARE,2008,7.
20 S ARNON, T DOLFIN, S BAUER, et al. Iron supplementation for preterm infants receiving restrictive rec blood cell transfusions:reassessment of practice safety[J]. J PERINATOL,2010,3
21常玉荣,葛庆峰,刘培光.血清铁、铁蛋白和铁染色对缺铁性贫血的诊断价值[J].华北煤炭医学院学报,2007,9(4)：500-501.
22安镜如,林金明,陈曦.电化学发光研究及其在分析化学上的应用[J].分析化学,1991,19(11)： 1340-1346.
23李桂彩,陈德平,肖静坤.电化学发光免疫法与放射免疫法测定血清铁蛋白的评价[J].实用医技杂志,2006,5(6)：853-855.
24蒋理,徐建.散射比浊法测定血清铁蛋白的方法学评价[J].放射免疫学杂志,2008,21(2)：182-183.
25 KOHGO Y, NISHISATO T, KONDO H, et al. Circulating transferrin receptor in human serum[J]. Br J Haematol,1986,64:277.
26 HUEBERS HA, BEGUIN Y, POOTRAKUL P, et al. Intact transferrin receptors in human plasma and their relation to erythropoiesis[J]. Blood,1990,75(1):102-107.
27 CH FLOWERS, B S SKIKNE, A M COVELL,et al. The clinical measurement of serum transferrin receptor[J]. J Lab Clin Med,1989,114(4):368-377.
28 Cook J D, Flowers C H, Skikne B S. The quantitative assessment of body iron[J].Blood,2003,101:3359-3367.
29李强,苟清,廖清奎,等.单克隆及多克隆双抗体夹心ELISA法测定血清转铁蛋白受体[J].华西医大学报,1995,26(4)：398-402.
30吴鹏,王学文,庄昱洲,等.可溶性转铁蛋白受体及其复合参数在缺铁性贫血及慢性病贫血鉴别诊断中的意义[J].医学研究生学报,2003,16(6)：438-441.
31 PAULI SUOMINEN, KARI PUNNONEN, ALLAN RAJAMAKI, et al. Automated immunoturbidimetric method for measuring serum transferrin receptor[J]. Clin Chem,1999,45(8):1302-1305.
32卢忠,沈俊娅sTfR对慢性炎症患者并发缺铁性贫血的诊断价值[J].放射免疫学杂志,2007,20(1)：66-68.
33 RIIKKA VIKSTEDT, PIIA VON LODE, TIMO TAKALA, et al.Rapid one-step immunofluorometric assay for measuring soluble transferrin receptor in whole blood[J].Clin Chem,2004,50(10):1831-1833.
34 WHO/UNICEF/UNU Iron Deficiency Anemia:Prevention Assessment and Control, A guide for programme managers. World Health Organization, Geneva, Switzerland.2001.
35 CLYNEB,JONATHAN S OLSHAKER.The C-reactive protein[J]. J Emer Med,1999,17(6):1019-1025.
36陈杭,杨洁.C反应蛋白——天然防御蛋白研究进展[J].细胞生物学杂志,2003,25(5)：264-268.
37 S EDA, J KAUFMANN, M MOLWITZ, et al. A new method of measuring C-reactive protein, with a low limit of detection, suitable for risk assessment of coronary heart disease[J]. Scand J Clin Lab Invest Suppl, 1999,230:32-35.
38 M E WARIS, N J MELTOLA, J T SOINI, et al. Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein[J]. Anal Biochem,2002,309(1):67-74.
39 ALLEN J, BACKSTROM K R, COOPER J A, et al. Measurement of soluble transferrin receptor in serum of healthy adults[J]. Clin Chem,1998,44(l):35-39
40 SUOMINEN P, PUNNONEN K, RAJAMAKI A, et al. Evaluation of new immunoenzymometric assay for measuring soluble transferrin receptor to detection deficiency in anemic patients[J]. Clin Chem,1997, 43(9):1641-1646
41 BARRY S SKIKNE.Circulating transferrin receptor assay-coming of age[J].Clin Chem,1998,44(1):7-9
42庞鹏宇,揭玉丽,刘秀川等.血清可溶性转铁蛋白受体在缺铁性贫血诊断中的应用[J].中国热带医学,2007,7(8)：1483-1487.
43李剑虹,朴建华,杨晓光.转铁蛋白受体评估铁过量状况最佳临界值的研究[J].营养学报,2007,29(5)：431-434.
44张海晨,宋云霄.血清可溶性转铁蛋白受体参考范围的建立及其在缺铁性贫血诊断中的意义[J].诊断学理论与实践,2008,7(3)：339-340.
45杨振修.C-反应蛋白的检测[J].上海医学检验杂志,1999,14(5)：261-263.
46 ROBERTS WL, MOULTON L, LAW TC,et al. Evaluation of nine automated high-sentivity C-reactive protein method:implications for clinical and epidemiological applications Part 2. Clin Chem,2001 47(3):418-425.
47简序,王金和,程佩兰.C反应蛋白的临床研究进展[J].国外医学临床生物化学与检验学分册,200425(5)：471-473.
48鞠晓红,方德洋,马爱新等.C-反应蛋白水平的性别和年龄差异[J].广东医学,2007,28(6)：923-924.