【作者】 殷继永；

【导师】 霍军生；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2011， 博士

【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。更多还原

【Abstract】 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.更多还原