中文摘要：

目的探讨基于直接标记法的尿微量白蛋白检测抗体微阵列（也称抗体芯片）构建方法，并评价其技术可行性。方法通过玻片处理、活化、固定等程序构建白蛋白抗体芯片。摸索用生物素标记标准品白蛋白及尿液样本的最佳条件。经封闭后加入生物素标记的标准品白蛋白，孵育，洗涤后加入链酶亲和素-Cy3，再经过孵育、洗涤后，用共聚焦激光扫描仪获取荧光强度值。根据不同浓度标准品白蛋白的荧光信号强度值做出浓度-信号强度曲线，并评价其检测灵敏度、特异性、重复性。收集临床糖尿病患者尿液标本，对尿液样本同时行免疫比浊法和抗体芯片法检测白蛋白值。结果生物素标记标准品白蛋白和样本的最佳总蛋白／生物素质量比为2：1。根据标准品白蛋白所做出的标准曲线其R^2〉0．99；捕获抗体浓度为1g／L时，最低白蛋白检测浓度为0．0617mg／L。阵列内变异系数为3．35％～7．59％；阵列间变异系数为6．78％～9．22％。抗体芯片检测值与免疫比浊法检测值呈正相关（R^2=0．9199，P〈0．01）。结论成功构建基于直接标记法的抗体芯片，其具有简单、快速、高效、高灵敏度和重复性好等特点；具有开发应用于微量白蛋白尿大规模人群筛查的潜力。

英文摘要：

Objective To build an antibody microarray based on direct labeling strategy for microalbuminuria measurement, and evaluate it＇s technical potentiality for clinical application. Methods Urine samples of diabetic patients were collected. Antibody microarrays were constructed by preparation of array support, array fabrication, then protein assay and data analysis were performed. Procedure conditions for each step especially the labeling of samples were optimized. The set-ups were evaluated in terms of sensitivity, specificity and reproducibility. Urinary albumin excretion in the samples was detected by fabricated protein array, which was compared to that detected with immunoturbidimetry. Results The signal intensity was best when protein quality ratio of pure albumin or urine sample against NHS-biotin was 2：1. A calibration curve with a correlation coefficient of 0.9995 was established. The lower limit of detection was 0.0617 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs as 6.78%-9.22% and 3.35%-7.59%, respectively. Compared with the immunoturbidimetry, the antibody microarray was able to detect the extremely lower grade albumin in urine samples. The correlation coefficient of the results obtained by the two methods was 0.9199 （P 〈0.01）. Conclusion An antibody microarray based on direct labeling strategy for microalbuminuria measurement is successfully established, which is comparable to immunoturbidimetry in its accuracy and will have great potential for clinical use with its high throughput, sensitivity, specifity and reproducibility.