基于直接标记法抗体微阵列的初步构建-东南大学学报医学版

基于直接标记法抗体微阵列的初步构建

单位：东南大学肾脏病研究所, 附属中大医院, 江苏南京 210009

分类号：Q503

出版年·卷·期（页码）：2009·28·第二期（85-89）

摘要：

目的: 初步构建基于直接标记法的抗体微阵列,并评价其应用于临床的可能性。方法: 选择白蛋白作为目标蛋白,通过琼脂糖铺片、玻片活化、机器点样、点样后固定等步骤构建抗体微阵列,经封闭非特异位点、标记抗原、加样后对白蛋白进行检测,对关键步骤——抗原标记进行最佳条件摸索,通过对临床标本的检测评价其潜在应用价值。结果: 应用BCA蛋白定量试剂盒测定尿液样本总蛋白浓度做出的标准曲线,其R²均大于0.99;温度为37℃时抗原标记效果最佳;标记后抗原存放1周对信号的影响无统计学意义(P <0.01);本方法能够检测出临床检测为阴性的尿微量白蛋白浓度。结论: 本研究构建了基于直接标记法的抗体微阵列,并对关键的抗原标记环节进行了最佳工作条件优化,有应用于临床检测的潜力。

Objective To build an antibody microarray based on direct labeling strategy and evaluate it's technical potentiality for clinical application.Methods Constructed antibody microarrays by preparation of array support,array fabrication,then undertook protein array assay and data analysis.Optimized the experimental condition of key procedure for labeling of samples.Urinary albumin excretion in the samples were detected by using of the fabricated protein array,which were compared to that detected with immunoturbidimetry.Results Color response curves for BSA using the standard test tube protocol had calibration curves with a correlation coefficient over 0.99.The signal intensity was best when the protein labeling temperature was 37℃ compared with 0,4 and 25℃.The conservation time of labelled samples has no effect on the signal intensity(P <0.01).Compared with the immunoturbidimetry,the antibody microarray was able to detect the extremly lower grade albumin in urine samples.The correlation coefficient of the results obtained by the two methods was 0.919 9（P <0.01.Conclusion We have successfully established an antibody microarray based on direct labeling strategy and have optimized the experimental conditions for the key step that will have great potential for clinical use.

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