基于夹心免疫分析的抗体微阵列的构建

目的：建立一种基于夹心免疫分析的抗体微阵列构建的优化方法。方法：将MCP-1的捕获抗体点样于修饰后的玻片，标准抗原加样覆盖所点阵列，生物素标记抗体和链酶亲和素-cy3依次加样孵育, 激光共聚焦扫描仪获取图象并进行数据分析。对捕获抗体浓度、封闭液种类、系统可重复性和定量检测能力、两种因子平行性检测对信号分析的影响及点样后玻片稳定性进行分析和评价。结果：随着捕获抗体浓度的升高，信号强度逐渐增加；2℅ BSA/PBS和5℅ 酪蛋白可作为本系统的封闭液；所构建系统具有较好的可重复性（组内变异 1.3%，组间变异8.7%）和定量分析能力（所建立的抗原浓度-相对信号强度标准曲线相关系数达0.9995）；并实现了两因子的平行性分析和点样后玻片的稳定性。结论：确立了基于夹心免疫分析的抗体微阵列构建的优化方法，为进一步构建多因子定量检测抗体微阵列奠定了基础。

Construction of Antibody Microarray Based On Sandwich Immunoassay

Objective: To establish a method of constructing antibody microarray based on sandwich immunoassay. Methods: Modified glass slides were robotically printed with capture antibodies against MCP-1, then dilutions of the cytokine were applied to the arrays and the protein was detected with biotin-labeled antibody coupled with Cy3- conjugated streptavidin. A laser confocal scanner was used to obtain the images and the signal intensity was analyzed subsequently. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, blocking buffers, reproducibility and quantitative ability of the system, two-cytokine analysis and shelf life of the post-printing slides. Results: The signal intensities increased with increasing capture antibody concentration; 2%BSA and 5% casein were proper as the blocking buffer for this system; The results revealed high reproducibility with regard to intra-array (1.3%) and the inter-array (8.7%) variation at the capture antibody concentration of 125ug/ml and good qualitative ability with a correlation coefficient of 0.9995 between the antigen concentration and the signal intensity; Besides, an antibody microarray for the parallel analysis of two cytokines was established and the printed arrays could be stored for at least two months without any apparent change of the performance parameters. Conclusion: A method for constructing antibody microarray based on sandwich immunoassay was established which might lay a foundation for fabrication of antibody microarray with multiplexing and quantitative capacity.