Construction of an antibody microarray based on agarose‐coated slides

Abstract

Abstract The antibody microarray, a high‐throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose‐coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (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. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near‐quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra‐array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 µg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.