中国生物工程杂志

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.