Quantification of 500 human protein biomarkers using antibody arrays and its applications in immunology research (TECH2P.921)

Abstract

Abstract Proteomic approach continues to be the trend for biomarker discovery through profiling as many proteins as possible in a biological sample. Though LC-MS technology can accurately detect medium-to-high abundance proteins (> ng/ml), many cellular modulators such as cytokines are present at pg/ml level. Immunoassays continue to be the most utilized tools for biomarker discovery and validation due to their convenience, sensitivity and specificity. Here we validated the sandwich detection of 500 most analyzed human protein biomarkers with a pair of antigen-specific antibodies. Subsequently, capture antibodies were fixed on glass slides with non-contact arrayer, and multiplex ELISA detection of these protein biomarkers was achieved through planar antibody array technology with laser fluorescence detection. To overcome the potential cross reactivity among different reagents, multiple non-overlapping arrays were developed based upon their cellular abundance, functionality, and cross reactivity. Most of the markers can achieve pg/ml detection - similar to single ELISA detection but with much wider dynamic range. These arrays were further validated with normal human sera, where over two thirds of these proteins are within the detection range. Separate array panels were developed for those proteins with high abundance in sera, where high sample dilutions are needed. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.