Development of non-overlapping multiplex antibody arrays for the quantitative measurement of 400 human and 200 mouse proteins in parallel (TECH1P.849)

Abstract Accurate detection of multiple cytokines in complex biological samples is essential to progress in immunological research. However, development of high-density multiplex sandwich-based ELISA panels is hampered by nonspecific cross-reactivity between and among target-specific reagents (i.e., capture antibodies, detection antibodies, and/or antigens); this is particularly challenging for bead-based assays, in which all three are free to interact in solution. To overcome this bottleneck, we tested for nonspecific cross-reactivity among reagents used on our quantitative antibody arrays (in which capture antibodies are affixed to a glass slide). Combinations of reagents exhibiting nonspecific interactions were separated on discrete arrays. Using this divide-and-conquer strategy, we developed and validated 10 human and 5 mouse non-overlapping 40-plex arrays for parallel, quantitative detection of 400 human and 200 mouse proteins respectively. These markers cover the most analyzed secretory proteins such as cytokines, chemokines, growth factors, soluble receptors, and other regulatory factors. To our best knowledge, this is the highest density multiplex antibody arrays developed to date. Such a system hold a great promise in determining key factors and mechanisms in immune cell physiology and pathology, in identifying novel drug targets, and in screening for putative biomarkers from patients' samples.