Array-based ELISAs for high-throughput analysis of human cytokines

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multi plexed, sandwich;vpe ELISA that measures the concentration of seven differe nt human cytokines-TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10-from a single sample in each,well of a 96-well plate. The m ini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 p attern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mA bs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light producted from HRP-c atalyzed oxidation of the substrate is measured at each spot in the array b y imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characterstics that make them suitable for the high-throughput screening o f anti-inflammatory compounds.