1. Array-based ELISAs for high-throughput analysis of human cytokines.
- Author
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Moody MD, Van Arsdell SW, Murphy KP, Orencole SF, and Burns C
- Subjects
- Anti-Inflammatory Agents pharmacology, Antibodies, Monoclonal, Cell Line, Cytokines immunology, Humans, Interferon-alpha analysis, Interferon-alpha immunology, Interferon-gamma analysis, Interferon-gamma immunology, Interleukin-1 analysis, Interleukin-1 immunology, Interleukin-10 analysis, Interleukin-10 immunology, Interleukin-6 analysis, Interleukin-6 immunology, Monocytes cytology, Monocytes drug effects, Monocytes immunology, Sensitivity and Specificity, Tumor Necrosis Factor-alpha analysis, Tumor Necrosis Factor-alpha immunology, Cytokines analysis, Enzyme-Linked Immunosorbent Assay methods
- Abstract
In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different 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 mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.
- Published
- 2001
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