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 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.
Moody
M D
MD
Pierce Endogen, Inc., 30 Commerce Way, Woburn, MA 01801-1059, USA. mmoody@endogen.com
Van Arsdell
S W
SW
Murphy
K P
KP
Orencole
S F
SF
Burns
C
C
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002460:Cell Line; D016207:Cytokines / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016898:Interferon-alpha / Q000032:analysis / Q000276:immunology; D007371:Interferon-gamma / Q000032:analysis / Q000276:immunology; D007375:Interleukin-1 / Q000032:analysis / Q000276:immunology; D016753:Interleukin-10 / Q000032:analysis / Q000276:immunology; D015850:Interleukin-6 / Q000032:analysis / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000276:immunology
D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D016207:Cytokines; D016898:Interferon-alpha; D007375:Interleukin-1; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma