Detection of human cytokines with both chemiluminescent and fluorescent plate‐based protein arrays

Abstract

We have developed chemiluminescent and fluorescent multiplexed protein assays on the SearchLight® platform for the quantitative analysis of 12 human cytokines that are involved in the Th1/Th2 and inflammatory responses. Twelve different capture antibodies for human cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α) were printed in each well of 96-well microtiter plate. The assay was carried out as a typical sandwich ELISA assay until the last step. After adding streptavidin-horseradish peroxidase (SA-HRP) conjugate and a chemiluminescent substrate, the image of the protein array was captured with a CCD camera. For the fluorescent assay, an infrared dye-labeled streptavidin was applied to the plate, and then the protein array was scanned with a laser imager. Serum samples from normal human controls and sepsis patients were assayed with both the SearchLight chemiluminescent and fluorescent arrays. The levels of several cytokines were elevated in serum from sepsis patients compared to normal controls, and the two signal detection systems produced comparable results. The results showed that the multiplexed protein assay is a powerful tool for quantitative screening of patient samples. We have also developed higher density fluorescent arrays (24 and 37-plex) for profiling cytokines, chemokines, angiogenesis factors, and other protein biomarkers in human serum samples.