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Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System

L-1 or TNF-, one of the cytokines secreted by EC is IL-8, which promotes the attachment of circulating neutrophils and induces B2-integrin expression. IL-1 and TNF- are also responsible for the upregulation of many cell surface proteins, including ICAM-1 (intercellular adhesion molecule)1, 2. Because cytokines and adhesion molecules are involved in the inflammatory response, they are important targets for the pharmaceutical industry. Although traditional ELISAs have been used to quantitate secreted cytokines, these assays require numerous wash and incubation steps, and are cumbersome to perform. In addition, quantification of cell surface proteins is difficult with current HTS technologies.

A new platform, the FMAT system, addresses these screening issues for immunosorbent assays and can simultaneously quantitate both the secreted cytokines and cell surface proteins. The FMAT system includes a macro-confocal scanner with a 633 nm helium/neon laser. The system images and quantitates cell- and bead-bound fluorescence in a mixand- read or homogeneous format, using 96- or 384-well plates. Two photomultiplier tubes (PMTs) detect the emitted fluorescence at different wavelengths from two separate red dyes (Dye A and Dye B). This enables multiplexed assays using two separate fluorophores.

Performing Immunoassays on the FMAT System

Bead-based immunoassays, called FLISAs, were previously developed for the FMAT system to quantitate the amount of secreted IL-6 and IL-8 in the tissue culture media of HUVEC treated with TNF- or IL-1. Because no wash steps are involved, FLISAs can be performed with the cells remaining in the microplate wells. Tissue culture supernatant need not be removed to fresh wells. Furthermore, the cell-surface expression of ICAM-1 on HUVEC can be simultaneously quantified by a fluorescent dye-labeled, anti-ICAM-1 antibody.

To quantitate secreted IL-8, beads conjugated with goa
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