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


Immunoassays are valuable and widely used tools in the biological sciences. In drug discovery research, immunoassays are used to measure secreted cytokines in the tissue culture supernatants of treated cells. However, scientists often avoid immunoassays in high throughput screening (HTS) because they require cumbersome manipulations. Applied Biosystems has developed a singlestep, mix-and-read bead-based immunoassay, termed FLISA (fluorescence- linked immunosorbent assay), for the FMAT 8100 HTS System. The instrument is a macro-confocal scanner that enables mix-and-read, bead- and live cell-based HTS assays in 96- and 384-well formats. Furthermore, multiplexed assays, based on color or bead size, can be performed in a single well.

The unique features of the FMAT system allow bead-based FLISAs to be performed in the same well in which tissue culture cells are grown. A simple multiplexed protocol can simultaneously quantitate secreted cytokines and detect the upregulation of a cell surface marker on human umbilical vein endothelial cells (HUVEC) in a mix-and-read assay. After induction with IL-1 or TNF-, the cells are either stained with CentriRed Stain, a nucleic acid staining dye from Applied Biosystems, or with an anti-ICAM-1 antibody to measure upregulation of the cell surface adhesion molecule. At the same time, reagents used in the FLISA are added for quantification of secreted IL-8. Using the FMAT system for simultaneous measurement of secreted metabolites and cell surface markers simplifies data collection and enhances information obtained from a high throughput screen.


Cytokines are important mediators in the inflammatory response of vascular endothelial cells (EC). Their presence promotes the secretion of additional pro-inflammatory cytokines and expression of cell-surface adhesion molecules. Upon activation with I 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 t anti-mouse IgG are first coated with monoclonal anti- IL-8 antibodies. The coated beads and dye A-labeled anti-IL-8 detection antibodies are then added to the tissue culture media. An antibody/peptide sandwich will form and a fluorescent signal will be detected when the IL-8 peptide is present in the media and the bead/antibody/peptide complexes settle to the bottom of the well. To measure cell surface expression of ICAM-1, a dye B-labeled anti-ICAM-1 antibody is added simultaneously with the beads and IL-8 detection antibodies (Figures 1 and 2). Alternatively, HUVEC cells are detected by the addition of CentriRed Stain from Applied Biosystems (Figures 3 and 4). A standard curve, generated by addition of known amounts of IL-8 to wells containing untreated cells in tissue culture media, enables accurate determination of IL-8 concentration in test samples (Figure 5).

Only the fluorescence associated with the beads and cells is registered by the software. Because the depth of focus is within 100 μm from the well bottom, most of the unbound dyelabeled antibodies remain outside the focus area and are undetected, eliminating the need for wash steps.

To simultaneously quantitate IL-8 and cell surface ICAM-1 after TNF-α or IL-1 stimulation:

1.Plate 5,000 HUVEC cells/well in a 96-well TC-treated (tissue culture) plate.

2.Incubate overnight.

3.Replace media with 100 μL fresh media (with or without 10 ng/mL TNF-α or IL-1) and incubate overnight.

4.Add 50 μL PBS with .01% NaN3 containing:

Beads coated with capture monoclonal anti-IL-8 antibody

Dye A-labeled detection polyclonal anti-IL-8 antibody

Dye B-labeled anti-ICAM-1, antibody (Figures 1 and 2) or CentriRed Stain (Figures 3 and 4) 5.Incubate overnight at room temperature and scan.

Conc lusion

The increasing demands for higher throughput in HTS assays has resulted in greater exploration of the ability of homogeneous, or mix-and-read assays to eliminate time-consuming or hard-to-automate wash steps but still provide quality leads. The FMAT 8100 HTS System addresses these needs by enabling both fluorescent live-cell and bead-based assays in a mix-and-read format. Thanks to the FMAT systems simple format, miniaturization in 384-well plates is now easy. Higher throughput can be achieved by the instruments color- and size-based multiplexing capabilities.

A wide variety of HTS assays have already been adapted for the FMAT system, including live-cell receptor/ ligand binding4, apoptosis5, immunofluorescence5, and bead-based fluorescent immunoassays (FLISAs)3.

In the experiments discussed here, immunofluorescence and FLISA assays were combined in a single well, and the secreted cytokines and cell surface expression of an inducible marker were quantitated simultaneously. The generation of standard curves in buffers that match likely test samples, demonstrates that quantitative FLISA measurements can be obtained using tissue culture media with cells in the well. The ability to perform mix-and-read, beadand cell-based assays simultaneously greatly simplifies HTS while increasing yield and data quality.


1 Pober, J.S. and Cotran, R.S. (1990) Physiol. Rev. 70, 42751.

2 Chen, C.C. and Manning, A.M. (1996) Cytokine 8, 5865.

3 Swartzman, E. E., Miraglia, S. J., Mellentin-Michelotti, J., Evangelista, L., and Yuan, P. M. (1999) Anal. Biochem. 271, 14351.

4 Mellentin-Michelotti, J., Evangelista, L.T., Swartzman, E.E., Miraglia, S.J., Werner, W.E., and Yuan, P.M. (1999) Anal. Biochem. 272, 182190.

5 Miraglia, S., Swartzman, E. E., Mellentin-Michelotti, J., Evangelista, L., Smith, C., Gunawan, I., Lohman, K., Goldberg, E., Manian, B., and Yuan, P. M. (1999) J. Biomol. Screen. 4, 193204.



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