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Ligand Binding to Transmembrane Receptors on Intact Cells or Membrane Vesicles Measured in a Homogeneous 1-Microliter Assay Format

EVOTEC Biosystems AG, Applied Assay Development, Hamburg, Germany

Andreas Scheel

EVOTEC Biosystems AG, Applied Assay Development, Hamburg, Germany

Eloisa Lopez-Calle

EVOTEC Biosystems AG, Applied Assay Development, Hamburg, Germany

Michael Busch

EVOTEC Biosystems AG, Applied Assay Development, Hamburg, Germany

Kenneth J. Murray

Molecular Screening Technologies, SmithKline Beecham, Harlow, Essex, UK

Andrew J. Pope

Molecular Screening Technologies, SmithKline Beecham, Harlow, Essex, UK

We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1,l without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.

Journal of Biomolecular Screening, Vol. 6, No. 3, 159-170 (2001)
DOI: 10.1177/108705710100600306


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