Ligand binding to transmembrane receptors on intact cells or membrane vesicles measured in a homogeneous 1-microliter assay format.

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 microl 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.

A novel and robust homogeneous fluorescence-based assay using nanoparticles for pharmaceutical screening and diagnostics.

We have established a new type of homogeneous immunoassay based on nanoparticles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA). This method allows the characterization of single fluorescently labeled molecules or particles with respect to their molecular brightness and concentration. Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the complex scales with the number of bound conjugates. The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present various examples of NPIAs where capture antibodies were linked to the nanoparticles, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quantitate free antigen through competition experiments, and second, an antigen-directed antibody was labeled to establish an assay similar to a sandwich ELISA setup. The major advantages of a NPIA are the robustness and high signal-to-noise ratio at short measurement times, as demonstrated with a miniaturized experiment in a Nanocarriertrade mark holding a volume of 1 microl/well. In addition to the good data quality, NPIAs are straightforward to perform because they require no washing steps. NPIAs open new dimensions for high throughput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is applicable to most types of assays.