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Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophyco-cyanin as acceptor (XL665). A double-tagged substrate, biotinyl-E-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-L-alanyl-L-propyl-N∊-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH2), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

CD28 has been demonstrated to provide the major costimulatory signal for CD4-positive T cells. Ligation with its natural ligands CD80 (B7-1) and CD86 (B7-2) leads to signals during activation that are required for the production of interleukin-2, and this process has been implicated in the regulation of T-cell anergy and programmed cell death. This article describes the assay development, assay validation, and primary screening for small molecule antagonists of this interaction, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from excited europium ions to cross-linked allophycocyanin, which then subsequently emits a fluorescent signal. An "indirect" approach was taken, whereby the cross-linked allophycocyanin (XL665) is covalently linked to an antihuman antibody that binds to a human immunoglobulin (Ig) domain fused to CD28. The CD86 that is expressed as a fusion protein with a rat Ig domain is bound to biotinylated sheep antirat antibody, which is complexed with streptavidin-europium cryptate. This "cassette" format facilitates the development of related assays using CTLA-4 in place of CD28 and/or CD80 in place of CD86, allowing easy determination of the selectivity of active compounds. When the CD28 and CD86 are in close proximity (i.e., bound), there is a specific time-resolved emission at 665 nm that is largely absent in either unbound partner. Experiments to optimize the reagent concentrations, incubation time, solvent effects and quench effects by colored compounds are discussed, as are the results from robustness testing and data from primary screening.

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.