Development of a CD28/CD86 (B7-2) binding assay for high throughput screening by homogeneous time-resolved fluorescence

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 anti-rat antibody, which is complexed with streptavidim-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.