Development of a CD28/CD86 (B7-2) Binding Assay for High Throughput Screening by Homogeneous Time-Resolved Fluorescence

Abstract

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.

Allophycocyanin 1 as a Near-Infrared Fluorescent Tracer: Isolation, Characterization, Chemical Modification, and Use in a Homogeneous Fluorescence Resonance Energy Transfer System

“…Replacing APC XL by APC1 XL as acceptor in the FRET pair used in the HTRF technology would not affect the high efficiency of the technology: the exceptionally large R o distance of 8.5 nm obtained with APC remains unchanged with APC1, allowing molecular interactions in large complex assemblies to be probed (28). Moreover, the significant increase of the acceptor quantum yield obtained with APC1 XL conjugates would ensure a better restitution of the FRET signal through the fluorescence acceptor emission at 665 nm.…”

HTRF®: pioneering technology for high-throughput screening

“…Calculation of the ratio for each well is, therefore, critical in decreasing false positives. This patented ratio [101] is an essential tool for preventing interference due to variability of the medium or of chemical compounds [24].…”

“…HTRF assays have also been developed for indirect detection (via secondary antibodies) of tagged complexes such as CD28 and CD86 [24].…”

A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence

“…Measurement of the ratio of fluorescence at 665 nm to that at 620 nm, which is emitted from europium cryptate (internal reference), makes it possible to correct for media absorbency in real time. Because of these advantages, HTRF has been utilized for high-throughput screening of various biomolecular targets [14][15][16][17][18][19][20][21].…”