Heterogeneous binding assays can be performed with radio-labeled or lanthanidelabeled ligands. Virtually every heterogeneous binding assay utilizes a filtration step to remove unbound labeled ligand. This technology has the advantage of removing compounds that could interfere with the detection of the bound tracer. The main disadvantage of this nonhomogeneous assay is the difficulty in automating the separation step, which results in lower throughput of the screen.
Fluorescent labeling of ligands can prove quite a challenge especially when small molecules are involved; maintaining functional activity or affinity after labeling requires additional testing. A limited number of europium-labeled ligands including galanin, bombesin, neurokinin-A, neurotensin and substance P are available from PerkinElmer for use with its DELFIA® assay platform. DELFIA is a heterogeneous time-resolved fluorometric assay method in which an enhancement step assures high sensitivity and wide response range.
After binding equilibrium of receptor and ligand is attained, the contents of the wells are filtered into an Acrowell® (Pall Life Sciences, Ann Arbor, Michigan), a patented low fluorescent background membrane. An enhancement solution is added to the wells and the fluorescence is measured in a time-resolved fluorescence reader. This assay format eliminates the need to use radioactive ligands although few receptor ligands tagged with europium are available for HTS, resulting in very limited use of this technology.
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