The use of biotin labeled ligands in receptor binding assays is proposed as a substitute for radiolabled ligands. High affinity biotinylated ligands are used in immunochemistry studies but are not generally employed in receptor binding experiments. Biotinylated ligands also have high affinities for the proteins avidin and streptavidin. These proteins can be coupled with various enzymes which catalyze spectrophotometric, fluorometric, or chemiluminescent detection in microliter plates. Using standard receptor binding protocols, biotinylated ligands can be incubated with the appropriate receptor and possible competitors for the receptor. Free or receptor bound biotinylated ligands are detected by binding to the avidin or st reptavidin enzyme conjugates. This mechanism makes it possible for one molecule of biotinylated ligand to be responsible for the generation of many molecules of product, providing an amplification of the initial signal. Several strategies for selected receptor binding assays, utilizing biotinylated ligands and a microliter plate assay format assays will be examined. The knowledge gained in these investigations should aid in the development of additional high throughput nonradioactive receptor binding assays.
NIH ExPorter
NIH ExPorter