Radioligand-binding Assay

There should be specific, saturable binding to the receptor, accompanied by pharmacological characteristics appropriate to the functional effects, demonstrable using a radioactive, eg, tritium or iodine-125, ligand to label the receptor. Radioligand binding assays (1,6) have become a significant means by which to identify and characterize receptors and enzymes (see Immunoassays Radioactive tracers). Isolation of the receptor or expression of the receptor in another cell, eg, an oocyte can be used to confirm the existence of a discrete entity. 

Ansell, RJ Mosbach, K, Magnetic Molecularly Imprinted Polymer Beads for Drug Radioligand Binding Assay, Analyst 123, 1611, 1998. 

The selectivity for 5-HT4 vs. other 5-HT receptor subtypes was examined in a set of radioligand binding assays available at the time (5-HT1A, 5-HT1D, 5-HT2A., 5-HT2c and 5-HT3) for some of the new compounds. While most derivatives exhibited a high selectivity for 5-HT4 vs. 5-HT3 receptors, structural adjustments of 5 led to profound effects on selectivity vs. other 5-HT receptors. 

Radioligand binding assays are a relatively simple but at the same time a very important and efficient tool to study target-ligand interactions. Since they are very videly used in drug discovery and have been described extensively, this section only discusses their fundamental aspects and those aspects that are important for the description of the MS binding assays below . More detailed information can be found in the relevant literature [6, 7, 16-21]. 

The affinity constant of the test compound K can in turn be calculated from the IC50 value, the Kj of the marker and the concentration of M according to Cheng-Prusoff [see Eq. (3)] [22]. Since the concentration of free marker (M) is usually not determined in radioligand binding assays, it should be noted that the calculation of Ki according to Eq. (3) based on Mtot yields only reliable results if the depletion of the marker is negligible (i.e. <5% to 10% [7, 17, 21]). In competition experiments performed under typical conditions (Ttot Mtot this criterion is fulfilled. 

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