Saturation radioligand assays

This interpretation is supported by data from radioligand studies 8). According to saturation binding assays with [ Hjthiamethoxam and competition assays with [ Hjimidacloprid, all the compounds under discussion exhibit about the same affinity to aphid membranes. The data, given as Kq and /Cj values, respectively, for M persicae are (5) ... 

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. 

In the following, an example of this new kind of MS binding experiment is presented as a straightforward alternative to conventional radioligand binding assays and suitable for the performance of saturation, competition and kinetic binding assays [80]. 

The results of this saturation assay were validated by direct comparison to conventionally conducted radioligand binding assays using [ H]NO 711 as marker [80, 100]. Not only due to financial considerations, hot/cold dilutions had to be used in the radioligand binding assays, in contrast to the MS binding assays... 

Two main types of assay are usually undertaken, namely, competition and saturation assays. The former is used to measure the binding affinity of an unlabelled compound. A range of concentrations of the compound are mixed with a constant concentration of a carrier-free receptor specific radioligand and... 

Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [ H]SR141716A (a CBi-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nAf) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 jig/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 pM unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [ H]SR141716A.

In all radioligand experiments, it is critical to minimize nonspecific binding. Due to the hydrophobic nature of many proteins (e.g., membranes and chemokines) and small molecules, the assay buffer can be supplemented with nonspecific protein (e.g., BSA) or mild detergents (e.g.. Tween, CHAPS) to saturate nonspecific adsorption sites or to solubifize membranes while preserving structural integrity, respectively both supplements minimize nonspecific binding. 

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