Radioligand competitive binding assay

Secondary assays depend on the project. Where the primary screen was a cell-based assay, the secondary assay may be a radioligand competition binding assay. In other cases, such as where the primary screen was a biochemical assay, the secondary assay may be a cellular assay, and may be functional or mechanistic. One of the issues that may arise at this stage is that compounds with reasonable activity in the primary assay may not show activity in the secondary assay. There can be a number of reasons for this, including insufficient potency, inability of the compound to get into cells, or a higher intracellular concentration of the natural ligand (e.g., ATP) if the inhibitor is a competitive inhibitor. It is often necessary at this stage to prepare additional compounds in the series to get compounds of sufficient potency and/or permeability so that cellular activity can be demonstrated. 

The development of the warfarin immunoassays illustrates several points that are of value in development and use of enantioselective assays. In assays of this type, not only must enantioselectivity be considered, but also the cross-reaction with metabolites is still of importance. As in any RIA, high-specific-activity radioligand is required for the best sensitivity. The use of optically pure radioligand is a further advantage in enantioselectivity. The standard samples used for competitive binding assays must also be essentially optically pure. Otherwise, misleadingly high cross-reacrions may be observed. 

Neonicotinoids do not act as a homogenous class of insecticides. Radioligand receptor binding assays revealed two classes of neonicotinoids described here as competitive and non-competitive , respectively, relative to [ H]imidacloprid. Differences in affinity, mode of displacement, number of binding sites and temperature sensitivity suggest that thiamethoxam binds in a way unique among the commercial neonicotinoids. Metabolic transformation is not relevant for its insecticidal effects. 

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. 

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]. 

Fig. 7.19 Correlation between pK, values from competitive MS binding assays and radioligand binding assays.

ASSOCIATION CONSTANTS AND SITE DENSITIES CALCULATED FROM COMPETITIVE RADIOLIGAND BINDING ASSAYS USING MIPs... 

The MIPs have also been utilized as the recognition elements in pseudoimmunoassays. " In this approach, MIPs are substituted for antibodies to quantify the amount of analyte in a biological sample, such as blood plasma. Most MIP immunoassays are competitive binding studies in which a radio- or fluorescent-labeled analyte is added to a mixture of the MIP and imlabeled analyte. After equilibrium is reached, some fraction of the labeled species is bound to the polymer surface and thus can be separated from the supernatant. The supernatant is then analyzed via scintillation or fluorescence techniques to determine the concentration of the original unlabeled analyte. Mosbach et al. have demonstrated that MIP-based immunoassays can rival the selectivity of antibody-based assays. Imprinted polymers for the opioid receptor ligands enkephalin and morphine were prepared and showed submicromolar (pM) level selectivity in a radioligand competition assay in aqueous buffers. The analysis... 

In laboratories which use an NSILA bioassay, however, somatomedin activity has been expressed in units of insulinlike activity, generally p,U/ml, a convention which has to some extent continued as radioligand assays were introduced. Expressed in these units, normal human samples have a mean value of 350 66 pU/ml by competitive protein-binding assay (Z4). In the same laboratory, a mean normal value ( SD) of 148 45 ng/ml was determined by IGF-I RIA (Z5). The close correspondence between reference values obtained by RIA in different laboratories, when expressed as ng/ml of SM-C or IGF-I, suggests that mass units, if uniformly adopted, would allow better comparison of results. With the increasing availability of highly pu-... 

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