Competitive receptor assays

The development of maraviroc (21), much like other chemokine receptor antagonists, started with a high-throughput screen employing a competition binding assay and led to the hit compound UK-107,543. Chemical optimization of this compound led to the development candidate UK-427,857 during this optimization phase, a parallel characterization of the compounds was performed... 

The discovery process for all of these development candidates was similar. Antagonists exhibiting low nanomolar affinities were discovered through competition binding assays, and the antagonists were able to inhibit receptor func-... 

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-... 

Ballerstadt R, Schultz JS. Competitive-binding assay method based on fluorescence quenching of ligands held in close proximity by a multivalent receptor. Analytica Chimica Acta 1997, 345, 203-212. 

Stoessel S, Leclercq G (1986) Competitive binding assay for estrogen receptor in monolayer culture measure of receptor activity potency. J Steroid Biochem 25 677-682... 

Binding affinities for experimental kappa antagonists and the standard kappa antagonist norbinaltorphimine at the p, 8, and k opioid and receptors using competitive binding assays were performed using the method previously described by the authors (1). Testing results are provided in Table 1. 

Androgen receptor binding affinities were determined using competitive binding assays described by Kirkovsky (1) and summarized in Table 1. 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Antibodies can have distinct advantages over the natural receptor molecules that are used in some competitive binding assays. They are stable, soluble proteins with known chemical and physical properties. - Procedures for their purification are available, " but usually diluted antisera is used in fluid phase radioimmunoassays. It has been estimated that an individual animal has the potential to produce antibodies specific for approximately 10 diverse immunodominant moieties. These molecules may possess binding constants for individual antigens on the order of... 

For example, several strategies have been used for immunoassay techniques with fiber-optic biosensors. In the sandwich format, the receptor is immobilized on the stu"face of the fiber waveguide and a secondary or tracer antibody (which is labelled with a fluorescent dye) is added to the solution. In the absence of the analyte, the tracer remains in solution and little fluorescence is observed. However, after addition of the analyte, a molecular sandwich is formed on the sensor smface within the evanescent excitation volume. The sandwich assay is usually more sensitive than a competitive-binding assay because the fluorescence intensity increases with analyte concentration. 

Alternatively, MIPs have also been used in biological receptors for competitive binding assays. The assay principle is similar to that in other known biological assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) except that instead of antibodies, MIPs are utilized. This method is often called molecularly imprinted assay (MIA). Typically, in MIA methods, a marker molecule (a labeled analyte analogue) is incubated together with the sample and the MIPs. Analyte and marker molecules compete for the binding... 

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