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Binding Assays

Binding Assays

Ligand binding assays are a method to measure interactions between two molecules, such as a protein binding another protein, a small molecule, or a nucleic acid. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed which is usually determined via fluorescence or radiometric detection methods. This type of analytic test is a critical component in lead identification and later lead characterization processes. They are used to characterize most known drug targets and typically use filter-based separations technology to obtain necessary, “bound vs. free” fractions for assay validation.

There are two types of labeled ligand binding assays to detect a compounds binging to a target, radioactive and non-radioactive (fluorescent).

Radiometric

Radiolabeled ligand binding assays are a relatively simple yet an extremely powerful tool for studying binding of compounds to receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result radioligand binding assays are used by many and are considered the, “Gold Standard.” Often these assays are used by researchers in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. Radiometric assays are almost always required by the FDA.

Fluorescence

Fluorescence has wide spectrum of wavelengths, therefore multiple colors can be applied for detection of a specific target. There are many techniques used to measure fluorescence intensity, such as fluorescence anisotropy, fluorescence correlation spectroscopy, time-resolved fluorescence, fluorescence polarization, fluorescence and bioluminescence resonance energy transfer. All these techniques are effectively used as ligand binding assays.

Advantages Disadvantages of Radioactive and Fluorescence Assay

AssayAdvantagesDisadvantages
RadioactiveThe “gold standard”and most popular assay for membrane-bound targets.Potential hazards of handling low levels of radioactivity.
 Good robustness, precise determination of receptor density and distribution, ligand binding sites and affinity.
FluorescenceHave a broad spectrum of wavelengths, therefore it can apply multiple colors.Susceptible to different fluorescence interferences.
 Labeling of a ligand can lead to undesirable alterations in binding characteristics of the ligand.
 It is difficult to obtain good fluorescent ligands for many receptors.

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Binding Assays

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