Bindningsassays

Bindningsassays are experimental procedures used to measure the interaction between two or more molecules, typically a ligand and its binding partner such as a receptor, antibody, or protein. The assays quantify the amount of ligand that becomes bound to the target under defined conditions, often by separating bound from free ligand and detecting it with a labeled tag. Common formats include radioligand binding assays, enzyme-linked binding assays (ELISA-based), fluorescent or chemiluminescent binding assays, and label-free methods such as surface plasmon resonance (SPR) and bio-layer interferometry (BLI). A typical experiment involves incubating a fixed concentration of receptor with varying concentrations of ligand, then separating bound from free ligand and measuring bound ligand to generate a binding isotherm and derive parameters such as the dissociation constant (Kd) and maximum binding (Bmax). Competitive binding assays use an unlabeled ligand to displace a labeled tracer, from which affinity (Ki) can be calculated, often with the Cheng-Prusoffs correction.

Types of bindningsassays include direct binding (measuring binding of labeled ligand), competitive binding, and sandwich formats where a capture reagent and a detection reagent form a ternary complex.

Applications span pharmacology, drug discovery, immunology, and diagnostics, where bindningsassay data inform receptor occupancy, affinity, specificity, and screening outcomes. Design considerations include specificity, affinity range, nonspecific binding, effects of immobilization, and assay buffer conditions. Proper controls for nonspecific binding and total binding are essential.

Advantages include high sensitivity and quantitative output, particularly with radioactive or fluorescent labels. Limitations include potential alteration of binding by immobilization, hazards for radioligands in some formats, and that binding assays assess affinity rather than function. Data are typically analyzed by nonlinear regression to yield Kd, Bmax, and Ki values.