Ligand binding assay

A ligand binding assay (English ligand binding assay, LBA ) is a method of biochemistry and pharmacology to measure the affinity of the binding of two molecules to each other.

Principle

The binding tendency of two molecules together is called affinity. Each molecule can bind to each other with the appropriate affinity. The binding partner of a binding can be, for example, a ligand and a receptor. Small molecules are usually bound into a pocket of a protein, as a binding affinere is enabled by the increased contact area. The maximum ligand binding Bmax and the dissociation constant Kd may be determined by a Scatchard plot. The dissociation of high affinity binding are approximately 10-9 M-1 (for example, hormones, their receptors, stable protein complexes, antigen-antibody binding ).

In contrast to the following ligand binding assays, the surface plasmon resonance spectroscopy, bio -layer interferometry and the measurement of changes in impedance measure the change of the film thickness without the use of a label, but usually require higher concentrations of ligand.

Filter membranes

The filter test (synonym membrane binding assay ) is obtained by the addition of a labeled binding partner to a potential on a surface ( for example, filter paper, PVDF membranes, polyethyleneimine - coated glass fiber filters ) immobilized molecule. The label may be radioactive, be a reporter enzyme, biotin, a fluorophore, or an oligonucleotide. After several washes the coated surface determines the amount of residual bound molecules. Procedure without washing steps are sometimes referred to as mix-and -measure.

First, the measurement of the equilibrium constant is carried out at various concentrations of the labeled with the reporter molecule. In this case, the labeled molecule is incubated with the immobilized binding partner to reach chemical equilibrium. Subsequently, the membrane is dried and the amount of bound reporter molecule. After a predetermined number of washes, the amount of reporter molecules is measured again on the Memran, which provides information on the loss of reporter molecules. (English off- rate). Alternatively, the leaching can also be determined by determining the time course of the amount of reporter molecules.

Size Exclusion Chromatography

Unless receptor and ligand have different molecular weights, the binding partner bound to each other can also be isolated by size exclusion chromatography and the amount on the basis of the mark to be determined.

Isothermal Titration Calorimetry

By isothermal titration calorimetry, the affinity can be determined, provided that the binding is not driven only entropy (for a isenthalpic binding). Here, no markings are required, short-term bonds are not detected.

History

Original ligand binding assays have been used in the characterization of protein hormones, neurotransmitters and their receptors. In medical diagnosis of breast cancer, a variant was used is measured in cell extracts bound to radiolabeled estradiol, after an adsorption unbound estradiol to dextran - coated charcoal.