Ligand binding assay

A Ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor. A Ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor. There are numerous types of ligand binding assays, both radioactive and non-radioactive. As such, ligand binding assays are a superset of radiobinding assays, which are the conceptual inverse of radioimmunoassays (RIA). Some newer types are called 'mix-and-measure' assays because they do not require separation of bound from unbound ligand. Ligand binding assays are used primarily in pharmacology for various demands. Specifically, despite the human body’s endogenous receptors, hormones, and other neurotransmitters, pharmacologists utilize assays in order to create drugs that are selective, or mimic, the endogenously found cellular components. On the other hand, such techniques are also available to create receptor antagonists in order to prevent further cascades. Such advances provide researchers with the ability not only to quantify hormones and hormone receptors, but also to contribute important pharmacological information in drug development and treatment plans. Historically, ligand binding assay techniques were used extensively to quantify hormone or hormone receptor concentrations in plasma or in tissue. The ligand-binding assay methodology quantified the concentration of the hormone in the test material by comparing the effects of the test sample to the results of varying amounts of known protein (ligand). The foundations for which ligand binding assay have been built are a result of Karl Landsteiner, in 1945, and his work on immunization of animals through the production of antibodies for certain proteins. Landsteiner’s work demonstrated that immunoassay technology allowed researchers to analyze at the molecular level. The first successful ligand binding assay was reported in 1960 by Rosalyn Sussman Yalow and Solomon Berson. They investigated the binding interaction for insulin and an insulin-specific antibody, in addition to developing the first radioimmunoassay (RIA) for insulin. These discoveries provided precious information regarding both the sensitivity and specificity of protein hormones found within blood-based fluids. Yalow and Berson received the Nobel Prize in Medicine as a result of their advancements. Through the development of RIA technology, researchers have been able to move beyond the use of radioactivity, and instead, use liquid- and solid-phase, competitive, and immunoradiometric assays. As a direct result of these monumental findings, researchers have continued the advancement of ligand binding assays in many facets in the fields of biology, chemistry, and the like. Ligand binding assays provide a measure of the interactions that occur between two molecules, such as protein-bindings, as well as the degree of affinity (weak, strong, or no connection) for which the reactants bind together. Essential aspects of binding assays include, but are not limited to, the concentration level of reactants or products (see radioactive section), maintaining the equilibrium constant of reactants throughout the assay, and the reliability and validity of linked reactions. Although binding assays are simple, they fail to provide information on whether or not the compound being tested affects the target's function. Radioligands are used to measure the ligand binding to receptors and should ideally have high affinity, low non-specific binding, high specific activity to detect low receptor densities, and receptor specificity. Levels of radioactivity for a radioligand (per mole) are referred to as the specific activity (SA), which is measured in Ci/mmol. The actual concentration of a radioligand is determined by the specific stock mix for which the radioligand originated (from the manufactures.) The following equation determines the actual concentration: p m = C P M / S A ( C P M / f m o l ) V o l u m e ( m l ) × 0.001 ( p m o l / f m o l ) 0.001 ( l i t e r / m l ) = ( C P M / S A ) ( V o l ) {displaystyle pm={frac {CPM/SA(CPM/fmol)}{Volume(ml)}} imes {0.001(pmol/fmol) over 0.001(liter/ml)}={(CPM/SA) over (Vol)}}