Ligand binding assay
[4][5][6] Some newer types are called "mix-and-measure" assays because they require fewer steps to complete, for example foregoing the removal of unbound reagents.
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.
The first successful ligand binding assay was reported in 1960 by Rosalyn Sussman Yalow and Solomon Berson.
These discoveries provided precious information regarding both the sensitivity and specificity of protein hormones found within blood-based fluids.
[9] 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.
For instance, the Lois lab at Caltech is using engineered artificial ligands and receptors on neurons to trace information flow in the brain.
[13] Although binding assays are simple, they fail to provide information on whether or not the compound being tested affects the target's function.
[7] The Scatchard plot can be standardized against an appropriate reference so that there can be a direct comparison of receptor density in different studies and tissues.
If the ligand were to have bound to multiple sites that have differing radioligand affinities, then the Scatchard plot would have shown a concave line instead.
[16] EBDA performs the initial analysis, which converts measured radioactivity into molar concentrations and creates Hill slopes and Scatchard transformations from the data.
[16] Despite the different techniques used for non-radioactive assays, they require that ligands exhibit similar binding characteristics to its radioactive equivalent.
In order to measure process of ligand-receptor binding, most non-radioactive methods require that labeling avoids interfering with molecular interactions.
The measurements allow quantitation of the active ligand concentration and the binding constants (equilibrium, on and off rates) of the interaction.
[5] The device for which SPR is derived includes a sensor chip, a flow cell, a light source, a prism, and a fixed angle position detector.
The extract of disrupted tissue or cells is mixed with an antibody against the antigen of interest, which produces the antigen-antibody complex.
This method involves purifying an antigen through the aid of an attached antibody on a solid (beaded) support, such as agarose resin.
[18] Multiwell plates are multiple petri dishes incorporated into one container, with the number of individual wells ranging from 6 to over 1536.
[9] The multiwell plates are manufactured to allow researchers to create and manipulate different types of assays (i.e., bioassays, immunoassays, etc.)
[20] On-Bead Ligand Binding assays are isolation methods for basic proteins, DNA/RNA or other biomolecules located in undefined suspensions and can be used in multiple biochromatographic applications.
Direct analyzation methods based on enzymatic/fluorescent detection (e.g. HRP, fluorescent dye) can be used for on-bead determination or quantification of bound biomolecules.
A thicker filter is useful to get a more complete recovery of small membrane pieces, but may require a longer wash time.
The obtained signal is proportional to the number of ligands bound to a target structure, often a receptor, on the cell surface.
[27][28] The advantage of measuring ligand-receptor interactions in real-time, is that binding equilibrium does not need to be reached for accurate determination of the affinity.
[30] Drugs that act upon these receptors are incredibly selective in order to produce required responses from signaling molecules.
[16] Recently, an alternative dye solution and microplate system has been developed called FLIPR® (fluorometric imaging plate reader), which uses a Calcium 3 assay reagent that does not require a washing step.
As a result, change in dye fluorescence can be viewed in real time with no delay using an excitatory laser and a charge-coupled device.
A method called Scintillation proximity assay (SPA) has been recently developed, which eliminates this otherwise crucial step.
It works through crystal lattice beads, which are coated with ligand coupling molecules and filled with cerium ions.
[16] By nature, assays must be carried out in a controlled environment in vitro, so this method does not provide information about receptor binding in vivo.
The radiolabeled ligands are spatially located by a PET scanner to reveal areas in the organism with high concentrations of receptors.
