ENZYME / RECEPTOR BASED ASSAYS

KINDS OF ASSAYS:  ENZYME / RECEPTOR BASED ASSAYS (ALSO CALLED AS BINDING ASSAYS), CELL BASED ASSAYS (FUNCTIONAL ASSAYS)

https://www.slideshare.net/ankit_2408/radioligand-binding-studies

Compounds interacting with therapeutic targets like enzymes, cell-surface receptors, nuclear receptors, ion channels and signal transduction proteins are usually identified using in vitro biochemical assays.

LIGAND BINDING ASSAY: 

This is an analytical procedure which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. 

There are numerous types of ligand binding assays, both radioactive and non-radioactive. 

·         There are two typical assay formats used for analysis of receptor-ligand interactions in screening applications, filtration and scintillation proximity assay (SPA).  Both use radiolabeled ligand and a source of receptor.

·         Receptor binding assays using non-radioactive formats include fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer (FRET), and surface Plasmon resonance (SPR).  One of the largest differences between radioactive and non-radioactive ligand assays are in regards of dangers to human health. Radioactive assays are harmful in that they produce radioactive waste; whereas, non-radioactive ligand assays utilize a different method to avoid producing toxic waste.

v  Although binding assays are simple, they fail to provide information on whether or not the compound being tested affects the target's function.  Binding assays provide useful information with regard to the ability of compounds to bind to a receptor, essentially no information is provided on the efficacy of the compound, i.e. whether the compound elicits or inhibits a response at that receptor.

v  In general, these receptor binding assays are used to characterize known drug targets. 

In a typical filter-based separation technology what we obtain is “bound vs. free” fractions for assay validation.  In a filter binding assay, the filters are used to trap cell membranes by sucking the medium through them.  Washing filters with a buffer removes residual unbound ligands and any other ligands present that are capable of being washed away from the binding sites. The receptor-ligand complexes present while the filter is being washed will not dissociate significantly because they will be completely trapped by the filters. 

Competition or displacement binding studies allow determination of binding affinities for non-labelled ligands. These binding studies utilize a fixed concentration of a radiolabel and the affinity of non-labelled ligands are determined by a drugs ability to compete for the same binding site. Increasing concentrations of non-labelled ligand are used to displace or out-compete the fixed concentration of a radiolabel, generating an IC50 for the competing ligand.

Radiolabeled known drugs are used in these competitive binding assays.  Therefore, the assay is designed as a competitive inhibition assay using the radiolabeled known drug or ligand for a receptor to screen for more effective new chemical entities (NCEs).

SCINTILLATION PROXIMITY ASSAY:

SPA TECHNIQUE HAS BEEN WIDELY APPLIED TO RADIOIMMUNOASSAYS, RECEPTOR-LIGAND BINDING ASSAYS, ENZYMATIC ACTIVITY ASSAYS, RNA TRANSCRIPT DETECTION, PROTEIN-PEPTIDE INTERACTIONS, AMONG OTHERS.

Receptor-binding SPA assays are conducted by immobilizing receptors directly to SPA beads via a number of coupling methods.  In the SPA format, cell membrane or receptor is captured onto SPA beads.  The method utilizes scintillant-containing microspheres that are chemically treated to enable the coupling of molecules (e.g., antibodies, receptors, and enzymes) to their surface.

The type of beads that are involved in the SPA are microscopic in size and within the beads itself, there is a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labelled molecules interact and bind to the surface of the bead. This interaction will trigger the bead to emit light, which can be detected using a photometer. 

·         When the radio-labelled molecule is attached or is in proximity to bead, light emission is stimulated.

·         However, if the bead does not become bound to the radio-labelled molecule, the bead will not be stimulated to emit light. This is because the energy released from the unbound molecule is not strong enough to excite the SPA bead which is not stimulated to produce a signal.

SPA reagents are suitable for the detection of a range of isotopes including ³H, ¹²⁵I, ³²P, ³⁵S, and ¹⁴C, when used as radiolabels.  

The path length of the b particle is determined by its energy, and varies for different isotopes.  For example, b particles from ³H have an average path-length of approximately 1.5 mm, which easily meets the distance requirement for SPA.  The Auger electrons emitted by ¹²⁵I (g-emitting isotope) which have energies in the same range as b-particles and travel between 1 and 17.5 mm in aqueous media, also satisfy the distance requirement.  Therefore, ³H and ¹²⁵I are ideal isotopes for labeling ligands in SPA.    

Other isotopes of interest, such as ¹⁴C, ³²P and ³⁵S have longer path-lengths with mean ranges of approximately 58, 126 and 66 mm, respectively, and are less suited to application of the proximity principle.  Although SPA has recently been adapted to these isotopes as well.

The beta particles emitted by ³H and the Auger electrons released from ¹²⁵I by isotopic decay have average energies of 6 and 35 keV, respectively, and thus have short path lengths in water. This property makes them ideal for use with the SPA technology.

·         When ³H, ¹⁴C, ³²P, and ¹²⁵I radioisotopes decay, they release β-particles (or Auger electrons, in the case of ¹²⁵I). The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT-based scintillation counter or on a CCD-based imager, respectively. 

·         However, if the radioactive molecule does not associate with the SPA bead, the decay particles will not have sufficient energy to reach the bead and no light will be emitted.

The core of this technique is the support material: the SPA bead.  The most commonly used SPA beads are polyvinyltoluene (PVT) beads and yttrium silicate (YSi) beads.  High-efficiency scintillant has been incorporated inside the PVT matrix, which can be excited by the high-energy b-emission generated from the decay of radioactive isotopes when the SPA bead is within the effective path length for energy transference.

The surface of this SPA bead is coated with hydrophilic polyhydroxy film that reduces hydrophobicity of the bead to reduce nonspecific interactions.  This film has been chemically derived to covalently couple capture molecules, which can selectively bind molecules of interest including receptors, proteins or other disease targets. 

SPA beads with the following capture molecules are available commercially:  Protein A, avidin, streptavidin, wheatgerm agglutinin (GA), glutathione and various polyclonal secondary antibodies.

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