Competition binding assays

Finlayson K, Turnbull L, January CT, Sharkey J, Kelly JS (2001). 3H-Dofetilide binding to HERG transfected membranes  

Finlayson K, Pennington AJ, Kelly JS (2001) 3H-Dofetilide binding in SHSY5Y and HEK 293 cells expressing a HERG-like K+ channel Eur J Pharmacol 412(3) 202-212 

Wang J, Della Penna K, Wang H, Karczewski J, Connolly TM, Koblan KS, Bennett PB, Salata JJ (2003) Functional and pharmacological properties of canine ERG potassium channels. Am J Physiol-Heart circulat Physiol 284(1) H256-H267 

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Fig. 5.11 L-Alanine scan of CGRP Y-28-37, a selective antagonist for the human calcitonin gene-related peptide 1 receptor (CGRPi). The analogues were tested In binding-competition assays against ( l-lodohlstldylj-CGRP. Whereas the replacement of some residues...

A) Binding competition assays. These are mostly receptor binding assays, in which the receptor proteins are immobihzed on the surface of the SPA beads and a radiolabeled ligand is used as a tracer. This tracer competes with the test compound for binding to the receptor, which can be seen by a change in the S PA signal [23-26],... 

Table 1. SPA assay formats (A) binding competition assays for receptors and (B) enzymatic assays with signal increase, signal decrease, and product capture.

Size increase Binding of a tracer to a larger molecule, e. g. Hgand-receptor binding competition assays. 

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

This same experimental approach can be used to determine the appHcabiUty of the aDAS—AP to a competitive assay for DAS. As shown in Eigure 6, increasing amounts of free DAS were used to define the 50% inhibition level (ID q) of DAS for binding of two aDAS—AP conjugates to immobilized DAS. This approach was also used to determine the sensitivity of an EIA, as well as the specificity of the assay, as shown in Table 2. Increasing amounts of trichothecene mycotoxins closely related to DAS were added to microtiter plate wells containing a constant amount of prereacted DAS—aDAS—AP. After 30 min, excess toxin and any free toxin—aDAS—AP were washed out, and substrate was added. Quantification of the color produced was directly related to the abihty of the added toxin to displace aDAS—AP from the immobilized DAS, which is an indication that the aDAS also has an avidity for that toxin. 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Figure 11. Comparison of different assay types using a direct detection scheme were the receptors immobilized to the surface and the analyte is recognized at the surface (direct optical detection and using labelled systems), a competitive test scheme were labelled analyte molecules compete with the non-labelled sample, and thirdly a binding inhibition assay were analyte derivatives (ligand derivatives) are immobilized at the surface, in a preincubation phase the ligands block receptor molecules, non-blocked receptors go to the surface being either labelled or optically detected.

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

Fig. 12 Cartoon representing two versions of a kinase assay, (a) The tum-off version is a direct assay the extent of phosphorylation of the substrate is reflected in the binding of the quencher to the bead and hence to the quenching [88]. (b) The tum-on version is a competition assay increased phosphorylation of the substrate leads to it competing with the quencher in binding to the beads, and hence to increased emission [44]. Emissive polymers are represented by green lines quenched polymers by grey lines...

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

The competitive assay is another format used to quantitate an analyte. An unlabeled analyte competes with a labeled analyte (enzyme-conjugated molecule) for binding to a specific capture antibody (Figure 11.1c). 

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