Displacement assays

The mechanism of interaction with DNA is suggested. Ethidium bromide (EB) displacement assay was performed. We determine the binding constant of Tb-E to DNA to be in the order of Ig K = 6.47 0.4. The bathochromic and hypsochromic effects in the absorption spectra of investigated complex were observed and the interaction is assumed to be mainly of the mono-intercalating type. 

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... 

Displacement Assay for TTX and STX. Davio and Fontelo (45) and Richie et al. (46) describe methods for detecting STX by measuring displacement of radiolabelled STX from brain membranes. The sensitivity of this assay is approximately 1 ng STX/ml. TTX can also be detected since STX and TTX share the same biological receptor on the sodium channel. 

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Screening of chemokine receptors has focused mostly on ligand displacement assays. These assays use a radiolabeled ligand and cells or membranes... 

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],... 

Table 6.2 Training set molecules for a1A adrenergic receptor and their affinity measured in a radioligand displacement assay.

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent indicators, F-IDAs - are based on the next alternative concept described here. A receptor with an affinity for a given analyte is loaded with an indicator, usually a fluorescent or colored dye, whose spectral properties undergo a change upon complexation with the receptor. Treatment of this indicator-receptor complex with the analyte results in the displacement of the indicator from the receptor and a restoration of the indicator s original spectral properties, indirectly reporting analyte coordination (Fig. 27). For effective detection, two main requirements have to be fulfilled (1) the receptor/indicator interaction must be reversible and weaker than the interaction of the receptor with the designated analyte and (2) the indicator must show significantly different optical properties when bound to the receptor and when freely dissolved in solution. 

Fig. 27 (a) Representative scheme for a displacement assay protocol in which first a fluorescent indicator is coordinated to a host. As a consequence, its optical properties are altered Second upon analyte addition, the higher affinity of the analyte for the host leads to dissociation of the complex and displacement of the indicator. The original optical properties of the fluorophore are restored, signaling indirectly the presence of the analyte (b) Some examples of receptors and fluorescent indicators reported in the literature for F-IDAs... 

Horie S, Kubo Y (2009) Fluorescence-based indicator displacement assay for phospho-sugar detection using zinc(II) dipicolylamine-appended phenylboronic acid. Chem Lett 38 616-617... 

Comes M, Aznar E, Moragues M et al (2009) Mesoporous hybrid materials containing nanoscopic binding pockets for colorimetric anion signaling in water by using displacement assays. Chem Eur J 15 9024—9033... 

Figure 13.IS. Glucose displacement assay using an insoluble polymeric acceptor AMCA-Con A J.exc = 356 nm / = 224 mHz, Inset a-melbyl-D-mannoside.

Glutathione S transferases bind bile acids in vitro but doubt has been cast over whether this happens in vivo as these enzymes were not labelled by fluorescently labelled bile acids in experiments to identify the carrier proteins but may play a role with the raised levels in cholestasis. Liver fatty-acid-binding protein has been shown to bind bile acids by using a displacement assay with fluorescent fatty-acid ligand. This work clearly showed displacement to be directly related to hydrophobicity, such that lithocholate conjugates had the greatest effect. This may indicate a mechanism to minimise toxicity within the hepatocyte. 

Buryak, A. Pozdnoukhov, A. Severin, K. Pattern-based sensing of nucleotides in aqueous solution with a multicomponent indicator displacement assay. Chem. Commun. 2007, 2366-2368. 

Minunni, M., Mascini, M., Carter, R. M., Jacobs, M. B., Lubrano, G. J., and Guibault, G. G. (1996). A quartz crystal microbalance displacement assay for Listeria monocytogenes. Anal. Chim. Acta 325,169-174. 

The conventional competitive or non-competitive assays do not allow continuous detection so that, for on-line measurements, the so-called displacement assays are usually applied. Typically, the antibody is immobilized onto a solid support and packed in a column, and the corresponding antigen is labeled. The antibody binding sites are saturated with labeled antigens and the sample, containing the free (unlabeled) analyte, is injected into the column, resulting in displacement of the bound labeled analyte, as the affinity of the antibody for the labeled analyte is usually much lower than its affinity for the unlabeled analyte. The displaced labeled analyte is eluted and detected at the outlet of the column and the measured signal is directly proportional to the analyte concentration in the sample. 

Greene and Shimizu [77] have reported a displacement assay using a colorimetric dye f/V,/V-dimethyI-/V./V-("7-nitro-2.1,3-benzoxadiazol-4-yl)-l, 2-ethanediamine) with absorption maximum at 460 nm. This assay allowed the determination of seven different aromatic amines. Nevertheless, although the dye seems to be more sensitive in the polymer than in solution, after thorough optimization of the assay conditions, the displacement is only significant for the mmol L 1 amine concentration range. 

Kinkaid, A. and Wilton, D.C. 1991. Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay. Biochem. J. 278, 843-848. 

The binding of citrate by 4.28 has been investigated by the indicator displacement assay (IDA) method (cf. EDTA titrations, Section 3.1.3). Binding of an organic dye indicator with comparable structure to citrate competes with the substrate for the same binding site. The binding of the indicator... 

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