Ligand radiolabeling

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

The introduction of tritium into molecules is most commonly achieved by reductive methods, including catalytic reduction by tritium gas, PH2], of olefins, catalytic reductive replacement of halogen (Cl, Br, or I) by H2, and metal pH] hydride reduction of carbonyl compounds, eg, ketones (qv) and some esters, to tritium-labeled alcohols (5). The use of tritium-labeled building blocks, eg, pH] methyl iodide and pH]-acetic anhydride, is an alternative route to the preparation of high specific activity, tritium-labeled compounds. The use of these techniques for the synthesis of radiolabeled receptor ligands, ie, dmgs and dmg analogues, has been described ia detail ia the Hterature (6,7). 

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

We have also used a non-radiometric-binding approach based on fluorescence polarisation [29], where a fluorescent label is used in place of a radiolabel. As the fluorescently tagged oxytocin binds to the receptor, its rotational velocity is reduced and the polarisation of the fluorophore increases. The displacement of the ligand may be measured by a decrease in polarisation. 

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

Hence, a plot of log (pAR /(I -pAR)) against log [A] should give a straight line with a slope of one. Such a graph is described as a Hill plot, again after A. V. Hill, who was the first to employ it, and it is often used whenpAR is measured directly with a radiolabeled ligand (see Chapter 5). In practice, the slope of the line is not always unity, or even constant, as will be discussed. It is referred to as the Hill coefficient (%) the term Hill slope is also used. 

L Radiolabeled ligand whose binding is directly measured L can be an agonist or antagonist or even a channel blocker, etc. 

Of course, saturation experiments are only possible when a radiolabeled form of the ligand of interest is available. Competition experiments, on the other hand, are particularly useful in allowing the determination of dissociation constants for unlabeled drugs which compete for the binding sites with a ligand that is available in a labeled form. This approach has been widely adopted by the pharmaceutical industry as a rapid means of determining the affinity of novel compounds for a particular receptor for which a well-characterized radioligand is available. 

Fig. 7. General scheme for radiolabeling antibodies with rhenium using N2S2-ligand (N3S-ligand)...

A very simple technique is to use a radiolabeled ligand (usually a well-known substrate) of the specific transporter of interest. A recent suggestion for functional quantitation of the apparent affinities (fQ values) to P-gp using Caco-2 cells and the substrate taxol has been published [143], The method can be described simply as (1) determination of b —> a transport of3 H -taxol in normal, untreated Caco-2 cells and (2) determination of b —> a transport of 3H-taxol in the presence of verapamil (0.2 mM). The difference between these two components represents the active transport via P-gp. The two concentrations of the test compound are chosen as approximately 0.25 x K, and 4.0 x K, and for the inhibition of taxol transport, and in the study of Gao et al. [143], 16 pM and 250 pM of the test compound were used... 

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