Radioligand high specific activity

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

The radioligand should also have a high specific activity so that very small quantities of bound ligand can be accurately measured. The specific activity, simply defined as the amount of radioactivity, expressed in becquerels (Bq) or curies (Ci) per mole of ligand, is dependent on the half-life of the isotope used and on the number of radioactive atoms incorporated into the ligand molecule. A radioisotope with a short half-life decays rapidly so that many disintegrations occur in unit time,... 

Since oocytes are a transient, small-scale expression system, they are not the best suited for radioligand binding studies as (usually) hundreds of oocytes must be injected to harvest sufficient membranes for such measurements. This is both time-consuming and expensive. However, with high affinity and highly specific activity ligands, the expression of binding sites on the membranes of intact oocytes can be readily quantified (see later). 

The development of the warfarin immunoassays illustrates several points that are of value in development and use of enantioselective assays. In assays of this type, not only must enantioselectivity be considered, but also the cross-reaction with metabolites is still of importance. As in any RIA, high-specific-activity radioligand is required for the best sensitivity. The use of optically pure radioligand is a further advantage in enantioselectivity. The standard samples used for competitive binding assays must also be essentially optically pure. Otherwise, misleadingly high cross-reacrions may be observed. 

The radioligand [ H] t-butylbicycloorthobenzoate, [ HJTBOB, is prepared at high specific activity from the 4-C1 and 3,4-Cl2 analogs of TBOB by reductive dechlorination with tritium gas (19, 0). ... 

S]TBPS, with similar properties to [ H]TBOB as a radioligand, is synthesized from the corresponding phosphite by addition of sulfur at high specific activity (11). 

H]Mepyramine was introduced in 1977 as the first selective radioligand for the histamine H,-receptor [61, 93] and there are now a large number of studies in the literature which vindicate its use for this purpose. Since that time, a number of other compounds have been introduced as ligands for the histamine H,-receptor and these include the antidepressants [3H]doxepin [94, 95] and [3H]mianserin [96]. More recently, a quaternary derivative of diphenhydramine, ( + >A,4-[3H]methyldiphenhydramine [97], and a high specific activity [U5I]iodobolpyramine [98] have been developed. The high selectivity of [3H]mepyramine, however, has meant that it has been the ligand of choice for the majority of studies on H, -receptors in membrane preparations. 

Radioligands and Photoaffinitv Probes (Figure 51. pH]l of high specific activity has been prepared from 2 (13,28) or tribromopyrrole derivative 19 (28) by catalytic reduction with tritium. Q,o-3- or 4-Substituted-benzyloxycarbonyl-j8-alanyl derivatives with (31) or N3 (32,33) are proposed alternative radioligands and photoaffinity probes, respectively. The latter conversions used the carbodiimide-mediated coupling first established with 27 (14). 

Choice of Radioligand. A 11+C radiolabel will probably exist for most pesticides which will be considered for radioimmunoassay development. Such an intrinsic radiolabel will prove very valuable in titering antisera and possibly in numerous other steps from antigen synthesis through assay development. Unfortunately, for the actual assay, the commonly available 11+C radiolabels may not be of high enough specific activity. The theoretical limit on the specific activity of a single carbon atom is 63 mCi/mmole, and few pesticides have a specific activity of over 50 mCi/mmole even when they are labeled in... 

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