Radioligand 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). 

Radioligand, [reference] R Radiochemical yield (%) Synthesis time (min) Specific activity (GBq/pmol)... 

Mainly two types of reactions are possible for fluorinations with 18F Electrophilic and nucleophilic fluorinations. However, electrophilic fluorinations are not suitable for PET radioligands, since in that case the 18F is isolated as [18F]F2 with a specific activity which is too low for receptor research. 

The classical histamine H3 antagonist thioperamide has been labelled and evaluated as radioligand for the H3 receptor by Alves-Rodrigues et al [27], [3H]thioperamide was synthesised from [3H]4-(lH-imidazol-4-yl)piperidine, labelled with tritium in the 2 or 5 position of the imidazole and at the 4 position of the piperidine, and cyclohexylisothiocyanate (scheme 10) [28], It was purified with a semipreparative HPLC method and isolated with a specific activity of 6 Ci/mmol. 

H]GR 168320 was developed as a (stably labelled) tritiated H3 antagonist and evaluated for its in vitro use as a radioligand for the H3 receptor [29], As has been shown for thioperamide, the tritium labels at the 2 and 5 position of the imidazole moiety and at the 4 position of the piperidine moiety are sensitive to exchange with hydrogen from the storage solution. [3H]GR 168320 is labelled at the cyclohexane, by a catalytic reduction with 3H2, no details about the synthesis have been published (scheme 11). [3H]GR 168320 was isolated with a specific activity of 4.8 Ci/mmol. 

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... 

Fig. 6 also lists the per cent cross reaction which has been observed in their own laboratories by the producers of the antiserum. The displacement ability of A9-THC is taken as a standard at 100%, and for reasons discussed later, the radioligand used was tritium labeled A8-THC with specific activity of about 50 Ci/ mmole. 

In the case of analysis of A -THC not only must the specific activity of the radioligand be considered, but also its stability. When A9-THC was prepared at a specific activity of about 50 Ci/mmole, it had a very short shelf life. On the other hand, tritiated A -THC of similar specific radioactivity has proven to be a quite stable entity. Its binding characteristics with the antisera raised to either A8- or A9-THC make it a useful radioligand in spite of the heterologous nature of the assay thus introduced. 

This potential radioligand 192 for in vivo visualization of human cholinergic muscarinic receptor sites on smooth-muscle tissue by PET has been synthesized250-252 by methylation of the d-demethyl derivative 193 (equation 121). The r.y. of 192 based on [UC]CH3I was 66%, with a specific activity of 110-300 Ci mmol-1, and optical and radiochemical purities... 

Radioligand 125I-fibrinogen specific activity 3.7 Mbq/mg fibrinogen... 

H]-prazosin, 128, which is an ai-adreno-radioligand, has been prepared117 in a four-step synthesis starting from furoic acid and involving reductive catalysed denomination with tritium gas (equation 50). The specific activity and radiochemical purity of the product 128 were 22.6 Ci mmol-1 and 98%, respectively. 

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. 

Perhaps the major determinant in deciding on an appropriate volume for assay is the specific activity of the radioligand. For example, if in a 2 ml incubation volume total specific radioligand binding is 1000 cpm/mg protein, only 100 cpm/mg protein will be bound using a 0.2 ml volume. 

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