Radioactive assays

Several methods are available today to test candidate ion channel active drugs (ICADs) electrophysiology (patch clamp), binding assays, radioactive flux assays, membrane potential-sensitive fluorescent dyes, ion-sensitive dyes, and voltage sensing based on fluorescence resonant energy transfer (FRET). 

Stable isotopes can yield more information more quickly than radioactive ones. Although rapidly applied, the methods of analysis (n.m.r. spectroscopy and mass spectrometry) are much less sensitive than the method used to assay radioactivity. So a high enrichment is necessary and much more precursor needs to be fed. There is thus a real danger of so much being administered that the normal metabolism of the organism under study is disturbed, resulting in false conclusions. This is not the case with radioactive precursors because they are almost always fed in trace amounts with minimum disturbance of normal metabolism. 

DNA duplexes can be quantitatively modified with an alkanethiol linker at the 5 terminus through a combination of solid-phase and solution-phase methods, and deposited on gold surfaces [45]. Electrochemical assays, radioactive tagging experiments, and atomic force microscopy (AFM) all indicate that the derivatiza-... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

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... 

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

There should be specific, saturable binding to the receptor, accompanied by pharmacological characteristics appropriate to the functional effects, demonstrable using a radioactive, eg, tritium or iodine-125, ligand to label the receptor. Radioligand binding assays (1,6) have become a significant means by which to identify and characterize receptors and enzymes (see Immunoassays Radioactive tracers). Isolation of the receptor or expression of the receptor in another cell, eg, an oocyte can be used to confirm the existence of a discrete entity. 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

Assay of Radioactive Compounds. The radioactive samples were counted on steel planchets in a Nuclear Chicago Model D-47 low-background gas-flow counting chamber with an absolute counting efficiency (estimated by comparison with a standard) of about 20%. 

In the carbon-14 expts, HMX/RDX product was isolated qualitatively, separated Into its components, and each component assayed for carbon-14 beta radioactivity using a liquid scintillation counting technique (Ref 11). DPT-l4C was isolated as an intermediate product from the reaction mixt and similarly radioassayed. For the nitrogen-15 tagged AN expts, HMX and RDX were assayed mass spectrometrically for i5N/i4N ratios from which atom %1SN contents were calcd. In die course of these expts, each tagged species was added initially and also at subsequent stages of the reaction process. The important observations and results are summarized as ... 

FIGURE 5. Assay for Met(0)-peptide reductase. The assay for Met(OJ-peptide reductase is carried out in a two-step incubation. The first incubation mixture contains Met(OJ-L12, DTT and the Met(OJ-peptide reductase. At the end of this incubation, purified L12 transacetylase and [3H]acetylCoA are added and the amount of radioactivity incorporated into L7 is determined by its ability to be retained on a nitrocellulose filter. The latter represents the amount of Met(0)-L12 reduced since the oxidized protein is not a substrate for the transacetylase. 

Limitations encountered in routine use include (a) the use of radioactive materials, (b) limited sensitivity in the presence of high protein concentrations, (c) long assay time of up to 5 days,... 

Purification of the radioactive tracer was modified to include a fractional sublimation before a single extraction—recrystallization cycle to conserve the tracer material. Microgram samples were prepared in melting point capillaries for assay by mass spectroscopic analysis (Table III), made by direct probe injection of the sample into the ion source (18). The probe was heated rapidly to 200°C, and mass spectra were obtained during vaporization of the sample. Tri-, tetra-, and pentachlorodibenzo-p-dioxins vaporized simultaneously with no observed fractionation. 

Figure 9. Anti-PbTx antiserum inhibition of [ H]PbTx-3 binding to its receptor site in rat brain membrane preparations. Labeled toxin (0.5 nM in 1 ml PBS) was incubated with rat brain membranes (125 fig total protein) and increasing amounts of anti-PbTx antiserum (- -) or pre-immune serum (- -) for 1 hr at 4 C. Membrane-bound radioactivity was then measured in a centrifugation assay as previously described (8),...

The basic theory of competitive protein binding assays employing a radioactive label is as follows ... 

This equation illustrates the components of a competitive protein binding assay system. That is, the reaction system contains both radioactive and non-radioactive free ligand (P and P) and both radioactive and non-radioactive protein bound ligand (P Q and PQ). This type of assay assumes that binding protein will have the same affinity for the labeled or non-labeled material that is being measured. Although this assumption is not always completely valid, it usually causes no problems of consequence with most radioassays or radioimmunoassays. 

PMT assays were performed as described by Vannier et al. [3] by adding an equal volume of an enzyme preparation to a 0.1 M Tris-HCl buffer containing 3.36 pM of [ C]SAM (1.8 GBq mmol, 740 kBq ml", NEN), 1% (WA ) BSA and 12% sucrose, with or without 0.2% pectic acceptor. The incubation was run at 28°C for 12 h. After precipitation of the reaction product in 70% ethanol, the methylated polymers were selectively extracted with 0.5% ammonium oxalate and radioactivity was measured in a Tricarb 2250 CA Packard scintillation counter. 

FIGURE 8. The reduction of fMet(O)-Leu-Phe by a human neutrophil and purified E. coli Met(O) peptide reductase. Each assay contained in a final volume of 30 /j1 25 mM Tris-HCl (pH 7.4), lOmM MgCl, 15 mm dithiothreitol 540pmole fMet(O)-[ H]Phe-Leu and Met(0)-peptide reductase. After incubating at 37 °C for 60 min, the incubation mixture is acidified and extracted with ethyl acetate. After centrifugation, an aliquot of the organic phase is removed and assayed for radioactivity. Reproduced by permission of Academic Press from Fliss and coworkers ... 

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