Radioactivity assay, activation analysis

The post-bombardment processing of the activated sample may follow either a nondestructive assay of the radioactivity in the sample (gamma-ray scintillation spectrometry is used most often for this) or a chemical processing of the sample prior to the radioactivity assay. Techniques involving either precipitation, electrodeposition. solvent extraction, and ion exchange or some combination of these form the basts of the radio-chemical separation techniques used in activation analysis. 

If ferrous ions are present during assays of DNA and RNA polymerases in which radioactive newly synthesized polynucleotides are precipitated on to filter paper discs in trichloroacetic acid, spuriously high results are obtained, apparently due to acid-insoluble complexes of Fe with substrate nucleotides precipitating on the paper. Clearly ferrous ions should be avoided in assays of this type. The complex Al. ATP is a potent inhibitor of hexokinase, and neutron activation analysis of many commercial samples of ATP has shown that Al is ubiquitous, and the most common metal contaminant It is best removed by passing the ATP preparation over a cellulose polyphosphate column at pH 5. Chelates of ATP with divalent metal ions have been separated from non-chelated ATP using reverse-phase h.p.l.c. ... 

Traditionally, HAT activity is measured with a discontinuous radioactive filterbinding assay, which uses pH]acetyl-CoA as a histone acetyltransferase substrate [46]. The transfer of [ H]acetyl-groups to the histone substrate by histone acetyltransferases is detected by liquid scintillation counting of pHjacetylated histones, which are retained on a phosphocellulose disk. Due to its discontinuous character, this assay is technically problematic and not ideal for kinetic analysis. Hence, other assays that work with radiolabeled acetyl-CoA have been described that are suitable for a higher throughput. These work with streptavidin-covered beads [47] or a variant of the SPA with microtiter plates that contain a scintillant (FlashPlates) [48]. But as all these protocols are based on radioactively labeled substrates, they apparently show the same disadvantages that were described for the radioactive HDAC assay protocols. Therefore, nonradioactive assays have been developed to study histone acetyltransferase activity. 

Split-and-mix libraries have found numerous applications in the search for a substrate for a given receptor and vice versa. A typical affinity assay encompasses labeling of the host of interest with, e.g., a dye, a fluorophore, or radioactivity and equilibrating the labeled host with the bead-supported library of potential guests. The labeled host will be concentrated by those beads that carry molecules with affinity for the receptor. These beads are easily identified by visual inspection of the assay under a low-power microscope. Isolation and structural analysis reveals the structure of the active compound [3]. Recently, split-and-mix libraries are also successfully applied in the search for catalysts (see Chapter 5.4). 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

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. 

Experiments have been carried out to demonstrate the method described above by means of a multienzyme complex. This complex was assayed first for the AMP kinase activity. A reaction mixture was prepared containing unlabeled ATP (1 mM) and radioactive [3H]AMP only. The reaction was started by the addition of the complex, and samples were removed and analyzed by HPLC. Chromatographic profiles, each representing the analysis of a sample removed from an incubation mixture at increasing times after the start of the incubation, are shown in Figure 10.4. Both optical density and radioactivity were determined. 

There are other examples of HIV protease substrates that utilize the fluorescence energy transfer technique. Perhaps the strongest characteristic of these assays is that they provide a continuous readout of enzyme activity. Another advantage is their sensitivity (they use small concentrations of enzyme). The disadvantage of these assays is that they are susceptible to interference by some compounds (inhibition artifacts) because of inner and outer filter effects (see Notes 8-10). Other assays, for example, those based on radioactivity or high-performance liquid chromatography (HPLC) analysis of products are tedious to run, but are less susceptible to interference or inhibition artifacts. 

The development of various EIA procedures has led to confusing terminology and classifications, which are often misleading concerning their fundamental features and tend to obscure their relative merits. Not surprisingly, many comparative studies produced inconclusive results. The frequent comparison of the relative merits of radioactive and enzyme labels, as evaluated in RIA by saturation analysis and in ELISA by immunometric analysis, is basically faulty since the underlying principles of these assays are different (Ekins, 1980). Here, EIA will be classified according to their essential differences to expose the inherent limitations and potentials of each assay EIA are based either on Activity Amplification (AA) or Activity Modulation (AM). 

For each herbal drug preparation, a comprehensive specification must be submitted. This must be established on the basis of recent scientific data and must give particulars of the characteristics, identification tests, and purity tests. This has to be done, for example, by the appropriate chromatographic methods. If deemed necessary by the results of the analysis of the starting material, tests on microbiological quality, residues of pesticides, fumigation agents, solvents, and toxic metals have to be carried out. Radioactivity should be tested if there are reasons for concerns. Quantitative determination (assay) of markers or of substances with known therapeutic activity is required. The content must be indicated with the lowest possible tolerance. The test methods must be described in detail. 

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