Radioassay methods

The enzymatic radioassay method for the analysis of acetylcholine and choline in brain tissue has been reported by Reid et al. [210]. The method describes the determination of nanogram amounts of acetylcholine and choline in as little as 10 mg of brain tissue, involves isolation of acetylcholine by high-voltage paper electrophoresis, alkaline hydrolysis of acetylcholine to choline, and conversion of this into [32P]-phosphoryl choline in the presence of choline kinase and [y32P] ATP. The labeled derivative is isolated by column chromatography on Bio-Rad AG1-X8 resin, using Tris buffer solution as the eluent. Cerenkov radiation from 32P is counted (at 33% efficiency) in a liquid scintillation spectrometer. The amount of phosphorylcholine is proportional to the amount of choline over the range of 0.08-8.25 nmol. 

Bluth et al. described a simplified radio-enzymatic assay method for the determination of acetylcholine and choline in discrete structures of rat brain [211], 

Eckernas and Aquilonius described a simple radioenzymatic procedure for the determination of choline and acetylcholine in brain regions of rats sacrificed by microwave irradiation [215]. The methods are based on acetylation (in phosphate buffer) of free choline with [14C]-acetylcholine using purified choline acetyltransferase. After ion-pair extraction of [14C]-acetylcholine with tertiary phenyl borate contained in toluene-based scintillation cocktail, the radioactivity was measured. 

Spector et al. developed a specific radioimmunoassay method for the determination of acetylcholine [216]. The described method is suitable for determining 2-137 pmol of acetylcholine the radioligand used is 

Ladinsky and Consolo used an enzymatic radioassay method for the determination of acetylcholine and choline [218], The method was based on the electrophoretic separation of acetylcholine and choline, hydrolysis of acetylcholine to form choline, and acetylation of the choline with labeled AcCoA and choline acetyltransferase. The labeled acetylcholine formed was isolated and quantitated. The method was sensitive and specific, and permitted the routine handling of a large number of samples in a single experiment. The standard curves were linear up to at least 42.5 ng (0.4 nmol) choline and 45 ng (0.3 nmol) acetylcholine. The lower limit of sensitivity was 2ng, and the recovery of acetylcholine was 95% when carried through the entire procedure. 

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Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

Recently, radioassay methods have been refined to measure folates in biological samples. These techniques use radioactively labeled folates and competitive protein binding.80 Johnson et al.81 compare this method with traditional microbiological assay with L. casei. 

In classical activation analysis a material to be analyzed is bombarded with neutrons, charged particles, or photons (gamma-rays). By this bombardment radionuclides are produced from elements of the target material. These radionuclides can be analyzed qualitatively and quantitatively by radioassay methods. From the results and the knowledge of the nuclear reactions during the bombardment an analytical determination of the target material can be achieved. The radioactive products of the bombardment can be measured after the irradiation and the emitted types, energies, and half-lives provide information used for qualitative analysis and the radiation intensity supplies data for the quantitative composition of the material to be analyzed. 

A radioassay method for measurement of the products of proteolytic cleavage of isolated cartilage [ S]proteoglycan has been reported. The method was used to measure the chondroitin sulphate peptides separated from... 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

Four types of techniques for separating the bound fraction P Q from the reagent mixture are in common usage, loosely termed double antibody, solid phase, charcoal adsorption and solution precipitation. The first type is used with radioimmunoassay methods specifically, while the other three types can be used with both radioassay and radioimmunoassay methods. 

Of the radioassays that are commonly used to quantify americium, a-spectroscopy is used when isotopic analyses of americium must be conducted (e.g., 241Am and 243Am). 243Am is often added as a tracer to estimate the efficiency of the sample preparation method when quantifying 241Am in biological matrices. 

Table 21 HPLC Methods for Quantitating B l2 Vitamers in Foods (C8 Columns Detection by Radioassay)... 

The isotope dilution technique makes possible the analysis of mixts in which complete separation of the components is very difficult or impossible, since it is only necessary to isolate a small amt (enough for radioassay) of each component in the pure form. The method is based on the principle that the change in initial specific activity (counts per minute per unit wt) of the tracer will be proportional to the amt of inactive form of the same compd in the mixt that is, the change in counting rate of the original radioactive material is a function of the amt of dilution by the chemically equivalent inactive material... 

The availability of MIP microparticles through this synthetic method has also stimulated the development of analytical techniques that make use of them as sensing elements. Apart from competitive radioassays [30] and immunoassays [32], which were already performed with ground bulk polymers, the small, regular size of the beads prepared by dispersion/precipitation polymerisation enables their use in CEC [45, 46], scintillation proximity assays [35], fluorescent polarisation assays [47], and chemiluminescence imaging [48]. 

T3. Tibbling. G., A method for determination of vitamin B12 in serum by radioassay. Clin. Chim. Acta 23, 209-218 (1969). 

Nelson, N. (1987) A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay. Anal. Biochem. 165, 287-293. 

The colour quenching effect of carotenoids in liquid scintillation spectrometry has been studied in detail. The most efficient quenchers were lycopene [i/>, -carotene (225)] and echinenone [j8,/3-caroten-4-one (226)]. Use of the external standards ratio method to correct for colour quenching in the radioassay of carotenoids is justified.84... 

Due to the effident manner by which MHC ternary complexes can be formed, this method has potential for rapid screening of peptide analogues, possibly by radioassay, or for use in equilibrium and kinetic studies. 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

This material, which is said to have potential antidepressant activity, was administered to human subjects and the metabolites (recovered from urine samples) identified by radioassay and chromatographic methods [178]. 

Douglas. G. S., Ed.. "Radium by Radon Emanation Method." Radioassay Procedures for Environmental Samples, U.S. Public Health Service Publication No. 999-RH27. Rockville. MD. 1967. pp. (4-36H4-45I. 

There are several methods to assess degree of protein biotinylation. For example, titration of protein amino groups with TNBS before and after biotinylation allows estimation of the total number of biotin residues coupled per protein molecule (12). However, only streptavidin-accessible biotin residues localized on the surface of biotinylated protein contribute to its conjugation with streptavidin. To determine such accessible biotin residues we utilize direct solid-phase radioassay of binding of radiolabeled streptavidin to immobilized biotinylated protein (or vice versa) (17). 

The specific radioactivity of the labeled product can be obtained either by solid-sample counting of mercury(I) chloride or by gas-phase radioassay of the volatile halide itself. In the former method it is necessary to distill an aliquot of the volatile halide into aqueous base and to isolate the chloride formed on hydrolysis by precipitation with mercury (I) nitrate. The gas-phase nondestructive radioassay yields a specific activity in terms of pressure rather than in terms of weight of mercury (I) chloride. 

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