Radioassay procedure

Radioassay is a competitive protein binding assay which employs a natural binding protein instead of an antibody. For example, transcortin is the binding protein for cortisol in nature and also in the radioassay procedure for cortisol. 

Besch, P.K. Clinical Radioassay Procedures A Compendium. AACC (1975). 

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. 

RH-27) Radioassay Procedures for Environmental Samples. Jan. 1967. 497 p. Public Health Service, Washington, D.C. 21 30352... 

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. 

Cu (5.1 min). Instrumental radioassay was performed with a similar nuclear counting system as for the airborne gunshot residues (Ref 17) described above with the addition of a programmable computer coupled to the multichannel analyzer for data processing. Using these procedures, it was possible to detect Ba levels above 2 x 10 g/cm and Sb levels above 1.5 x 10" g/cm of floor surface... 

Procedures for the more sensitive ELISA and radioassays are similar to those previously described for anti-Tg antibodies a number of quantitative commercial kits are available. A combined micro-ELISA for simultaneous measurement of TgAb and TPOAb antibodies in a single test has also been described. Microsomal antibody concentrations are usually expressed in units per mfililiter with reference to the MRC IRP for Microsomal Autoantibody 66/387. The mean TPOAb activity, measured with an IRMA kit, is 280 60 lU/mL in normal sera. ... 

BaA and BaP is added and the entire sample is extracted with methylene chloride (250-, 100-, and 100-mL portions). The extracts are combined, 10 mL of isooctane added, and the solution evaporated to about 10 mL. When the solution approaches 10 mL, a second 10-mL volume of isooctane is added. This is repeated three times. The isooctane solution is made up to 75 mL and extracted with three 100-mL portions of dimethyl sulfoxide (DMSO) that contains 20% phosphoric acid. The DMSO extracts are each washed with three 30-mL portions of fresh isooctane, combined and diluted with 500 mL of water. This solution is extracted with three 80-mL portions of isooctane. The isooctane extracts are combined, rinsed once with water, and evaporated to 25 mL. At this point, a 1-mL portion of the solution is removed for intermediate radioassay and a UV scan. Usually the concentrate is sufficiently free from interferences and is evaporated down to about 0.05 mL and an aliquot injected into the GC. If further purification is indicated, the solution is chromatographed on a 1.1-cm X 90-cm column containing 75 g of Woelm Neutral Alumina deactivated with 2% water. Before deactivation, the alumina is dried in an oven for 1 hr at 150°C. The solvent elution schedule is as follows 25 mL cyclohexane prewash, 25-mL sample in cyclohexane, 100 mL cyclohexane, 100 mL cyclohexane-methylene chloride (9 1), 100 mL cyclohexane-methylene chloride (1 1), 100 mL methylene chloride. After the first 125 mL has eluted, 10 cuts are then radioassayed and combined into a single PNA fraction. This fraction is evaporated to about 0.05 mL in preparation for the GC-UV finishing step. Figure 2 is a diagram of the wastewater procedure. 

The isooctane solution is eluted through a long, narrow (6 mm X 90 cm) column of 2% partially deactivated alumina. The solvent elution procedure is as follows 5 mL prewash of cyclohexane, 10 mL sample, 30 mL 5% methylene chloride in cyclohexane, 30 mL 10% methylene chloride in cyclohexane, 30 mL 50% methylene chloride in cyclohexane, and 30 mL of methylene chloride. Ten-milliliter cuts are collected, counted, and combined according to the radioassay data. The combined cuts are reduced to approximately 0.05 mL on a steam bath under nitrogen after adding 5 fxL of n-hexadecane to prevent going to dryness. An aliquot (10 fxL) of this concentrate is used for the GC-UV finishing step. 

As mentioned in the Section 1, physico-chemical methodology for quantitative analysis of plant hormone focuses primarily on GC-SIM, although HPLC with selective fluorescence detection continues to be used for lAA analysis in some laboratories. Procedures, such as the 2-methylindolo-a-pyrone assay for lAA analysis [82], are now rarely utilised. With the exception of ethylene quantification [2] there is little use of non-MS-based GC detection techniques, despite the fact that selective analysis at the picogram level is achieved for ABA with an electron capture detector [83], and lAA and cytokinins with a nitrogen phosphorus detector [84,85]. The reason for the demise of these GC procedures is that the detectors are destructive and this precludes the reliable recovery of labelled internal standards for radioassay and isotopic dilution analysis. The usual compromise was to take two aliquots of the purified samples, one for GC analysis and the other for the determination of radioactivity. The accuracy of this approach is dependent upon the questionable assumption that the radioactivity in the purified sample is associated exclusively with the compound under study. In an attempt to circumvent this problem, a double standard isotope dilution procedure was devised for the quantitative analysis of lAA in which one internal standard was used to correct for losses during sample preparation and a second for GC quantification [86]. This procedure was used in several... 

Separation and Assay. Procedures for the separation, purification, and assay of carotenoids and retinoids by h.p.l.c., g.c., and g.c.-m.s. are given in an extensive article." Another, general, review includes information on the h.p.l.c. separation of retinoids.A particularly useful method has been developed for resolution and analysis of some carotenoid optical isomers.For example, (3R,3 R)-, (3S,3 S)-, and (3/ ,3 5)-astaxanthin were converted into the diastereomeric (-)-camphanic acid diesters, which were separated by h.p.l.c. This procedure has been used to analyse the isomeric composition of a natural astaxanthin sample. An h.p.l.c. procedure for separation of a-, P-, and y-carotenes (173)—(175) and lycopene (176) has been described." Several papers describe methods for the h.p.l.c. separation and purification of various retinal and retinol isomers and derivatives.A procedure for the preparative t.l.c. of oxidation products of retinyl acetate has been described,and a competitive protein-binding radioassay for retinoic has been reported. 

Snyder, F. (1969). Review of instrumentation and procedures for C and H radioassay by thin layer chromatography and gas liquid chromatography. Isotop. Radiat. Tech-nol. 6 381-400. 

Table 4.4 procedures for the radioassay of paper and thin-layer chromatograms... 

Table 4.7 procedures for rendering biological material suitable for radioassay... 

The enzyme and radioactive substrate are incubated for a known period of time together with appropriate blank samples. The reaction is stopped, often by acid precipitation of the enzyme, and the unreacted substrate and product separated from one another. An aliquot of the isolated product is then taken for radioassay. In only a few cases have continuous assay methods been used in radioisotopic enzyme procedures [310]. 

Glass fiber-paper chromatography (Hamilton and Muldrey, 1961) and paper chromatography (Rouser et al., 1961) can also be used for radioassay. However, paper chromatography is used for lipid separations only in reversed-phase systems (Malins and Mangold, 1960) or after silicic acid impregnation (Marinetti, 1962). Radioassay of lipids with these procedures has been limited. 

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