Radioisotope-based assays

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... 

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61], Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions ... 

Third, the hazards of handling and disposing of radioisotopes have made it desirable to find alternative methods of assay. When the assay involves fluorescent or intensely absorbing substrates or products, the sensitivity of HPLC-based assays rivals that of radiochemical assays. 

Various CL-based analyses of vitamin B12 are listed in Tables 27.2 and 27.3 for pharmaceutical and food samples. Compared with conventional analytical techniques, such as microbiological assay, high-performance liquid chromatography (HPLC), atomic absorption spectroscopy (AAS), radioisotope isotope assay, and fluorimetric detection, the current proposed CL method was more sensitive with a detection limit of 5 pg/mL. 

A radioisotope dilution assay and an assay based on animal growth are the most reliable methods for the determination of vitamin B12 activity. The tracer technique is highly specific for cyanocobalamin or analogs which are convertible to cyanocobalamin. The assay is specific for cyanocobalamin if cyanide treatment of the sample is avoided total cobalamins convertible to cyanocobalamin are determined if a given sample is first treated with cyanide. The method consists in the addition of a known amount of pure [ Co]-cyanocobalamin. A series of selective extractions and adsorptions to remove interfering substances is completed and the radioactivity and color of the purified sample are measured. From these data, it is possible to calculate the amount of cobalamin present in the original sample. The isotope dilution method is accurate and precise and can be used for both relatively pure samples and for crude extracts of low potency. 

Radioisotopic enzyme assays which conversely are based on the measurement of radioactivity in a product obtained through enzymatic conversion of a radiolabelled substrate for evaluating enzymatic activity. 

Radeke, H.S., Digits, C.A., Bruner, S.D., and Snapper, M.L. (1997) New tools for studying vesicular-mediated protein trafficking synthesis and evaluation of ilimaquinone analogs in a non-radioisotope-based antisecretory assay. J. Org. Chem., 62, 2823-2831. 

Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [84]. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyidopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported [462J. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. 

A small number of methods allow for simultaneous determination of FSH and LH in a single assay tube. Doublelabel RiAs using Co-labeled LH and I-labeled FSH as tracers are available as commercial kits. Following separation, each radioisotope is determined in the bound fraction by dual isotope counting or by repeat counting. A simultaneous immunofluorometric assay of LH and FSH, based on the use of the fluorescent lanthanides Eu " " and Tb, has also been described each is detected with a time-resolved fluorometer. ... 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

---