Radioisotope assays

Table 1 Hsts several of the chemical deterrninations and the corresponding reactions uti1i2ed, which are available on automated clinical analy2ers. With the exception of assays for various electrolytes, eg, Na", K", Cl , and CO2, deterrnination is normally done by photometric means at wavelengths in the ultraviolet and visible regions. Other means of assay include fluorescence, radioisotopic assay, electrochemistry, etc. However, such detection methods are normally required only for the more difficult assays, particularly those of semm or urine constituents at concentrations below )Tg/L. These latter assays are discussed more fully in the Hterature (3,4).

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. 

Selected entries from Methods in Enzymology [vol, page(s)] Radioisotopic assay of acetylcarnitine and acetyl-CoA, 123, 259 short-chain acylcarnitines identification and quantitation, 123,... 

Milner, Y. Avigard, G. A sensitive radioisotope assay for pectin methylesterase activity. Anal. Biochem., 1973, 51, 116-120. 

Enzyme kinetics are normally determined under steady-state, initial-rate conditions, which place several constraints on the incubation conditions. First, the amount of substrate should greatly exceed the enzyme concentration, and the consumption of substrate should be held to a minimum. Generally, the amount of substrate consumed should be held to less than 10%. This constraint ensures that accurate substrate concentration data are available for the kinetic analyses and minimizes the probability that product inhibition of the reaction will occur. This constraint can be problematic when the Km of the reaction is low, since the amount of product (10% of a low substrate concentration) may be below that needed for accurate product quantitation. One method to increase the substrate amount available is to use larger incubation volumes. For example, a 10-mL incubation has 10 times more substrate available than a 1-mL incubation. Another method is to increase the sensitivity of the assay, e.g., using mass spectral or radioisotope assays. When more than 10% of the substrate is consumed, the substrate concentration can be corrected via the integrated form of the rate equation (Dr. James Gillette, personal communication) ... 

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 ... 

J3. Jacobs, W. L. W., and Zondag, H. A., Radioisotope assay of vitamin B12 in human blood serum. Clin. Chim. Acta 24, 93-103 (1969). 

Pahuja, S. L., and Reid, T. W. (1982). Radioisotope assay for glutamine synthetase using thin-layer chromatography. J. Chromatogr. 235, 249—255. 

Folate deficiency leads to accumulation of Figlu, which is excreted in urine. The excretion is very pronounced after a loading dose of histidine, a test used to detect folate deficiency. More sensitive radioisotopic assays use folate binders to the vitamins. High urinary levels of Figlu may coexist with elevated levels of serum folate. Thus, in vitamin Bn (cobalamin) deficiency, since cobalamin participates in the following reaction, FH4 is trapped as... 

In 1979, the first SPA was described by Hart and Greenwald [15, 17]. It was an immunoassay using two polymer beads coated with antigens, one labeled with a fluorophore and the other with tritium ( H). In the following years, Amersham Biosdences Inc. further optimized this radioisotopic assay technology [18, 19]. 

Waxman, Schreiber, and Herbert first described a radioisotopic assay for the measurement of serum folate in 1971 (W14) which they claimed gave almost identical results to those obtained by with the L. casei microbiological assay and could separate low, borderline, and normal folate levels. Serum was mixed with PHjmethyltetrahydrofolic acid in a phosphate buffer and a folate binder was added which, in this case, was Carnation brand instant powdered milk. Following incubation, free vitamin was separated from that which was bound using hemoglobin-coated charcoal. The radioactivity of the supernatant was determined, and from this the folate concentration in the serum sample could be calculated using appropriate standards and blanks. 

Waxman, S., Schreiber, C., and Herbert. V., Radioisotopic assay for measurement of serum folate levels. Blood 38, 219-228 (1971). 

W22. Wood, W. G., Rohde, C, and Jacobs, A., A comparison of commercially available luminescence enhanced enzyme immunoassay with in-house non-radioisotopic assays for thyroxine binding globulin and total thyroxine. J. Clin. Chem. Clin. Biochem. 26, 135-140 (1988). 

DELFIA or time-resolved fluorometty (TRF) is a well-established technology that exploits the unique fluorescence properties of lanthanide chelates and provides a powerful alternative to radioisotopic assays in many high throughput-screening applications. 

McGarry, J.D. Foster, D.W. (1976) J. Lipid Res. 17, 277-281. An improved and simplified radioisotopic assay for the determination of free and esterified carnitine. 

Kelley RI, Segal S. Evaluation of reduced activity galactose-l-phosphate uridyl tiansferase by combined radioisotopic assay and high-resolution isoelectric focusing. J Lab Clin Med 1989 114 152-156. 

Kebabian, j. W., 1978, A sensitive enzymatic-radioisotopic assay for apomorphine, J. Neurochem. 30 1143-1148. 

Assay of adenosine deaminase and of other enzymes of purine and pyrimidine metabolism were performed by previously described radioisotopic assays (3). Electrophoresis of RBC ADA was performed on hemolysates after Spencer et al. (4). The enzymes of qlu-cose and glutathione metabolism were assayed after Beutler (5). Erythrocyte glucose consumption was assayed according to Cartier et al. (6). Adenosine deaminase was partially purified from human red blood cells the specific activity of the preparation was 1.2 pmol.min l.mg protein l. Rabbit antiserum to human adenosine deaminase was raised by immunization of white male rabbits with this preparation. 

KM Knights, R Drew. A radioisotopic assay of picomolar concentrations of coenzyme A in hver tissues. Anal Biochem 168 94—99, 1988. 

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