Radiometric assays

Fig. 1 Structure of BILN 2061 and initial lead peptide DDIVPC. Activities were determined in a radiometric assay against the NS3 protease domain...

Johnson CD, Russell RL. A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations. Anal. Biochem. 64 229-238, 1975. 

Several other methods, including a 31P NMR [39,40] and radiometric assay [28, 41] have also been used to monitor the PLCBc catalyzed hydrolysis of phospholipids. The assays that are currently available for PLCBc are compared in Table 1. 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Jerina, D. M., Dansette, P. M., Lu, A. Y. H., and Levin, W. Hepatic microsomal epoxide hydrase a sensitive radiometric assay for hydration of arene oxides of carcinogenic aromatic hydrocarbons. Mol. Pharmacol. (1977) 13 342-351. 

Discontinuous approaches are used through necessity in radiometric assays (Oldham, 1993) and in liquid- and gas-chromatography-based protocols (Syed, 1993). Many commercially available kits designed to measure a particular enzyme activity combine absorbance and fluorescence platereader-based technologies with assay protocols that could be done continuously. However, since many users of these kits have little experience in enzymology, assay instructions usually outline a simplified procedure whereby a... 

Oldham KG. 1993. Radiometric assays. Enzyme assays. A practical approach. Eisenthal R, Danson MJ, editors. Oxford Oxford University Press pp. 93-122. 

Even under seemingly ideal conditions, the steady-state concentration of the first reaction product may exceed the inhibitory constant for the binding of that product to the primary enzyme. In such cases, the linearity of the coupled assay can be misleading, and the investigator must validate the coupled enzyme kinetic data by direct comparison with the results obtained by another technique such as a stopped-time radiometric assay. This... 

Plasma CHE was determined by a radiometric assay (9) using acetylcholine and Btf 284c51. Plasma creatine kinase fCK) was determined by a spectrophotometric method (10). 

Potter WT, Garry VF, Kelly JT, et al. 1993. Short Communication Radiometric assay of red cell and plasma cholinesterase in pesticide appliers from Minnesota. Toxicol Appl Pharmicol 119 150-155. 

Potts JT, Jr., Bringhurst FR, Gardella TJ, et al. Parathyroid hormone Physiology, chemistry, biosynthesis, secretion, metabolism, and mode of action. In Degroot LJ (Ed.), Endocrinology. W.B. Saunders, Philadelphia, PA (1996) 920-965. Nussbaum SR, Zahradnik RJ, Lavigne JR, et al. Highly sensitive two-site immuno-radiometric assay of parathyrin and its clinical utility in evaluation patients with hypercalcemia. Clin. Chem. (1987) 33 1364-1367. 

Gerard, V. A., and DriscoU, T. (1996). A spectrophotometric assay for Rubisco activity Application to the icelp Laminaria saccharina and implications for radiometric assays. J. Phycol. 32, 880—884. 

Tulamo R M 1991 Comparison of high-performance liquid chromatography with a radiometric assay for determination of the effect of intraarticular administration of corticosteroid and saline solution on synoviai fluid hyaluronate concentration in horses. American Journal of Veterinary Research 52 1940-1944... 

Gan, T. E., Hallam, L., Pilkington, G. R., and Vanderweyden, M. B. (1981) A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells. Clin. Chim. Acta 116, 231-236. 

All radiometric assaying was performed by conventional liquid scintillation counting techniques using a Beckman LS-100 automatic scintillation counter and Ready-Solv GP scintillation solution. 

Panigrahi K, Delmas PD, Singer F, Ryan W, Reiss O, Fisher R, et al. Characteristics of a two-site immuno-radiometric assay for allcaline phosphatase in serum. Clin Chem 1994 40 822-8. 

Attaccalite, S., Carotenuto, P. and Laufer, R. (2005) Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well radiometric assay Drug Metabolism and Disposition The Biological Fate of Chemicals, 33, 812-819. 

BD Albertson, FP Haseltine. Non-radiometric Assays, Technology and Application in Polypeptide and Steriod Hormone Detection. New York Alan R. Liss, 1988. 

A radiometric and a spectrometric assay have been developed to measure PhaC activity. The radiometric assay measures the incorporation of isotope-labeled hydroxyacyl moieties into the polyester, which is present from the beginning as primer [17]. [3-14]ft-(-)-3-hydroxybutyryl-CoA or [3H]-P,S-3-hydroxybutyryl-CoA or in principle any other CoA thioester of a radioactively-labeled hydroxyacyl moiety could be used as substrate. Only the radioactivity that is really incorporated into the insoluble polyester is measured. The time course of the assay, the need to synthesize the substrates and the high costs make the assay very inconvenient and it is hardly used anymore. A more convenient assay is the spectrometric assay which measures the release of coenzyme A during the polymerization reaction in presence of Ellmann s reagent 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) yielding 5 -thio(2-nitrobenzoate) that absorbs at about 412 nm [16], Here, the enzyme activity is measured directly without delay. However, it is not the formation of the polymeric product that is measured, but the release of coenzyme A, which can also be due to the hydrolytic cleavage of the substrate by another enzyme that does not have any PhaC activity, like a thioesterase. Nevertheless, this assay is now most frequently used due to its convenience. 

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