Radioactive Assay Technologies

The earliest assays were based on the use of 32P as a label either on the ATP cofactor for kinases or on a peptide substrate for phosphatases. With kinases, the transfer of the 32P from the /position of ATP to a peptide or protein substrate resulted in a 32P-labeled peptide or protein that would be separated away from the ATP by capture on a filter and subsequent washing. The quantity of phosphoprotein could be quantified by scintillation counting. 

The availability of a new version of isotopically labeled ATP, [33P]ATP, provided benefits of safety and longer half-life. The lowered energy was also better suited for scintillation proximity assays (SPAs). The SPA was a major step forward because it eliminated the need for wash steps by capturing the [33P]-labeled peptide on a functionalized scintillating crystal, usually via a biotin-streptavidin interaction. 

All SPAs are based upon the phenomenon of scintillation. Scintillation is an energy transfer that results from the interactions of particles of ionizing radiation and the de-localized electrons found in conjugated aromatic hydrocarbons or in inorganic crystals. When the decay particle collides with 

Because of the complex and often overlapping principles behind kinase and phosphatase assays, I will review the principles of the various fluorescent and luminescent technologies. A textbook by Joseph R. Lakowitz (1999) titled Principles of Fluorescence Spectroscopy is recommended for detailed information on the biophysics of fluorescence. Olive (2004) and Von Ahsen and Boemer (2005) wrote good reviews on the advantages and disadvantages of various luminescent (including fluorescent) technologies for kinase assays. 

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The Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen ) developed by BioSignal, is a bead-based, non-radioactive assay technology. It uses the principle of luminescent oxygen channeling, which was first described in 1994 by Ullman [173], who two years later demonstrated a broad spectrum of applications for such immunoassays [174]. This principle senses the proximity of two beads, a donor and an acceptor bead, which is mediated by the interaction of molecules on the surfaces of the two beads. 

The driving force for the development of chemiluminescence-based assays (as well as any other optical or electrical detection methodology) is the replacement of radiolabels both for safety reasons and because of their intrinsic instability. Because the earliest high sensitivity immunoassays utilized antibodies with covalently attached as the label, this has served as a yardstick against which all subsequent assay technologies are measured. For this reason, it is important to understand the detection limits for I. Radioactive iodine is a y-emitter that eventually decays to a stable isotope of lead. The decay process exhibits first-order kinetics so that we can write... 

Nanosyn, Caliper Life Sciences, and most Carna catalytic assays rely on a microfluidics capillary electrophoresis (CE) technology commercialized by Caliper Life Sciences. These are non-radioactive assays that measure the change in electrophoretic mobility of the substrate (usually a fluorescent-labelled peptide or lipid) upon phosphorylation. Both substrate and product are measured in these assays enabling increased assay precision. Since... 

Considerable effort was expended in the development of alternative technologies that did not require the use and measurement of radioactivity. A number of nonisotopic assays for T4 were subsequently developed commercially for use on fuUy automated immunoassay systems or for use with existing chemistry analyzers. According to a 2002 College of American Pathologists Ligand Assay Survey, more than 95% of laboratories now use a nonisotopic T4 method. A variety of different labels were used to construct these nonisotopic assays. Enzymes such as horseradish peroxidase, alkaline phosphatase, and [3-D-galactosidase were the most... 

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