Luminescent enzyme assay

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Luminescent RE + chelates have been successfully developed as labels and probes for highly sensitive and selective bioassays in the past two decades. Time-resolved Inmines-cence detection has been widely applied in fluoroimmunoassay, DNA hybridization assay, enzyme assay, cell activity assay and fluorescence imaging microscopy. ... 

Nonradiometric methods iticlude absorbance, fluorescence. and luminescence spectroscopy. Enzyme assays are a common example. The as.suy is usually run at or below the value of thq substrate, with only about S% of the substrate consumed during the assay, and multiple enzyme turnovers occur during the a.ssay. Sometimes enzyme reactions arc coupled, especially if the target reaction does not produce a product that can be detected directly in the assay. An example is carboxypeptidase. which is coupled to the teduction of NADP to NADPH. giving ri.se to absorbance at 340 nm. 

The applications are succinctly described and include applications of luminescence in antioxidant research, phagocytosis, microbiology, ecology, food and environmental testing, immunoassay, enzyme assays, DNA probe assays, and reporter gene and gene expression assays. 

The method used for detection depends on the type of label used. Isotopic counting is employed for radioisotopes, colorimetry for enzyme assays, luminescence and fluorescence measurements can be achieved by means of photomultiplier tubes, while turbidimetry or nephelometry is used for particle enhanced assays. 

Biological Applications Calcium indicator assaying luminescent enzyme measuring luciferase activi-ty as a substrate for luciferase screening HIV-1 protease inhibitors ... 

Inoue, S. Sasaki, T. Method for assaying luminescent enzyme in presence of organic solvent. Jpn. Kokai Tokkyo Koho JP 2008000073, 2008 Chem. Abstr. 2008, 148, 115514. 

Minute amounts of coelenterazine can also be measured utilizing apoaequorin or apoobelin (Campbell and Herring, 1990 Thompson et ah, 1995). In this method, a sample containing coelenterazine is treated with an excess amount of apophotoprotein (apoaequorin or apoobelin) to convert it to a Ca2+-sensitive photoprotein (aequorin or obelin). The photoprotein formed is assayed by luminescing it with Ca2+ to determine the amount of coelenterazine originally existed. With this method, the luminescence reaction is fast and usually complete in a few seconds, in contrast to the slower luminescence reactions with luciferases that sometimes require a few minutes to complete. However, the formation of photoprotein from apoaequorin is slow and not necessarily quantitative, and the overall accuracy of the photoprotein method does not compare favorably with that of the luciferase method that directly measures coelenterazine. The author recommends using a luciferase if the enzyme is available. 

Since the pioneering work of Kleymann et al. (2002), Betz et al. (2002), Baumeister et al. (2007), and Crute et al. (2002), who showed that compounds identified as inhibitors of the helicase-primase enzyme complex could alleviate herpesvirus-induced disease in animal models, the attention of researchers developing antiviral compounds has been drawn more and more towards the virus-encoded helicases, particularly those of Herpes viruses and of RNA viruses such as Hepatitis C Virus (HCV) and SAKS coronavirus (SARS-CoV). Enzyme activity is usually assayed by measuring NTPase activity in the presence of an appropriate nucleic acid co-substrate although, more recently, novel fiuorimetric and luminescence principles have been applied to the measurement of strand unwinding and/or translocation of the protein along the nucleic acid (Frick 2003, 2006). 

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

A very versatile piece of equipment that is affordable for individual laboratories is the microplate reader. This allows multiple samples to be analyzed at once, commonly in a 96-well format, although 384- and 1536-well formats are available. Typical measurements that can be performed include UV-Vis absorbance, fluorescence, or luminescence, allowing a range of assays to be performed, such as cell growth, enzyme kinetics, enzyme stability, or enzyme-linked immunosorbent assay [60-62]. Functionality can be increased by the use of liquid dispensing systems or automatic plate handling. 

Figure 3 Example of a typical luminescent flow sensing device [manifold for pesticide chemiluminescent flow assay with one (A) or two (B) columns using immobilized enzymes]. A = immobilized dehydrogenase (Phe-DH) 1.0 m coil B = immobilized biolumi-...

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

Hamilton Umicon Lumicon chemi- and biolumium assay luminometer This equipment is used in test-tube scale luminescent immunoassays. With its sample compartment (thermostatted by means of Peltier elements, which allow the temperature to be set from 15°C to 40°C with a precision of 0.1°K) this instrument is suitable for the measurement of temperature-sensitive bioluminescence resulting from enzymic reactions and also in phagocyte-mediated luminescence measurements. 

R. A. Evangelista, A. Poliak, and E. F. G. Templeton, Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays, Anal. Biochem. 197, 213-224 (1991). 

To study drug-receptor/enzyme interaction, it is not always convenient or appropriate to use a living system of the target receptor. Instead, biochemical assays can be devised to mimic the target. Very often, the assays use multicolor luminescence or fluorescence-based reagents. In this way, the reaction path can be followed in space and time to enable quantitative evaluation of the reaction. 

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

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