Firefly bioluminescent assay

Wildish DJ (1976) Determination of adenosine 5 -triphosphate in estuarine water and sediments by firefly bioluminescence assay. Technical report of the fisheries maritime service research division, no. 649... 

His research with Grace Picciolo led to his invention of a method for the detection of adenosine triphosphate (ATP). The technique takes advantage of the naturally occurring luciferase enzyme and the chemical lu-ciferin. Both are obtained from the lantern of a firefly hence, this technique is sometimes referred to as a firefly bioluminescent assay. In combination with ATP and magnesium ions, luciferm and luciferase fluoresce, generating a light intensity that is proportional to the amoimt of ATP present. 

However, such a technique can be applied to any determination of ATP. If extraterrestrial life exists, then there is the possibihty that it will employ either ATP or a similar energy molecule. If that energy molecule is introduced into a firefly bioluminescent assay, then light will be produced and it can be measured. The detection of ATP or a similar molecule would be good evidence for the presence of extraterrestrial hfe. 

Trajkovska S, Dzhekova-Stojkova S, and Kostovska S (1994) Determination of validity for blood for transfusion by firefly bioluminescent assay for ATP. Analytica Chimica Acta 280 246-248. 

Ca2+-sensitive photoproteins, 367 coelenterazine, 362 coelenterazine enol-sulfate, 364 coelenterazine luciferase, 363 Cypridina luciferase, 366 Cypridina luciferin, 365 dehydrocoelenterazine, 365 stabilized coelenterazine, 364 Asteroidea, 231 Astronestbes, 338 Atolla, 91,140, 334 ATP, 3-5,10-16, 23-29 Aracbnocampa luminescence, 26 assay of firefly luciferase, 11 firefly bioluminescence, 3-5, 10-16... 

RAPID AND SIMULTANEOUS BIOLUMINESCENT ASSAY OF AEQUORIN AND FIREFLY LUCIFERASE... 

We have developed a highly sensitive simultaneous bioluminescent assay of firefly luciferase and aequorin. Firefly luciferin-luciferase reaction is specific and sensitive for the determination of ATP, and this reaction has been widely used, e.g. for hygiene monitoring. Aequorin bounds specifically to Ca and then emits blue light, thus aequorin is useful to study intercellular We thought that these photoproteins... 

Brovko Y, Romanova NA, Ugarova NN. Bioluminescent assay of bacterial intracellular AMP, ADP and ATP with the use of coimmobilised three-enzyme reagent (adenylate kinase, pyruvate kinase, firefly luciferase). Anal Biochem. 1994 220 410-4. 

P. J. Worsfold and A. Nabi, Bioluminescent Assays with Immobilized Firefly Luciferase Based on Flow Injection Analysis. Anal. Chim. Acta, 179 (1986) 307. 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

A good example of the possibility of sensitivity enhancement is a bioluminescent assay by firefly luciferase. The transfer of the process from water into the colloid system Brij 96-water-cyclohexane leads to increased catalytic activity of the enzyme and sensitivity of the corresponding analysis by two decimal orders [69,70] (see Fig. 12). 

Tani H, Morisaki A, Ishida A, Tokeshi M (2012) On-chip bioluminescence assay of ATP and kinases using immobilized firefly luciferase in three dimensional microfluidic chip. In Proceedings of the 16th international conference on miniaturized systems for chemistry and life sciences, Okinawa, 28 Oct-1 Nov... 

If it is desired to use the quenched scintillation cocktail as a reference source for the firefly bioluminescent reaction for ATP assays, it is only necessary to react a known number of firefly luciferln molecules with a large excess of luciferase and adenosine triphosphate, to obtain a new constant for Eq. (25)... 

The most obvious activator assays are those in which cofactors are analyzed all the common cofactors can be determined in this way. and in many cases their own properties provide simple means of measuring reaction rates. The analyses are usually very selective. ATP in red blood cells and many other samples is often determined by using its. specific participation in the well-known firefly bioluminescence reaction ... 

Bioluminescence in vitro chemosensitivity assays are now used to assess the sensitivity of tumor cells (obtained by surgical or needle biopsy) to different dmgs and combinations of dmgs. Cells are grown in microwell plates in the presence of the dmgs at various concentrations. If the tumor cells are sensitive to the dmg then they do not grow, hence total extracted cellular ATP, measured using the bioluminescence firefly luciferase reaction, is low. This method has been used to optimize therapy for different soHd tumors and for leukemias (306). 

Laetmogone, 301, 337 Latnpadena, 163, 339 Lampito, 216, 234, 335 Lampteroflavin, 270 Lampteromyces, 267, 270 Lampyridae, 1, 2 See also Fireflies distribution, 2 morphology, 2 Lampyris, 2, 337 Latia bioluminescence, 189 activators and inhibitors, 189 light emitter, 191 luminescence spectrum, 192 reaction scheme, 190 Latia luciferase, 183-189, 343 assay, 184... 

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

Bioluminescence provides the basis for sensitive enzymic assay methods both for substrate assays and coupled enzyme assays. Firefly luciferase (EC 1.13.12.5) catalyses the production of light (540-600 nm) by the oxidation of luciferin (d-LH2) (Figure 8.18). 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

CIEEL is of particular interest for the development of modern chemiluminescent bioassays. The most popular clinical bioassays utilize thermally persistent spiro-adamantyl-substituted dioxetanes with a protected phenolate moiety. These designed 1,2-dioxetanes include an energy source, a fluorophore, and a trigger grouping, and are therefore structurally similar to bioluminescent substrates such as firefly luciferin. Three main commercial dioxetanes 75 are available as one-reagent assays for alkaline phosphatase and are sold under the name of AMPPD (R1 = R2 = H), CSPD (R1 = Cl, R2 = H), and CDP-Star (R1 = R2 = Cl) <2006S1781, 2003ANA279>. These substrates are sensitive to 10 21 mol of alkaline phosphatase in solution. 

Bioluminescence-based analytical assays were used to measure various analytes in nanoliter sample volumes. Nanoliter volumes of multiple bioluminescent analytical assays were deposited in an array format and lyophilized. ATP-firefly luciferase (FFL) and NADH-bacterial luciferase (BL) platform reactions were compared. We achieved parallel sample delivery via sample-hydrated membranes. A CCD camera measured the luminescent kinetics for each assay. These miniaturized assays and instruments can he prepared as micro-analytical systems to operate in point-of-care (POC) diagnostic devices. 

Fukoda S, Tatsumi H, Maeda M. Bioluminescent enzyme immunoassay with biotinylated firefly luciferase. J Clin Ligand Assay 1998 21 358-362. 

K. WulfT, F. Stahler, and W. Gruber, Standard Assay for Total Creatine Kinase and the MB-isoenzvme in Human Serum with Firefly Luciferase, in Bioluminescence and Chemiluminescence ieds. M. A. DeLuca and W. D. McElroy), Academic Press, New York, 1981, p. 209. 

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