Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
1998-09-04
2001-02-06
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
Reexamination Certificate
active
06183978
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to an assay format for the detection of the presence of luciferase, as a reporting molecule or target molecule, in an assay. The assay finds particular value in assays for the detection of luciferase present in a biological sample as the expression product of a luciferase gene transfected into a host cell as a reporter gene. Additionally, compositions useful in the assay method, and kits for performing the assay, are addressed.
2. Background of Prior Art
Chemiluminescent assays using luciferase as an enzyme to indicate the presence of ATP in a biological system or sample are widely described in the literature. Luciferase catalyzes chemiluminescent light release according to the following reaction system:
Although a variety of luciferase enzymes have been identified, and all that catalyze a similar reaction utilizing ATP may be used in conjunction with the invention of this application, the best characterized luciferase is firefly luciferase, the enzyme responsible for the characteristic “flash” of this particular beetle (
P. pyralis
). This luciferase is a 61-62 kD monomeric enzyme which catalyzes the light emission described above with a quantum yield of 0.88. The light naturally emitted at pH 7.8 is a green-yellow light (about 562 nm).
Luciferase assays for the quantitiation of luciferase enzyme are described in U.S. Pat. Nos. 5,652,289 and 5,283,179, Wood. A well known difficulty encountered in developing luciferase assays is the enzyme kinetics which produces a very rapid peak intensity, i.e., the “flash of light”, coupled with a rapid decay to a very low level, is discussed extensively in these patents. The patents also address prior art attempts to solve this problem, including the addition of coenzyme A (CoA) and/or a sulfhydryl compound such as dithiothreitol (DTT) to support greater overall light release.
A wide variety of literature has been developed concerning luciferase assays, and methods to increase both peak light intensity and total light emission. Among the various alternatives explored is the selection of specific solvents, Kricka, et al., Arch. Biochemistry and Biophysics 217, 674-681 (1982), other SH compounds such as N-acetylcystein, Lundin, Bioluminescence and Chemiluminescence, and the addition of sodium arsenate and other salts, DeLuca et al., Analytical Biochemistry 95, 194-198 (1978).
Specific assays employing these formulations to detect target molecules at as low as picomolar concentrations are described in Olsson, et al., Journal of Applied Biochemistry 5, 437-445 (1983), Kimmich, et al., Analytical Biochemistry 69, 187-206 (1975), Brovko, et al., Analytical Biochemistry 220, 410-414 (1994), Momsen, Biochemical and Biophysical Research Communications 84, 816-822 (1978) and Holmsen, et al., Analytical Biochemistry 46, 489-501 (1972). Many of these references describe the role of myokinase, also known as adenylate kinase, for measurement of an adenine nucleotide with a luciferase reaction, (see, in particular, Brovko, Momsen, Olsen and Holmsen, supra). Myokinase is a ubiquitous enzyme found in both prokaryotic and eukaryotic cells, with a molecular weight in its monomeric form of about 21 kDa. An important feature of myokinase is its high substrate specificity to adenine nucleotides, being reactive with ADP, AMP and ATP. Myokinase catalyzes a bidirectional reaction:
Just as ATP and Mg
++
are essential components to drive the luciferase chemiluminescent reaction forward, AMP is a known inhibitor of the luciferase light-yielding reaction.
U.S. Pat. No. 5,618,682, Scheirer, addresses a luciferase assay based on the luciferase-luciferin reaction which is described as providing light emission over a period of up to 8 hours. In this assay, a sample, which is suspected of containing luciferase, is combined with the luciferase substrate luciferin, ATP, AMP, and other co-factors for promoting catalytic activity. Essential to this patent is the combination of both ATP, to support the luciferase-catalyzed reaction, and AMP, to retard the enzyme kinetics, so as to produce a sustained light emission.
As mentioned by Scheirer, luciferase has become important as a reporter molecule outside of the traditional environment of measurement of ATP, as an indicator of ATP synthesis, metabolism, or as an indicator for the presence of bacterial cells. A number of genes for luciferase have been identified and been demonstrated to be effective, when transfected, in expression in heterologous hosts. Thus, the luciferase gene has become an important reporter gene, since detection of the presence of luciferase in a recombinant cell candidate can be used in a wide variety of environments, provided light emission intensity and duration can be supported so as to enable for facile detection, and importantly, automated detection.
Accordingly, it remains an object of those of ordinary skill in the art to develop a luciferase assay which provides, simultaneously, sufficient sensitivity and light intensity, over a sustained period of time, to permit automated chemiluminescent detection of the presence of luciferase in a sample, particularly as a reporter gene in recombinant cell assays.
SUMMARY OF THE INVENTION
The above objects, and other objects developed more completely below, are met by an assay for the presence of luciferase which both increases the signal intensity of luciferase detection over existing methods, and provides for prolonged light emission, such that enhanced automated detection can be achieved. In contrast to prior art formulations employing AMP to inhibit light signal decay, and an initial ATP charge to promote the luciferase reaction, the inventive assay employs, in addition to luciferin, ADP and myokinase. The assay composition also includes magnesium, an essential component for the luciferase and myokinase reactions, and advantageously employs dithiothreitol (DTT) or other sulfhydryl compound or mixture of compounds to enhance the stability of the luciferase that may be present in the sample.
A first buffer (Buffer A) includes as an essential ingredient myokinase. Optional ingredients that may provide a greater signal intensity include magnesium and a sulfhydryl reducing compound such as DTT or &bgr;-mercaptoethanol. This buffer also comprises conventional buffering agents, such as N-[tris(hydroxymethyl)methyl]glycine (tricine) or other zwitteronic buffers, EDTA, other surfactants such as Triton X-100, and may comprise support materials such as glycerol.
In conducting the assay, two reaction buffers are prepared, the second comprising ADP and D-luciferin as essential components. This reaction buffer may be further comprised of conventional stabilizing components that do not affect the luciferase reaction, such as dextran.
Buffer A, comprising myokinase and optionally magnesium as essential ingredients, preferably in the form of MgSO
4
, is added to a reaction vessel comprising the biological sample to be tested, typically cells in culture medium, although cell lysis preparations are also conventionally used. Thereafter, buffer B is added to the reaction vessel, and the vessel is incubated at ambient conditions for a brief period of time, about 10 minutes. Thereafter, the vessel is placed in a luminescence measuring device, such as a luminometer or other light detection device to determine light intensity. In an alternative embodiment, the two reaction buffers may be combined or “premixed” for a short period of time, and then added to the biological sample.
REFERENCES:
patent: 3962037 (1976-06-01), Mitchell
patent: 5283179 (1994-02-01), Wood
patent: 5618682 (1997-04-01), Scheirer
patent: 5648232 (1997-07-01), Squirrell
patent: 5650289 (1997-07-01), Wood
Analytical Biochemistry, 158, pp. 1-5 (1986), Ugarova, et al., “Bioluminescent Assay of Creatine Kinase Activity Using Immobilized Firefly Extract”.
Analytical Biochemistry, 46, pp. 489-501 (1972), Holmsen,e t al., “Determination of ATP and ADP in Blood Platelets: A Modification of the Firefly Luciferase Assay f
Bronstein Irena
Olesen Corinne E. M.
Voyta John C.
Yan Yu-Xin
Kelber Steven B.
Marschel Ardin H.
Moran Marjorie A.
Piper Marbury Rudnick & Wolfe LLP
Tropix, Inc.
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