Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
1998-01-29
2001-01-09
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S184000, C435S189000
Reexamination Certificate
active
06171809
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the use of luminescence in the analysis of biological materials. More particularly, it relates to methods involving reporter gene techniques in which cells are expressed containing a luciferase and then detected by reactions which produce luminescence.
Luciferases are found in a variety of organisms, including fireflies, photobacteria, jellyfish, and sea pansies, among others. Luciferases may be used to measure reporter genes. In this technology, a reporter gene, such as a luciferase encoding polynucleotide, is used as an indicator for the transcription and translation of a gene in a cellular expression system. The reporter gene is operatively linked to a promoter that is recognized by the cellular expression system. In a typical reporter gene assay, a DNA vector containing the reporter gene is transfected into a cell capable of expressing the reporter gene. After sufficient time has passed for the expression of the reporter gene, the cellular membrane is disrupted to release the expressed gene product. The necessary reagents are then added to permit measurement of the enzyme activity of the reporter gene. In the case where a luciferase is used as the reporter gene, the photons of light produced by oxidation of a substrate called a luciferin are measured.
While the most common luciferase used in analysis by luminescence is firefly luciferase, other luciferases may be used. One such luciferase is renilla luciferase, which is derived from sea pansies, a marine coelenterate of the class anthozoans. The present invention is related to a method of determining the presence of renilla luciferase, either alone or in the presence of firefly luciferase. Heretofore, the use of renilla luciferase as a reporter has been limited by the short period of light generation, as also had been the case with firefly luciferase.
In U.S. Pat. No. 5,618,682, assigned to Packard Instrument Company, it was shown that through the use of certain reagent compositions, the brief release of light (“a flash”) from firefly luciferase could be extended for many hours (“a glow”). The advantage of extending the time during which light is released is that it becomes possible to carry out screening of multiple samples simultaneously, which is not feasible if the flash of light lasts only a few seconds or minutes. Such reagent compositions have been very successful commercially under the trademark LucLite™.
The release of light by bioluminescence with firefly luciferase involves the oxidation of a substrate, i.e., a luciferin, in the presence of adenosine triphosphate (ATP) and oxygen to produce adenosine monophosphate (AMP), pyrophosphate, and carbon dioxide.
ATP
+
firefly
luciferin
+
O
2
→
firefly
luciferase
oxy
luciferin
+
CO
2
+
AMP
+
PP
i
+
light
This reaction is illustrated in U.S. Pat. No. 4,286,057 in which a method is disclosed for measuring creatine kinase via the reaction of adenosine diphosphate with creatine phosphate which is catalyzed by creatine kinase and produces ATP. The product ATP is measured by the firefly luminescence reaction and, thus indirectly, the activity of creatine kinase. An increase of the light duration was found to result from adding the reaction product AMP, although the patentees in the '057 patent did not indicate that they obtained a glow time of several hours, as did the patentee in the '682 patent.
The reaction of renilla luciferase differs from that of firefly luciferase in that the substrate is a different molecule, coelenterazine, rather than the firefly luciferin, and only oxygen is involved.
coelenterazine
+
O
2
→
renilla
luciferase
oxy
coelenterazine
+
CO
2
+
light
Carbon dioxide is produced as with firefly luciferase, but neither ATP nor AMP are required. Thus, the reagents used need not include ATP and AMP as used in the firefly luciferase reaction. It has now been discovered, however, that when the reagents used in the firefly luciferase reaction taught in the '682 patent are present when renilla luciferase is being detected, the extended glow time obtained with firefly luciferase is present with renilla luciferase as well. At the same time, the amount of light produced is increased by a factor of ten relative to that of firefly luciferase in a dual assay system.
In one commercially available system from Promega Corporation, a dual reporter technique is used in which an assay is made first with firefly luciferase, after which the first reaction is quenched and a second assay is made using renilla luciferase. The total time required for both assays is said to be about 30 seconds. Thus, the method does not depend on maintaining a long glow period, and the short time is considered an advantage of the system. For multiple screening tests using many samples simultaneously, however, such a short period is not desirable. Instead, much longer glow times are advantageous. The present invention is intended to provide a method for accomplishing that objective.
In WO 96/40988, assigned to Promega Corporation, both single and dual reporter assays are discussed. They are characterized by the use of quenching agents to prevent crosstalk between adjacent sample cells in a multiple cell sample plate or, in dual assays, to stop a first reaction so that a second reaction can be carried out. It is said that by the use of this method, more accurate single assays can be achieved and that dual assays can be carried out in a single sample cell. According to the patentees, it is possible to carry out a dual assay in about 30 seconds. Thus, it appears that the extended glow period desired by the present inventors was not present in either of the two reactions, nor was it considered desirable.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods for assaying biological samples for the presence of firefly luciferase and renilla luciferase together or for renilla luciferase alone. A reagent composition is added which will include compounds selected to produce an extended glow rather than flash luminescence. For detection of firefly luciferase, firefly luciferin, ATP and AMP are required. For detection of renilla luciferase, the corresponding luciferin, coelenterazine, is required. In addition, the composition may include free radical scavengers such as dithiothreitol (DTT), chelating agents such as ethylene diaminetetraacetic acid (EDTA), detergents such as Triton® N-101 (nonylphenoxypolyethoxyethanol), buffers such as HEPES, N-[2-hydroxyethyl] piperazine-N
1
-[2-ethane sulfonic acid], and protease inhibitors such as phenylacetic acid (PAA) and oxalic acid (OA).
In one embodiment, a sample suspected to contain renilla luciferase is mixed with a reagent mixture containing coelenterazine, as a free radical scavenger, DTT, and, as a chelating agent, EDTA, or functional equivalents of DTT and EDTA. Optionally, the mixture may include one or more detergents, buffers, and protease inhibitors. The luminescence is measured by methods familiar to those skilled in the art, such as the TopCount™ Microplate Scintillation and Luminescence Counter available from Packard Instrument Company, Inc., Downers Grove, Ill. In a preferred embodiment, each 100 mL of the reagent mixture contains about 0.2-30 mg of coelenterazine, about 200-2,000 mg of DTT, and about 0.05-100 mg of EDTA. The coelenterazine may be native coelenterazine or an analogue, such as m-, e-, v- or f-coelenterazine. The reagent optionally may contain a protease inhibitor such as phenylacetic acid (PAA) or oxalic acid (OA) and a detergent such as nonylphenoxypolyethoxyethanol.
In another aspect, the invention is a method for detecting the presence of both firefly and renilla luciferases in a single sample. The firefly luciferase is measured by adding to the sample a reagent mixture containing firefly luciferin, adenosine triphosphate (ATP), co-factors necessary
Jenkens & Gilchrist
Marschel Ardin H.
Moran Marjorie A.
Packard Instrument Company
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