Multiple enzyme assays

Details Multiple enzyme assays Multiple enzyme assays

1999-12-14

2003-07-01

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S014000, C435S023000, C435S975000, C435S004000

Reexamination Certificate

active

06586196

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention discloses multiple enzyme assays which measure the activity of at least one endogenous enzyme in a single aliquot of a sample or population of cells and multiple enzymes in a single aliquot of a sample provided that at least one of the enzymes is an endogenous enzyme. The present invention also discloses methods and kits for detecting the activity of multiple enzymes.
2. Background of the Prior Art
A wide variety of reporter gene assays are used in both biomedical and pharmaceutical research for the study of gene regulation and identification of cellular factors and chemical compounds that affect gene expression. Alam and Cook, Anal Biochem, 1990; 188:245-54; Bronstein, et al., Anal Biochem., 1994; 219:169-81. Reporter gene assays are useful in the study of gene regulatory elements because reporter gene activity, i.e., production of the reporter protein, is directly proportional to the transcriptional activity of the regulatory elements of the gene. A reporter gene construct for use in these assays contains one or more gene regulatory elements which are of interest, the minimal sequence requirements for transcription of a functional mRNA, and the coding sequence for a reporter protein. Alam. et al., Anal. Biochem., 188:245-254, 1990. Analysis of constructs containing various deletions within the regulatory region enables mapping of regulatory sequences necessary for transcription and cell specific expression.
Introduction of a reporter gene construct into cells, followed by quantitation of the expressed protein or its activity, provides an indirect measure of gene expression. For example, the sensitive quantitation of reporter gene products is important for analysis of gene expression, signal transduction pathways, identification of protein interactions, and drug discovery. Further, quantitation of the reporter gene enables mapping of the gene promoter and enhancer regions, analysis of gene expression mechanisms, and screening of chemical and natural product libraries for effectors of gene expression.
Chemiluminescent reporter gene assays combine high sensitivity with broad dynamic range, typically 6-7 orders of magnitude. Chemiluminescent 1,2-dioxetane substrates for several reporter enzymes, including &bgr;-galactosidase (&bgr;-Gal), &bgr;-glucuronidase and alkaline phosphatase (AP) are used in highly sensitive assays. These chemiluminescent assays provide superior alternatives to traditional colorimetric, fluorescent, and radioisotopic detection methods. 1,2-dioxetane chemiluminescent substrates have also been used in a dual assay for luciferase and &bgr;-galactosidase reporter enzymes. Enzymatic cleavage of each chemiluminescent substrate produces a destabilized dioxetane anion, which fragments and emits light.
Sensitive chemiluminescent assays, not limited to reporter gene assays, have been described using dioxetane substrates. Bronstein, U.S. Pat. No. 4,978,614, incorporated herein by reference. These dioxetane substrates emit visible light following,enzyme induced degradation. Enhancement of the chemiluminescent degradation of 1,2-dioxetanes by enhancer substances comprising certain water soluble molecules, such as globular proteins or synthetic polymers that have hydrophobic regions, has been described. Vovta et al., U.S. Pat. No. 5,145,772, incorporated herein by reference. These dioxetane substrates are also used in reporter gene assays for alkaline phosphatase, &bgr;-galactosidase, and &bgr;-glucuronidase. See e.g., Bronstein. et al., Anal. Biochem., 219:169-181, 1994, and citations therein. The use of dioxetane substrates and enhancers in reporter gene assays has been described in U.S. application Ser. No. 08/579,787, incorporated herein by reference. U.S. application Ser. No. 08/579,787 describes assays in which the products of multiple reporter genes are sequentially quantitated in the same aliquot of cell extract. Simple, rapid, and highly sensitive combined multiple reporter gene assays which detect commonly used reporter genes are described which do not use radioisotopes or require external light sources. These assays produce enhanced levels of light and therefore increase the dynamic range and sensitivity of the assay and enable the use of a wide variety of instruments.
1,2-dioxetane substrates have been incorporated into the GALACTO-LIGHT™, GALACTO-LIGHT PLUS™ and GALACTO-STAR™ assay systems available from Tropix, Inc., for quantitation of &bgr;-galactosidase reporter enzyme activity and have been used with mammalian cell cultures, tissue extracts, microinjected frog embryos, protozoan parasites, yeast and bacteria. Jain, et al., Anal Biochem., 1991, 199:119-24; Bronstein, et al. Bioluminescence and Chemiluminescence: Fundamental and Applied Aspects, (Campbell, et al., eds) Chichester:Wiley, 1994, 20-3; Martin, et al., Bioluminescence and Chemiluminescence: Molecular Reporting with Photons, (Hastings, et al., eds.), Chichester:Wiley, 1997, 525-8; Bronstein et al., Clin. Chem., 1996, 42:1542-6. The GUS-LIGHT™ system is used for &bgr;-glucuronidase reporter detection. Bronstein. et al., BioTechniques., 1994, 17:172-7. CSPD® substrate is utilized in the PHOSPHA-LIGHT™ assay system for quantitation of either secreted or non-secreted forms of the human placental alkaline phosphatase (PLAP) reporter enzyme. Bronstein, et al., Bioluminescence and Chemiluminescence: Fundamental and Applied Aspects, (Campbell, et al., eds.) Chichester:Wiley, 1994, 20-3; Bronstein, et al., Clin. Chem., 1996, 42:1542-6; Bronstein, et al., BioTechniques, 1994, 17:172-7. The DUAL-LIGHT® system by Tropix, Inc. combines a 1,2-dioxetane with luciferin in a single-tube assay for &bgr;-Gal and luciferase reporter enzymes. Martin, et al., BioTechniques, 1996, 21:520-4; Bronstein. et al., Bioluminescence and Chemiluminescence: Molecular Reporting with Photons, (Hastings, et al., eds.) Chichester:Wiley, 1997, 451-7.
Currently, multiple reporter gene assays are commonly used to provide controls for efficiency of transfection. In such assays, cells are transfected with a mixture of two separate plasmids, each having a different reporter gene. The expression of one reporter gene is controlled by different regulatory regions being studied while the other reporter gene, acting as a control, is generally constitutively expressed by a standard promoter or enhancer. The activity of the experimental reporter enzyme is normalized to the activity of the control reporter enzyme.
The measurement of multiple enzyme activities in a single assay provides several capabilities. Transfection normalization can be performed by quantitation of both experimental and control reporter enzymes. Pharmaceutical screening strategies benefit from multiple reporters to distinguish the effect of a compound on a specific transcription factor from a non-specific effect on gene expression, or for multiplex screening of several drug targets. These advantages are described in co-pending U.S. application Ser. No. 08/579,787, filed Dec. 28, 1995.
While reporter enzyme expression is useful for measuring gene regulation, it is also desirable to have a mechanism to measure cell number, cell adhesion, cytotoxicity, and cell proliferation. Reporter enzymes may have limited usefulness for performing these measurements because the promoter used for controlling such a reporter gene preferably acts independent of the exogenous compounds added to the cells for testing gene expression. One skilled in the art would need a gene construct that is expressed at a constant level by the cell regardless of what is added to the test cells. For example, one would have to use a reporter enzyme linked to a strong promoter that is not affected by the test compounds.
The measurement of both a reporter enzyme and endogenous cellular enzyme activity provides assays for normalization of reporter enzyme activity to cellular proteins, or potentially enabling simultaneous quantitation of the reporter activity and cell number, cell proliferation, cell adhesion, or cytotoxicity. It would be desira

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