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Luciferase Detection Assay System

a luciferase and assay technology, applied in the field of enzyme activity detection methods and kits, can solve the problems of reducing the time window, reducing the flash height, and reducing the amount of arsenate, so as to improve the kinetics of light production and total light, and the assaying of luciferase is simple and sensitive.

Inactive Publication Date: 2009-02-19
PERKINELMER LIFE & ANALYTICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent is about a new method for detecting the activity of a protein called luciferase, which is involved in bioluminescence. The method involves using phosphate and ammonium ions in a reaction mixture to enhance the light signal generated by the reaction. This results in a strong and stable light signal that can be measured using existing laboratory equipment, such as scintillation counters. The method also allows for the preparation and assay of a large number of samples in microplates, which is useful in high-throughput screening applications. Overall, the method simplifies and sensitizes the assay for luciferase, making it more accessible to laboratories without special equipment or procedures."

Problems solved by technology

These conventional “flash” type assays using firefly luciferase result in a flash of light, which decays rapidly with the addition of substrates to the enzyme.
Especially in automated (e.g. robotic) assay procedures, this is a major problem as it dramatically reduces the time window in which a signal, if present, can be detected.
Arsenate lowers flash height and tends to prolong the light emission period.
The decrease of the intensity of the light signal is considerable undesirable, in particular when microtiter plates or instruments capable of reading out strips are used.
In addition, the use of arsenate is not desirable from an environmental point of view.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Effect of Different Concentrations of Ammonium and Phosphate Ions

[0062]This example demonstrates the effect of different concentrations of Tris and phosphate on the performance of a luciferin-luciferase reaction.

[0063]A stock solution of 4.0 M Tris (source of ammonium ions) was neutralized with phosphoric acid (H3PO4) until a pH of 7.0 was reached. This stock buffer solution contained 2.0 M of phosphate. The stock solution was used to make a range of assay reagents comprising, respectively, 2.2, 1.1, 0.55, 0.275, 0.138, 0.069, 0.034 and 0.017 M Tris. The phosphate concentration in these assay reagents was 1.1, 0.55, 0.275, 0.138, 0.069, 0.034, 0.017 and 0.085 M phosphate, respectively.

[0064]Each reagent was checked for its pH to be 7.0 and was adjusted with small amounts of H3PO4 (several μl of a 2 M H3PO4 solution) if needed. The phosphate anions added in this manner had no significant effect on the above reported phosphate concentration. Each assay reagent was supplemented with D-...

example 2

Effect of Different Sources of Ammonium Ions

[0070]This example demonstrates the advantageous effects on light yield if ethanolamine or ammonium phosphate is used to provide the luciferin-luciferase reaction with ammonium ions.

[0071]Stock solutions of 4.0 M ethanolamine (H2NCH2CH2OH) and 3.0 M ammonium-phosphate ((NH4)2HPO4) were neutralized with phosphoric acid (H3PO4) until pH 7.0 was reached. Dilutions were made in the same manner as described in example 1. Furthermore, all methods and other chemicals used to create the assay mixture and to test this for its light yield in a luciferin-luciferase reaction, were standardized and thus the same as in example 1. Final ethanolamine concentrations in the assay were 1.0, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016 and 0.008 M, and 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 and 0.004 M phosphate, respectively. According to the present invention, a final concentration of 1M ethanolamine is assumed to provide the aqueous, neutral reaction mixtur...

example 3

Effect of Different Types of Reducing Agents

[0076]This example demonstrates the effect of various types of reducing agents used to protect the reactive sulfhydryl group of luciferase from oxidation. This can be important for a sustained activity of the enzyme. (DeLuca et al. Biochemistry 3:935 (1964); and Lee et al., Biochemistry 8:130-136 (1969)). Various protective agents were compared against a control sample not containing a reducing agent in a luciferin-luciferase reaction comprising ammonium and phosphate ions (0.5 M Tris and 0.25 M phosphate).

[0077]Stock solutions containing 100 mM of the following reducing agents were prepared: sodium-dithionite (Na2S2O4), dithiothreitol (DTT), Tris(2-carboxyethyl) phosphine (TCEP), sodium-thiosulfate (Na2S2O3) and sodium-sulfite (Na2SO3). The TCEP stock solution was prepared by dissolving the hydrochloride salt (TCEP-HCl).

[0078]A 3.5 M Tris / 1.8 M phosphate stock solution was diluted to a concentration of 1.2 M Tris / 0.62 M phosphate (or 1.11...

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Abstract

The invention relates to methods and kits for detecting enzyme activity using bioluminescence. In particular, it relates to a novel assay system with increased light yield for a sensitive and convenient detection of luciferase activity, such as luciferase reporter enzyme activity. Provided is a method of detecting luciferase activity in a sample, comprising incubating the sample in the presence of luciferin and ATP to allow the generation of a light signal, wherein said light signal is enhanced by performing the incubation in a reaction mixture comprising phosphate and ammonium ions, and measuring the light signal. The invention also relates to kits for use in such method.

Description

[0001]The present application is a continuation of application Ser. No. 11 / 433,791 filed on May 12, 2006, which claims priority from U.S. Provisional Application bearing Ser. No. 60 / 681,093 filed 13 May 2005, and European Patent Application bearing Serial No. EP 05076165.9 filed 18 May 2005. The foregoing applications are hereby incorporated by reference herein.BACKGROUND OF THE INVENTION[0002]The invention relates to methods and kits for detecting enzyme activity using bioluminescence. In particular, it relates to a novel assay system with increased light production for a sensitive and convenient detection of luciferase activity.[0003]Bioluminescence is a naturally occurring phenomenon that has been utilized for a number of applications, particularly in molecular biology where the enzyme associated with it have been used as genetic reporters. Bioluminescence is nearly ideal for use as a genetic marker. Typically there is no endogenous luminescent activity in mammalian cells, while ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/66
CPCG01N33/581C12Q1/66Y10S435/81
Inventor VAN LUNE, HARRYBRUGGEMAN, JOHAN JOCHEM
Owner PERKINELMER LIFE & ANALYTICAL SCI
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