JP4761150B2 - Stabilized composition and stabilization method of coelenterazine (Renilla luciferin) solution for high-throughput luminescence activity measurement - Google Patents

Description

  The present invention relates to a stabilization method for stably measuring the enzyme activity of various luciferases using coelenterazine (Renilla luciferin) or an analog thereof as a substrate at room temperature for a long time.

  In the field of life science, it is common to measure the transcriptional activity of genes that occur in cells, to evaluate the influence of foreign factors on cells, to propagate intracellular information transmission, or to analyze the expression of individual proteins. Etc. are used. In order to quantitatively measure gene expression over a wide range and analyze temporal kinetic changes, gene transcription activity measurement by a reporter technique using luciferase which is a luminescent enzyme is actively performed. On the other hand, evaluation and quantitative analysis of specific substances in living organisms is also active, especially in immunoassays using antibodies. Quantification of molecules to protein is performed. In this case, a labeling method using radioactivity or chromogenicity is generally used for detecting an antibody, but in recent years, a labeling method using luciferase has also been used. Therefore, the improvement and development of luciferase technology aimed at elucidating life phenomena is becoming an increasingly important theme.

  Currently, the main luciferase technologies are firefly, Cypridina, Renilla, copifera-derived luciferase and luminescent jellyfish-derived aequorin photoprotein. In order to utilize these luciferases, there are firefly luciferin, sea urchin luciferin, and coelenterazine (also known as Renilla luciferin, firefly icapreluciferin, oprophorus luciferin), which are substrates for Renilla, copepod luciferase and aequorin (Table 1). The three luciferins can be extracted directly from the luminescent organism, but they can also be chemically synthesized and are each commercially available.

Coelenterazine, which can be synthesized relatively easily, is already used in many products. For example, Promega's dual assay system inserts two transcriptional active regions into the firefly luminescent enzyme gene, simultaneously inserts the B transcriptional active region into the Renilla luciferase gene, and introduces two gene constructs into the cell. This is a system for measuring two transcriptional activities. In this method, after cells are crushed, firefly luciferin is added, and transcriptional activity of A is measured.
After quenching the light emission of firefly luciferase, coelenterazine is added and B transcriptional activity is measured.

On the other hand, recently attracted attention is a co-podal luciferase (Gaussia Luciferase US6436682) secreted extracellularly from Prolume. A system that measures the gene expression level by adding coelenterazine to the culture medium by utilizing the secretion of this gene introduced into the cell and the synthesized luciferase is suitable for a high-throughput assay.

Since the pKa value of the proton of the imidazopyrazinone ring of coelenterazine is around 7.4, the proton is easily dissociated in a neutral or weakly basic buffer. Therefore, coelenterazine reacts with oxygen and emits faint light while undergoing oxidative decomposition. In the known method, coelenterazine must be stored in a freezer at -20 ° C. or lower with an alcohol solution having a pH of 4-5 under acidic conditions (Non-patent Document 1). On the other hand, the optimum pH of the luciferase group using coelenterazine as a substrate is around 7-8.
There is a large difference between the stable pH values of coelenterazine. Moreover, in order to suppress the influence of the alcohol solution under acidic conditions, a small amount of luciferin (1/1000 v / v) is added to the luciferase solution (Non-patent Document 1).

Since the volume of the wells of the 96-well plate and 384-well plate used in the high-throughput assay is 0.2 mL or less, it is desirable to add 0.2 μL or less of coelenterazine acidic alcohol solution (Non-patent Document 1). Since the dispensing volume of the existing 96-well plate luminometer is 25 to 250 μL, it is impossible to add a small amount of coelenterazine acidic alcohol solution.
Furthermore, in the case of an immunoassay, it is desirable to add the coelenterazine solution directly to the washed well. Thus, in any case, it is necessary to dilute coelenterazine with a pH 7-8 buffer solution.

On the other hand, since coelenterazine is unstable in a weakly basic aqueous solution, when coelenterazine is diluted with a buffer solution of pH 7-8, variation in luciferin activity appears between the first sample and the last sample in the measurement of a large amount of samples. Therefore, accurate measurement cannot be performed (FIG. 1). The stability of coelenterazine in a pH 7-8 buffer solution is an important issue for high-throughput assays. In addition, coelenterazine is a factor that reduces the dynamic range due to self-luminescence, which is the background when measuring, and a reaction solution that reduces self-luminescence is also necessary.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 349-353 (1997)

  The purpose is to stabilize coelenterazine or an analog thereof capable of maximizing the luminescence activity of luciferase using coelenterazine as a substrate and corresponding to high-throughput analysis.

As a result of intensive studies to solve the above-mentioned problems, the present inventor did not reduce the activity of coelenterazine at room temperature in the vicinity of pH 7-8 where the luminescence activity of luciferase using coelenterazine as a substrate is maximized, and Established stabilization technology that can reduce the background.

The present invention relates to the following inventions.
1. A stabilized composition of coelenterazine or an analog thereof comprising coelenterazine or an analog thereof and an antioxidant.
2. A method for preserving coelenterazine or an analog thereof, comprising preserving coelenterazine or an analog thereof in the presence of an antioxidant.
3. A kit for use in a coelenterazine bioluminescent system, comprising coelenterazine or an analog thereof and an antioxidant.
4. A method for measuring coelenterazine bioluminescence, comprising measuring bioluminescence in the presence of an antioxidant in a bioluminescence measurement system of coelenterazine luciferase and coelenterazine or an analog thereof.
5. A method for measuring coelenterazine bioluminescence, characterized in that bioluminescence is measured in the presence of an antioxidant in a bioluminescence measuring system of a bioactive substance modified coelenterazine luciferase or coelenterazine recombinant luciferase and coelenterazine or an analog thereof. .
6. The measuring method according to item 5, wherein the physiologically active substance is at least one substance selected from the group consisting of antigens, antibodies, haptens, hormones, enzyme substrates, sugar chains, and nucleic acids.
7). Item 7. The composition, method, and kit according to any one of Items 1 to 6, wherein the antioxidant is selected from the group consisting of ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, and sulfite.

  In a particularly preferred embodiment of the present invention, a stabilized coelenterazine and coelenterazine analog stabilization method that maximizes the luminescence activity of luciferase using coelenterazine as a substrate and is compatible with high-throughput analysis is provided. By using this stabilization, the luminescence activity of luciferase using coelenterazine as a substrate can be measured with good reproducibility at room temperature. They can be used for the treatment of medical conditions, testing and new drug development.

  The structure of coelenterazine or its analog is shown below.

(Where
X is a 4-hydroxybenzyl group, a 4-SO ₃ H-benzyl group, a benzyl group, an aralkyl group which may be substituted such as a naphthylmethyl group, a cyclohexylmethyl or the like (cycloalkyl having 3 to 8 carbon atoms). Represents a methyl group.

  Y represents an aryl group which may be substituted.

Z is an optionally substituted aralkyl group, a cyclohexylmethyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or -CH2- (optionally substituted heterocycle) Represents.

  Aralkyl groups include benzyl, phenethyl and naphthylmethyl.

Examples of the aryl group include phenyl and naphthyl. Examples of the heterocyclic group include thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and indolyl.

Examples of the substituent for the aralkyl group, aryl group, and heterocyclic group include straight-chain or branched alkyl groups having 1 to 6 carbon atoms such as a methyl group, an ethyl group, a propyl group, and a butyl group (monosubstituted with a hydroxyl group). Or an alkanoyl group having 1 to 6 carbon atoms such as a halogen atom (chlorine atom, fluorine atom, bromine atom or iodine atom), acetyl group or the like. , A linear or branched alkoxy group having 1 to 6 carbon atoms such as a methoxy group, an alkyl group having 1 to 4 carbon atoms such as OH, SH, COOH, SO ₃ H, an amino group, a methylamino group, or a dimethylamino group Examples thereof include an amino group, a nitro group, and a cyano group that are mono-substituted or di-substituted with a group, and have 0 to 3, preferably 0, 1, or 2 of these substituents.

Coelenterazine and its analogs are known or can be easily produced from known methods. (Non-patent document 1, Non-patent document 2)
Non-Patent Document 2, Tetrahedron Lett., 2001, 42, 2997-3000.
In the present invention, coelenterazine or an analog thereof can be stabilized by using coelenterazine or an analog thereof and an antioxidant in combination. In addition, the bioluminescence system containing Renilla luciferase, copepod luciferase, hyodosievir luciferase and coelenterazine or its analogs can be measured in the presence of an antioxidant to enhance the luminescence signal and reduce the luminescence background. And the S / N ratio can be greatly improved.

Examples of the antioxidant include ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, sulfite, butylhydroxyanisole, polyphenols, alkali metal borohydride, and the like, and these can be used alone or in combination of two or more. . Preferably, ascorbic acid, erythorbic acid or a salt thereof is used. When combining two or more antioxidants, it is preferable to combine ascorbic acid with at least one other antioxidant.

  Examples of ascorbate, erythorbate, and sulfite include alkali metal salts such as sodium, potassium, lithium, and cesium, and alkaline earth metal salts such as ammonium salt, calcium, and magnesium.

  When an antioxidant such as ascorbic acid or a salt thereof, erythorbic acid or a salt thereof or sulfite is added to a bioluminescent system using coelenterazine or an analog thereof, it is preferably added at a concentration of about 0.005 to 1M.

  A composition comprising coelenterazine or an analog thereof and an antioxidant is suitable for stabilizing coelenterazine or an analog thereof (solution or solid matter such as powder, granule, crystal, etc.). In the composition, about 40000 to 800,000 parts by weight of an antioxidant is blended per 1 part by weight of coelenterazine or an analog thereof.

Therefore, a composition containing coelenterazine or an analog thereof and an antioxidant (especially ascorbic acid, erythorbic acid or a salt thereof, or sulfite) has not only the storage stability of coelenterazine or an analog thereof at room temperature, This is particularly preferable because the rise of the ground can be suppressed.

The kit for use in the coelenterazine bioluminescent system of the present invention includes coelenterazine or an analog thereof and an antioxidant, and further includes an assay buffer, a lysis buffer, and the like as necessary. Assay buffer includes a buffer solution having a pH of about 6-9, preferably about pH 7-8, and specifically, a buffer solution such as a Tris buffer solution, a phosphate buffer solution, an acetate buffer solution, and a Good buffer solution. . Examples of the lysis buffer include liquids containing UREA, Thiourea, DTT, DMSO, CHAPS, and the like.

  Examples of photoproteins that measure bioluminescence with coelenterazine or an analog thereof of the present invention include Renilla luciferase, copepod luciferase, and hyodosibiviriferase.

The stabilizing composition / measurement method used in the present invention can also be used in a system containing the above-mentioned photoprotein (luciferase) alone, and the photoprotein is modified by binding / complexing with a physiologically active substance. It may be a thing. By using such a complex, the amount of the physiologically active substance can be quantified. The physiologically active substance is not particularly limited, and examples thereof include antigens, antibodies, haptens, hormones, enzyme substrates, sugar chains, nucleic acids (DNA, RNA) and the like.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1 Luminescent activity of coelenterazine in the presence of a stabilizer and four types of solutions (1, 2, 3, 4) below the S / N value were prepared, and the final concentration of coelenterazine (0.001 mM)
And was measured as background activity. Next, Renilla luciferase was added to measure the luminescence activity (RLU: Relative Light Unit), and the measured values are shown in FIG.
Further, FIG. 3 shows the S / N value obtained by dividing the RLU by the first measured background value. As a result, it was found that the background of the sodium ascorbate salt solution 2 or the sodium sulfite solution 3 was low and gave a good S / N.
2, 3 and 4,
1: 0.1 M Tris-HCl pH 7.4 / 0.3 M NaCl
2: 0.1 M Tris-HCl pH 7.4 / 0.3 M Sodium ascorbate
3: 0.1 M Tris-HCl pH 7.4 / 0.2 M Na ₂ SO ₃
4: 0.1 M Tris-HCl pH 7.4 / 0.2 M Thiourea
Using Renilla luciferase, the remaining activity of coelenterazine in the above solutions 1, 2, 3, and 4 was examined, and the half-life of each coelenterazine solution in each solution was examined. As a result, the half-life of the ascorbic acid sodium salt solution was more than 10 times that of the other solutions. From the above results, it was found that the sodium ascorbate solution was the best in the high-throughput luminescence activity measurement using coelenterazine.

Example 2 Autoluminescent activity of coelenterazine in 10% FBS solution The following two types of solutions (1) and (2) were prepared, and coelenterazine was used at a final concentration (10 μM or 100 μM).
And mixed with animal cell culture medium (solution containing 10% FBS) at 1: 1 (volume ratio), and luminescence for 10 seconds was measured as background activity. The results are shown in FIG.
(1), 0.1 M Tris-HCl pH 7.4 / 0.3 M NaCl
( _2), 0.1 M Tris-HCl pH 7.4 / 0.3 M Sodium ascorbate
Example 3: Specific luciferase luminescence by luciferase and auto-luminescence Ratio of copepodal luciferase secreted from animal cells (with 10% FBS)
It was made to react with the coelenterazine solution (final concentration: 10 μM or 100 μM) prepared in Example 3, and the luminescence activity (RLU) was measured, and the ratio of the measured value to the respective autoluminescence was calculated and summarized in FIG. As a result, the ascorbic acid sodium salt solution showed an improvement in the ratio of luminescence to autoluminescence at least twice.

The present invention can be applied as follows.
1) High-throughput dual assay system using Cypridina luciferase / coped luciferase gene: A transcriptional active region is inserted into the Cypridina luciferase gene, and a B transcriptional active region is inserted into the copepod luciferase gene, and two gene constructs are introduced into the cell. After a certain period of time, a part of the culture solution is taken out and dispensed in half into a multi-sample plate, for example, two 96-well plates. Add Cypridina luciferin solution to one plate and measure A transcriptional activity. To the other plate, a coelenterazine stabilization solution is added and B transcriptional activity is measured. A or B transcriptional active region is a control gene sequence (
For example, SV40, CMV promoter) normalizes one transcriptional activity. 9
With a 6-well plate, it takes a long time to measure the luminescence activity of all samples, but a luminescent value with high reproducibility can be measured by using a coelenterazine stabilizing solution.
2) High-throughput immunoassay with copepod luciferase: Since copepod luciferase is stable at room temperature for a long time, for example, a biotin-labeled copepod luciferase / streptavidin complex is produced, and the target substance is detected with an antibody by the sandwich method or competitive inhibition method. An immunoassay is performed using a biotin-labeled copepod luciferase / streptavidin complex as the next antibody. Although it takes a long time to measure the luminescence activity of all samples, a luminescent value with high reproducibility can be measured by using a coelenterazine stabilizing solution.

Image of coelenterazine high-throughput assay Luminescent activity of coelenterazine in the presence of stabilizers. Luminescent S / N ratio of coelenterazine in the presence of stabilizers The half-life of coelenterazine in the presence of stabilizers. Autoluminescent activity of coelenterazine in 10% FBS solution Ratio between luminescence and autoluminescence by copepod luciferase luciferase

Claims (6)

A stabilizing composition of coelenterazine or an analog thereof comprising coelenterazine or an analog thereof and an antioxidant, wherein the antioxidant is selected from the group consisting of ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, or sulfite Composition . Coelenterazine or an analog thereof, characterized by storing coelenterazine or an analog thereof , wherein the antioxidant is selected from the group consisting of ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, or sulfite How to save . A kit for use in coelenterazine bioluminescence system, see containing coelenterazine or an analogue with an antioxidant, selected antioxidant, ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, from the group consisting of sulfites is, kit. Bioluminescence is measured in the presence of an antioxidant in a bioluminescence measuring system of coelenterazine luciferase and coelenterazine or an analog thereof, and the antioxidant is ascorbic acid or a salt thereof, erythorbic acid or a salt thereof, sulfite A method for measuring coelenterazine-based bioluminescence selected from the group consisting of salts . Bioluminescence is measured in the presence of an antioxidant in a bioluminescence measuring system of coelenterazine luciferase or coelenterazine luciferase modified with a physiologically active substance and coelenterazine or an analog thereof, and the antioxidant is ascorbic acid Or a coelenterazine-based bioluminescence measuring method selected from the group consisting of a salt thereof, erythorbic acid or a salt thereof, and a sulfite . 6. The measurement method according to claim 5, wherein the physiologically active substance is at least one substance selected from the group consisting of antigens, antibodies, haptens, hormones, enzyme substrates, sugar chains, and nucleic acids.

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