JPH0847399A - Method for measuring bioluminescence - Google Patents

Abstract

PURPOSE:To enhance luminescence intensity and to measure bioluminescence by the use of an inexpensive substance having excellent stability by treating a luminescent substrate with luciferase in the presence of both a polyphosphoric acid compound (salt) and a sulfhydryl compound and measuring luminescence. CONSTITUTION:In a method for treating a luciferase substrate with luciferase and measuring emitted bioluminescence, a luciferase substrate such as luciferin is mixed with adenosine triphosphate (ATP) and luciferase in the presence of both a polyphosphoric acid, e.g. an inorganic polyphosphoric acid such as tripolyphosphoric acid, metaphosphoric acid or pyrophosphoric acid or an organic polyphosphoric acid compound such as cytidine 5'-triphosphate (CTP) or cytidine 5'-diphosphate (CDP) or its salt and a sulfhydryl compound such as dithiothreitol and beta-mercaptoethanol or glutathione. Emitted bioluminescence is enhanced and emission time is sustained. Enzyme immunoassay or DNA probe assay is used to measure bioluminescence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bioluminescence reaction utilizing luciferase. Luciferase
(LUCIFERASE) is a generic term for oxygenases that catalyze bioluminescence, and is also called luminescent enzyme. This enzyme catalyzes the oxidation of luciferin by oxygen molecules, and luciferin emits light as the chemical change occurs. This reaction mechanism is known to differ depending on the species. Of these, the bioluminescence reaction by luciferase isolated from firefly is well studied. Firefly luciferase (EC.1.13.12.7)
Acts on adenosine triphosphate (hereinafter abbreviated as ATP) and a luciferase substrate typified by firefly luciferin to generate adenosine monophosphate (hereinafter abbreviated as AMP) and oxyluciferin, and at the same time emits light (LE in the formula).
(Indicated by 1) is generated (chemical formula 1).

[0002]

Embedded image

The bioluminescence reaction by this firefly luciferase requires magnesium ion. Looking at the reaction in more detail, first, AMP-luciferin (luciferyl adenylate) and pyrophosphate are produced from ATP and luciferin, and then luminescence occurs due to the oxidation reaction of AMP-luciferin. This reaction is said to be inhibited by pyrophosphate, amines, etc. (Kyoritsu Publishing's "Chemical Dictionary"). The amino acid structure of luciferase is very similar among fireflies of different species, and since the same luciferin also serves as a substrate as described below, its reaction mechanism is considered to be almost the same (G.Wien
hausen and M. DeLuca, Photochemistry and Photobiolog
y, 42,609-611; 1985). Luciferin, which is a substrate for luciferase, is a generic term for substances that emit light when oxidized by the catalytic activity of luciferase, and their structures differ greatly depending on their origin (MERCK & Co. Inc., THE MERCK
INDEX, 10th). Thus, for example, the same type of luciferin can be used as a substrate even in fireflies of different species, but cannot be a substrate when the organisms are different, and the luciferin of a luminescent organism can function as a substrate only for luciferases of closely related species. . Such bioluminescence of the luciferin-luciferase system includes firefly (Photinus pyralis) and click beetle (Click beetle; Pyrophorus plagiophthalam).
us), and other than these, luminescent bacteria (Photobacterium ph
otoreum, Vibrio harveyi, etc., Cypridina (Vargula h)
ilgendorfii), and Renilla reniformi
s) is known. Luciferase (EC.
1.14.14.3) differs from firefly luciferase in that it utilizes the chemical energy of reduced flavin mononucleotide (FMNH2). On the other hand, Cypridina and Renilla luciferase are simple reactions different from firefly luciferase and use only luciferin or coelenterazine and oxygen as substrates.

The bioluminescence reaction by luciferase is very useful as an analytical technique because it is advantageous in terms of sensitivity as compared with chemiluminescence using peroxidase (hereinafter abbreviated as POD). However, luciferase extracted from biomaterials such as fireflies is expensive and has a problem in stability, which has hindered its spread. In recent years, a luciferase having a highly stable amino acid sequence that has been mutated (Japanese Patent Laid-Open No. 5-244942) has been reported, and it has become possible to stably supply a large amount of luciferase by gene recombination technology.

It is expected that such an easy-to-handle luciferase will enable the application of the bioluminescence reaction to various fields which have been difficult to put into practical use in the past. The most typical application field of luciferase is as a reagent for analysis. As a specific example of an analytical reagent,
First, there are various binding analysis systems using luciferase as a labeling substance. Binding assay systems include immunoassays that use the antigen-antibody reaction, nucleic acid hybridization assays that use the affinity of complementary nucleic acids, and avidin-biotin, hormone-hormone receptors, and sugar-lectins. Analytical systems and the like that utilize the binding of substances with specific affinity are known. The use of luciferase as a labeling substance in these binding analysis systems makes it possible to trace the amount of the substance finally bound (or not bound). The technique of using luciferase as a labeling substance can easily achieve higher sensitivity than POD labeling. Specifically, it has been reported that firefly luciferase is directly labeled on a low molecule such as hapten or biotin and applied to an enzyme immunoassay. (Wannlund J. et al. Biochem. Biophys.Res.Commun.
(1980) 96,440-446, Eur. Patent Appl. 141581, 1985). Although it has not been reported that a high molecular weight antigen, antibody or DNA is directly labeled, it is considered that this is because the activity of the firefly luciferase is easily lost in the labeling method currently used. In order to solve this point, an analysis method using a stable ATP-producing enzyme as a labeling enzyme and firefly luciferase as a luminescent reagent has been developed, and thyroid stimulating hormone, human chorionic gonadotropin, and 17-α hydroxyprogesterone Applied to bioluminescent enzyme immunoassay
(Clinical examination, 38,190-195, 1993). In addition, an assay method using Renilla luciferase as a labeling enzyme has been reported. Recombinant and biotinylated luciferase are already commercially available and used for enzyme immunoassay and DNA probe assay (Stults N et al. In: Biolumine.
escence and Chemiluminescence: Current Status, West
Sussex, pp533-536, 1991). In addition to this, the bioluminescence reaction by luciferase may be used for detecting luciferase or ATP itself. For example, it is necessary to trace luciferase activity in screening foreign genes using the luciferase gene as an index and in analyzing gene mutations.

[0006] Detection of ATP using firefly luciferase is carried out by measuring the number of cells or by using ATP as an enzymatic reaction product.
It is used for the tracking of enzyme reaction using as an index, and for the binding analysis system using ATP itself as a label. That is, water,
It has been reported to be applied to an indirect method for measuring the number of microorganisms contained in foods, cosmetics, etc., a method for measuring the activity of cell tissues such as blood cells and spermatozoa, and a degree of influence of stress such as drugs. Further, the bioluminescence reaction by luciferase can be applied to other than the analytical reagent. For example, it can be applied to a luminous body that does not require electricity or fuel. Although a portable luminescent material utilizing a chemiluminescent reaction has already been commercialized, it would be useful if the bioluminescent reaction was similarly put to practical use as a portable luminescent material.

[0007]

[Problems of the prior art] The stability of the enzyme has been a major obstacle to the practical application of the bioluminescence reaction by luciferase. However, the stability of the enzyme is being solved by gene recombination technology as described above.

The second problem is that bioluminescence due to luciferase disappears within a short period of time.
The bioluminescence reaction by luciferase is very strong in vitro, but disappears within a few seconds. Therefore, when using bioluminescence reaction for analysis, a luminometer equipped with strict control of reaction time to maintain sensitivity and accuracy and auto-injection function to capture instantaneous luminescence was required. . As a technique for stabilizing luminescence over a long period of time, a method of adding coenzyme A (hereinafter abbreviated as CoA) to the reaction system has been proposed (Table 6-500921). CoA emission time by the addition of stably come to continue for more than a few minutes, yet the emission intensity itself is also enhanced 10 times or more, simply measuring luciferase traces of resulting 10 ^{-2 0} M or less I can do it now. However, since CoA has an SH group in its structure, it is easily oxidized and has a problem in stability. It is also pointed out that commercially available CoA is expensive because it is extracted from yeast and the like, and that ATP and the like may be mixed. The mixing of ATP is
When ATP is used as a measurement target, it leads to an increase in background, which is not preferable. Against this background, Co
The provision of a substance having a luminescence-enhancing effect in place of A is awaited.

In addition to sulfhydryl compounds such as CoA, a method utilizing pyrophosphate has been reported (Arch.
Biochem. Biophys. 46,399-416; 1955). As described above, pyrophosphate is known to inhibit the bioluminescence reaction by firefly luciferase. However, in this report, under the special condition that pyrophosphoric acid is supplied to the reaction solution at the time when the bioluminescence reaction proceeds to some extent and the luminescence intensity decreases, a phenomenon in which a strong luminescence is temporarily generated occurs. Has been observed. If these phosphoric acid compounds are added before the addition of ATP, the initial luminescence amount will decrease even if the reaction is started by adding ATP. Therefore, an increase in luminescence due to pyrophosphoric acid, etc. can be expected only during the reaction, and the initial luminescence can be expected. It was thought that it could not be used for amplification of luminescence including. In any case, it was not sufficient as a technique for maintaining the bioluminescence reaction at a high level immediately after the initiation of the reaction.

Further, in Japanese Patent Laid-Open No. 55-13893, D- which acts as a competitive inhibitor together with pyrophosphate
It has been shown that a luciferin derivative (L-luciferin etc.) is added to stabilize the luminescence reaction. 10 ^-4 M or less in this report, in particular 10 ^- by ⁶ M of the combined effect of pyrophosphate and competitive inhibitor, has achieved a stable emission level in exchange for reduction of the emission intensity itself. As a mechanism of action, it is said that stable luminescence can be obtained as a result of suppressing abrupt progress of luminescence reaction by competitive inhibition reaction. However, although this reaction can be expected to stabilize the luminescence level, it does not enhance the luminescence intensity itself, so it is difficult to realize a highly sensitive measurement system using luciferase.

[0011]

DISCLOSURE OF THE INVENTION The present invention provides a technique for extending the luminescence time of bioluminescence by luciferase while maintaining a sufficient luminescence intensity without using an expensive substance having stability problems such as CoA. Offering is an issue. Another object of the present invention is to provide a bioluminescence enhancing substance having a performance exceeding CoA.

[0012]

The object of the present invention is solved by the following means. That is, firstly, the present invention is a method for measuring bioluminescence generated by allowing luciferase to act on a luciferase substrate, which comprises performing a bioluminescence reaction in the presence of a polyphosphate compound or a salt thereof and a sulfhydryl compound. I will provide a.

In the present invention, the bioluminescence reaction by luciferase is a polyphosphoric acid compound or a salt thereof (hereinafter, simply referred to as a polyphosphoric acid compound including a salt: "feature 1") and a sulfhydryl compound (hereinafter abbreviated as SH compound: The feature is that it is performed in the presence of "feature 2".

Characteristic 1: Polyphosphoric acid compound The polyphosphoric acid compound is present in the reaction at least about 10 times.
^-6 M or more, preferably 10 ^-6 -10 ^-2 M is added. The effect of sustaining the luminescence time and the effect of enhancing luminescence by the polyphosphoric acid compound become large depending on the concentration, and therefore a sufficient effect may not be obtained at a too low concentration. On the contrary, if the concentration is too high, it may cause an inhibitory action depending on the luciferase used and the reaction conditions. A particularly practical concentration range is 10 -5 -10 ^-3 M
Can be mentioned. This concentration range is a sufficient range in which the effect of enhancing the luminescence reaction is not affected by fluctuations in other reaction conditions, and is also a safe range in which reaction inhibition is unlikely to occur due to unnecessary addition. Polyphosphoric acid compounds useful in the present invention include inorganic pyrophosphoric acid compounds such as tripolyphosphoric acid, metaphosphoric acid, hexametaphosphoric acid, and pyrophosphoric acid. These compounds may be used in the form of salts. Known salt types include sodium, potassium and iron. Among them, the effects of tripolyphosphoric acid and pyrophosphoric acid are excellent and can be shown as preferable polyphosphoric acid compounds. On the other hand, when attention is paid to the type of salt, for example, in pyrophosphoric acid, potassium salt is particularly excellent in the effect of enhancing the emission intensity and the effect of extending the emission time. The polyphosphoric acid compound of the present invention may be present in the reaction solution when measuring luminescence. Therefore, it does not always have to exist from the start of the reaction, and it can be added when the luminescence reaction is progressing due to other components to start luminescence measurement. But,
It is advantageous to avoid coexistence in advance, so that it is advantageous to add components other than the components at the same time as much as possible, because the reagent composition is simplified and the measurement operation and reaction time management are easy. For example, in the case of pyrophosphate, coexistence with luciferase may lead to a decrease in enzyme activity in advance, so it is preferable to avoid contact between the two before the reaction. There is no.

Characteristic 2: SH compound The SH compound includes dithiothreitol (hereinafter abbreviated as DTT), dithioerythritol, β-mercaptoethanol, 2-mercaptopropanol, 3-mercaptopropanol, 2,3-dithiopropanol, Glutathione, CoA, etc. can be used. In the present invention,
These compounds are added to the reaction solution at 0.1-200 mM, preferably 8-100 mM. It is needless to say that this concentration range is a more practical concentration range in which the effects of the present invention can be surely obtained in the same manner as the concentration range exemplified for the polyphosphoric acid compound, and the possibility of adverse effects is low. . The SH compound of the present invention only needs to be present in the reaction solution when measuring luminescence. Therefore, it does not always have to exist from the start of the reaction, and it can be added when the luminescence reaction is progressing due to other components to start luminescence measurement.
However, in order to fully utilize the effect of maintaining the emission intensity of the SH compound, it is effective to act when the emission intensity is as high as possible. Therefore, preferably, for example, it is made to coexist with luciferase and substrates in advance so that the luminescence reaction can be started in the presence of the SH compound. In addition, it is easier to add the necessary components at the same time, which simplifies the reagent composition.
This is advantageous because it is easy to perform measurement operations and control of reaction time.
In particular, SH compounds such as DTT can be expected to have a protective action against the enzyme, and therefore coexistence with luciferase seems to bring about the improvement of storage stability. Of course these S
There is no problem in allowing the H compound to coexist with the substrates.

The bioluminescence reaction in the present invention means a luminescence reaction catalyzed by various luciferases.
Firefly luciferase is a typical luciferase of the present invention. In addition to these, luciferases derived from luminescent bacteria, Cypridina and Renilla are known. Regarding the utilization of the bioluminescence reaction by luciferase, there is utilization as a reagent for analysis as described above, but there are also the following utilizations.

That is, it is used as a reporter gene. The gene for firefly luciferase has already been isolated and its nucleotide sequence has been determined (T.Masu
Da et al. Gene 77,265-270; 1989), this luciferase gene can be used as a reporter gene to clarify the mechanism of action of various genes and intracellular metabolic mechanism (Alam, J., and Cook). , JLAnal.Biochem.188,245
-254; 1990). Luciferase has the following characteristics and is said to be extremely excellent as a reporter gene. (1) The sensitivity of activity measurement is extremely high, which is quick, convenient, and inexpensive (de Wet, JR et al. Mol. Cell. Biology,
7,725-737; 1987), (2) Highest quantum efficiency among various luminescence reactions (Seliger, HH and McElroy, WDA
rcheves of Biochem. and Biophys. 88,136-141; 196
0), (3) Luciferase functions as a reporter gene immediately after translation of mRNA since it is a single polypeptide having a molecular weight of 62,000 and does not require special post-translational modification for enzyme expression (de Wet, JR et al. Proc.
Natl.Acad.Sci.USA 82,7870-7873; 1985) Further analysis of gene expression such as transcriptional activity analysis of promoters and enhancers (Williams, TM et al. Anal.Bioch
em.176, 28-32; 1989), elucidation of structure and mechanism of action of mRNA in cells (J. Callis et al. Genes and Development,
1,1183-1200; 1987), structure and mechanism of action of proteins with gene regulatory functions (MLWaterman et al. Mol. Endorc., 2,14-
21; 1988), analysis of organ-specific expression patterns in transgenic plants / animals (Ow, D, et al. Science 234,
856-859; 1986), markers for viruses and cells (Rodrigue
tz, D., Proc.Natl.Acad.Sci, USA 86,1287-1291; 1989)
It can be used for a wide range of applications.

Among firefly luciferases, those from American firefly (Photinus pyralis) are the most studied. Genji firefly, Heike firefly, click beetle and the like have different amino acid primary structures from American firefly, but they are the same in that they emit light using ATP and firefly luciferin as substrates, and their reaction mechanisms are similar. Therefore, the method of the present invention can be applied to the bioluminescence reaction catalyzed by luciferase derived from different species of firefly. The structures of many of these luciferases have already been determined, and those obtained by gene recombination are known. Also known are mutants in which a part of amino acids are substituted to modify stability and emission wavelength. In the present invention, as long as a luciferase substrate similar to a natural enzyme can be used as a substrate, these recombinants can be used similarly to the natural one. In particular, luciferase whose stability is improved by substituting amino acids contributes not only to improving the storage stability of the reagent components but also to improving the measurement sensitivity, and thus can be exemplified as a preferred luciferase. In this type of luciferase, a luciferase of Genji firefly or Heike firefly is thermostable (Japanese Patent Laid-Open No. 24494/1993).
2) is known.

On the other hand, the luciferase substrate of the present invention is not particularly limited in structure and origin as long as it serves as a substrate for the luciferase constituting the reaction system. Luciferin is a generic term for substances that emit light as substrates of reactions in bioluminescence, and is also called luminescent element. It is excited by free energy when it is oxidized by molecular oxygen in the presence of luciferase, emits visible light, and returns to the ground state. At this time, one photon is emitted by the oxidation of one molecule of luciferin. For example, firefly luciferin is chemically 4,5-Dihydro-2- [6-hydroxy-2-benzothiz
It is known to be olyl] -4-thiazocarboxylic acid. In addition to this, 4-methyl-D-luciferin (Mi
ller, JL et al. (1990) J.Clin.Chem.Clin.Biochem., 2
8,471-474), D-luciferin derivatives such as D-lucyphenyl-L-methionine (Table 63-501571) and aminoluciferin derivatives (Table 01-50).
As described above, luciferin and luciferase, which can also be used for H.2431, etc., may change the emission wavelength depending on the mutation of the structure (or amino acid sequence). Taking advantage of this feature, it is preferable to select a combination of the two so that the emission wavelength can be easily obtained. When luciferase is used as a labeling enzyme in the binding analysis method, multi-item simultaneous measurement can be performed by combining luciferases having different emission wavelengths depending on the items.

When the present invention is used for measuring luciferase activity, the components other than the polyphosphate compound and the SH compound, that is, the luciferase substrate, is used according to a known application form. These substrates are provided in sufficient concentration for luciferase. Specifically, the concentration is about 0.1-1 mM in the reaction solution containing luciferase. Since the concentration of the substrate depends on the origin and specific activity of luciferase, or the enzyme activity value expected to exist in advance, it is advisable to select the concentration that gives the optimum conditions experimentally. In addition to luciferase substrates, some luciferases require coenzymes or metal ions depending on the luciferases used. Therefore, it is necessary to combine the components required for activity expression in accordance with the enzyme. For example, luciferase extracted from fireflies requires ATP and magnesium ions for the luminescence reaction. ATP is about 0.1-1.0 mM in the reaction solution,
Further, magnesium ion may be added so as to have a concentration of 2-15 mM. The bioluminescence measuring method of the present invention is carried out at a temperature and pH suitable for the luciferase used. For example, when using American firefly luciferase, the temperature can be 20 to 30 ° C. and the pH can be 6 to 8. It goes without saying that such basic reaction conditions can be changed as appropriate by changing the enzyme or using a recombinant having a modified amino acid sequence. Furthermore, as an enzyme stabilizer, bovine serum albumin is added to 0.0
It is recommended to add 1-5 mg / ml. The same conditions can be used in the case of Genji firefly and Heike firefly, and the reaction at a higher temperature is also possible in the case of using luciferase having thermostability.

The bioluminescence in the present invention can be detected by a known method. Specifically, a method of quantitatively counting the emission intensity with a commercially available luminometer, or a method of visually detecting with a light-sensitive material is generally known. In the present invention, since the luminescence is maintained at a high level and stably, it is not always necessary to perform the strict time management required for the bioluminescence measurement up to now. As confirmed in the examples, in the luminescence reaction of the present invention, strong and stable luminescence is produced when the conditions of the polyphosphoric acid compound and the SH compound are aligned, and therefore the emission intensity can be measured after these conditions are aligned. It can be done in the time zone. Therefore, the luminescence intensity may be traced at a constant time period while the luminescence reaction continues. At this time, it is advantageous to integrate the emission intensities over as long a time as possible because improvement in sensitivity can be expected.

The following examples can be given as specific timings of the light emission measurement. One of the most basic timings is to simultaneously add other components necessary for the luminescence reaction to luciferase and trace the luminescence immediately after that. In this example, since the reagent components can be collected together, simple measurement is possible, and since the measurement is performed immediately after the start of light emission, a long counting time can be taken, which is also advantageous in terms of sensitivity. Of course, it is not exact even after the start of the luminescence reaction, and in the present invention, some time lag does not have any influence on the actual measurement result. To start the luminescent reaction in the presence of polyphosphoric acid and an SH compound,
It suffices to add polyphosphoric acid together with at least one of the components essential for the reaction. Specifically, for example, in the case of firefly luciferase which requires ATP, the luminescence reaction can be started by adding ATP + polyphosphate to luciferase + luciferin. In this case, the SH compound may be added to any component. In addition to this, a method in which a polyphosphate compound and an SH compound are added later to a conventional luminescence reaction system configured by adding basic substrates to luciferase and the luminescence that is enhanced at this time is traced is adopted. You can also
In this method, the number of reagents constituting the reaction is increased by one, but it is a simple method in that commercially available bioluminescent reaction reagents can be used as they are.

The method for measuring bioluminescence according to the present invention can be used for measuring luciferase activity. Luciferase has the significance of activity measurement as a gene marker and a labeling enzyme. Particularly, when measuring luciferase activity as a labeling enzyme, higher sensitivity and accuracy are required. The substance to be labeled is an immunologically active substance,
Examples include nucleic acids and specific binding ligands. More specifically, antibodies, antigens, and haptens as immunologically active substances, DNA and RNA as nucleic acids, biotin for avidin as a specific binding ligand, hormone receptors for hormones, lectins and boric acid compounds for sugars. Such combinations are known. There are various application forms of the assay system using luciferase as a labeling enzyme. For example, the following example can be shown as a reaction principle adopted in an immunoassay.

Reaction Principle 1: Competitive Method The luciferase-labeled antigen and the analyte antigen in the sample are reacted competitively with the antibody. In this case, the antibody may be immobilized, or a second antibody may be used for B / F.
It may be separated. The amount of luciferase-labeled antigen that binds to the antibody shows an inverse relationship with the amount of the analyte antigen. A principle called a binding inhibition reaction method, which is an application of the principle of the competitive method, is also known. According to this principle, the antigen to be measured is first bound to the antibody, and then the labeled antigen is reacted. As in the competitive method, the amount of labeled antigen bound is inversely proportional to the amount of antigen to be measured. In any of the principles, antibody analysis can be performed by exchanging the relationship between antigen and antibody.

Reaction Principle 2: Sandwich Method The luciferase-labeled antibody is reacted with the antigen to be measured which has reacted with the solid phase antibody. The condition is that the antigen to be measured has at least two binding sites. Finally [solid phase antibody] + [measured antigen] + [luciferase labeled antibody]
The sandwich-like complex is formed, and the amount of bound luciferase-labeled antibody is proportional to the amount of the antigen to be measured. In the sandwich method, a one-step method in which a solid phase antibody and a luciferase-labeled antibody are simultaneously reacted with an antigen, a two-step method in which a solid phase antibody or a luciferase-labeled antibody is reacted with the other, and then a solid phase antibody is used. A method is known in which a luciferase-labeled antibody is brought into contact after reacting with an antigen to be measured and washing. The sandwich method includes not only the measurement of an antigen but also a method of reacting an antibody to be measured with a solid-phased antigen and detecting the antibody with an antibody that recognizes a luciferase-labeled antibody. In any case regardless of the reaction principle, the analysis is completed by separating the luciferase-labeled component bound (or not bound) by some means and measuring the luciferase activity. In measuring the luciferase activity, the method for measuring bioluminescence according to the present invention can be applied.

In the immunological measurement based on these reaction principles, the substance which can be labeled with luciferase contains an antigen, an antibody, or an active fragment thereof. That is, it may be a domain peptide containing an antigenic determinant of an antigen, or an antigen-binding site of an antibody such as F (ab ') 2, Fab', Fab or Facb. A monoclonal antibody can also be used as the antibody. Many methods are known for binding an enzyme protein such as luciferase to an immunologically active substance. (Clinical pathology, Extra edition, Special issue No. 53, P39-42, 1983, etc.) For convenience of explanation, the antigen-antibody reaction was taken as an example, but it goes without saying that the present invention can be applied to other various binding assay systems. That is, if the antigen-antibody reaction is replaced with a hybridization assay utilizing the affinity of a nucleic acid having a complementary sequence, it can be applied to the analysis of nucleic acid. Enzyme labeling methods for nucleic acid molecules are also known. Luciferase can be directly introduced into the nucleic acid, or can be indirectly bound by avidin-biotin reaction or an antigen-antibody reaction.

Secondly, the present invention provides a reagent capable of carrying out the method for measuring bioluminescence according to the present invention described above. Examples of such a reagent include a bioluminescence measuring reagent containing luciferase, a luciferase substrate, a polyphosphate compound, and an SH compound. In addition to this, as the reagent according to the present invention, a reagent for measuring luciferase activity by a bioluminescence reaction containing a luciferase substrate, a polyphosphate compound, and an SH compound can be shown. As the reagent based on the present invention, a reagent for measuring ATP, which utilizes the fact that firefly luciferase requires ATP for the luminescence reaction, can be shown. The reagent for measuring ATP according to the present invention comprises firefly luciferase, a luciferase substrate, a polyphosphate compound, and an SH compound. The amount of each component used in these reagents is set so that a necessary and sufficient concentration can be obtained in the final reaction solution. The reagent for measuring bioluminescence, the luciferase activity or the reagent for measuring ATP according to the present invention may be supplied in a solution state in which necessary components are dissolved, or may be lyophilized and supplied by a known method. In any case, it goes without saying that the luciferase and the components other than the luciferase are kept out of contact with each other so that the luminescence reaction does not proceed during storage. Of the polyphosphate compounds, pyrophosphate is preferably not allowed to coexist with firefly luciferase in advance, but other polyphosphate compounds may be able to coexist with luciferase.
In addition, SH compounds can coexist with luciferase, and regarding substrates, polyphosphate compounds and S
No problem occurs even if any component of the H reagent is added. If all reagents are distributed in a dry state, there is no possibility that the luminescence reaction will proceed during storage even if luciferase and the substrates coexist. Therefore, for example, a simple commercial form in which a reaction container is filled with all necessary reagents and a luminescence reaction is started only by adding a sample is considered.

These reagents may be preliminarily added with additional components which give the above-mentioned preferable reaction conditions. Specific examples include substrates such as ATP necessary for luciferase activity expression, cofactors such as magnesium ions, buffers that provide a pH suitable for the reaction, salts that provide the required salt concentration, and the like. . Further, a known stabilizer may be added to enhance the storage stability of luciferase and substrates. As an example of the buffer, tricine, glycylglycine, tris-acetic acid, tris, etc. can be used under a suitable pH. As stabilizers, sugars such as sucrose and galactose, polyhydric alcohols such as ethylene glycol and glycerol, inactive proteins such as bovine serum albumin, and chelating agents such as EDTA are known. Sugars and inactive proteins also function as excipients during freeze-drying. The reagent for measuring luciferase activity according to the present invention can be used in various binding analysis methods using luciferase described above as a labeling enzyme. For application to this type of binding analysis method, it is convenient to combine necessary reagents with a standard sample into a kit.

Thirdly, the present invention provides a method for enhancing bioluminescence, which comprises performing a bioluminescence reaction in the presence of a polyphosphate compound and an SH compound in a method of measuring the bioluminescence generated by allowing luciferase to act on a luciferase substrate. provide. The method for enhancing bioluminescence according to the present invention means a method for enhancing the luminescence intensity obtained by a known method by at least 2 times or more by adding a polyphosphoric acid compound and an SH compound. Not only bioluminescence but luminescence intensity is generally displayed as a result of counting the number of luminescence per unit time (unit: CPM; Count).
Per Minute), it is possible to increase this value 10 times or more in the bioluminescence enhancing method of the present invention.

Fourthly, the present invention provides a luciferase and a luciferase substrate-enhancing agent for bioluminescence, which comprises a polyphosphate compound and an SH compound.

Fifth, the present invention provides a method for measuring the bioluminescence generated by allowing luciferase to act on a luciferase substrate, wherein the bioluminescence reaction is carried out in the presence of a polyphosphate compound and an SH compound to extend the luminescence time of bioluminescence. Provide a way. The method of extending the luminescence time of bioluminescence according to the present invention means a method of extending the luminescence time obtained by a known method by at least double (60 seconds or more) by adding a polyphosphoric acid compound and an SH compound. The light emitted by bioluminescence generally decays rapidly within a few seconds. The method of extending the light emission time of the present invention suppresses the decay of the light emission and maintains the strong light emission intensity for a long time.

Sixth, the present invention provides a luciferase, and a luminescence time extender for bioluminescence by a luciferase substrate, which comprises a polyphosphate compound and an SH compound. INDUSTRIAL APPLICABILITY The bioluminescence enhancing method, the enhancing agent, the method for prolonging the luminescence time, and the agent for prolonging the luminescence time of the present invention can be applied to a wide range of reactions as long as it is a bioluminescence reaction that proceeds by luciferase and a luciferase substrate. Examples of such bioluminescence reaction include various binding analysis reactions using the labeling enzymes described above,
Examples thereof include luciferase and ATP themselves, which are detection methods, and portable light emitters, which are applications other than analysis methods.

Seventh, the present invention relates to a method for allowing a luciferase substrate to act on a luciferase substrate in the presence of a polyphosphate compound and an SH compound to carry out a bioluminescence reaction, and a polyphosphate compound, an SH compound, a luciferase substrate, and a luciferase. A bioluminescent reaction composition comprising: A method of performing a bioluminescent reaction of the present invention,
Alternatively, the bioluminescent reaction composition is useful for use as the previously mentioned portable light emitter. In order to start the luminescence reaction at an arbitrary timing, it is preferable that the luciferase and the substrates are filled in a pre-partitioned container. If the compartment of this container is made to be rupturable by pressure from the outside, a simple luminescent material can be obtained in which the luminescent reaction starts only by applying pressure from the outside. Or with particles or membranes with luciferase immobilized,
If the solution containing the substrates is used, the luciferase can be repeatedly used in the luminescence reaction only by exchanging the solution containing the substrates, which is economical. Since the bioluminescence reaction composition according to the present invention can obtain luminescence of various wavelengths by combining luciferase and luciferin, it is possible to select the luminescence color by utilizing this feature.

[0034]

The polyphosphoric acid compound of the present invention, in combination with the SH compound, can be used in the bioluminescent reaction shown in Formula 1
It has the effect of increasing the emission intensity and the effect of extending the emission time. Among the polyphosphoric acid compounds, pyrophosphoric acid is A
It is one of the reaction products of the luminescence reaction using TP and firefly luciferin as substrates. Therefore, strictly speaking, it can be said that the conventional bioluminescence reaction also proceeded in the presence of pyrophosphate. However, in the bioluminescence reactions reported so far, only a very short luminescence time and not enough luminescence intensity were obtained. It is considered that the reason is that the amount of pyrophosphoric acid existing in the sample from the beginning or the amount of pyrophosphoric acid accumulated in the reaction solution as a luminescent reaction product was extremely small. The pyrophosphoric acid existing in such a state does not constitute the present invention. Moreover, it does not bring about the excellent effects of the present invention. The polyphosphoric acid compound in the present invention means a compound which is artificially added to the reaction system in an amount exceeding the concentration normally expected to exist.

Although the mechanism of action of the polyphosphate compound is unknown, the present inventors artificially supplied the polyphosphate compound together with the SH compound to the field of the luminescence reaction to enhance the luminescence intensity of bioluminescence and the luminescence time. It was confirmed that a completely new effect of prolongation of the above can be obtained and the present invention has been completed. Considering the equilibrium state of the enzymatic reaction, the reaction product, pyrophosphoric acid, should rather be removed from the reaction system. This is because removal of the reaction product may possibly shift the reaction equilibrium, that is, eliminate product inhibition. The prior art documents actually referred to above also describe that pyrophosphate acts as an inhibitor in the reaction of firefly luciferase. Therefore, it must be said that the addition of a polyphosphoric acid compound such as pyrophosphoric acid has the effect of enhancing the emission intensity and extending the emission time, which is far beyond the expected range.

Regarding the polyphosphoric acid compound, the bioluminescence enhancing mechanism is described as follows in the above-mentioned literature. That is, none of the polyphosphoric acid compounds such as pyrophosphoric acid serves as a luminescent reagent in place of ATP. The bioluminescence reaction is "ATP-luciferin-luciferase-
It emits light through the complex of "Mg ⁺⁺ ", but at the same time, an inactive substance is generated and the luminescence reaction is suppressed (almost 97% of ATP).
Is no longer used in the reaction). It is described that when a polyphosphoric acid compound is added in this state, the inactivated state is converted into the activated state and the luminescence is temporarily increased.
Regarding the luminescence enhancing effect of CoA introduced above, C
It is explained that oA suppresses the decay of luminescence due to the production of oxyluciferin. Dethio CoA lacking a sulfur atom does not affect the luminescence reaction itself and acts as a competitive inhibitor of CoA. From this, it is considered that dethio CoA and CoA bind to the same site of luciferase, but dethio CoA lacking a thiol group does not contribute to luminescence enhancement. Since the thiol group at the terminal of cysteamine is effective for the activity expression of CoA, the effect of CoA is considered to be different from the effect of compounds such as pyrophosphate.

On the other hand, S which is the second feature of the present invention
The H compound is a component often used as an enzyme protective agent in the field of enzyme reaction. However, the magnitude of the effect realized by the combination with the polyphosphoric acid compound in the present invention is far beyond the range expected from the conventional purpose of use, as shown in the examples. The general concentration when used as an enzyme protector is, for example, 5 mM for DTT.
The following are considered preferable. If it exceeds 5 mM, luciferase may be inhibited. Higher concentrations of SH compounds have been shown to be utilized when utilizing CoA, but the exemplified concentrations are 30-80 mM. On the other hand, the SH compound in the present invention has a preferable concentration range of 8-100 mM. Considering the difference in the optimum concentration and the magnitude of the effect of the present invention, it is considered that the action mechanism of the SH compound of the present invention is different from that of the conventional SH compound in the presence of CoA. As described above, the mechanism of action of the polyphosphate compound and the SH compound used in the present invention can be expected to be different from the conventional bioluminescence enhancer.

[0038]

Industrial Applicability According to the present invention, it is possible to obtain two novel effects of enhancing bioluminescence and extending luminescence time without utilizing CoA or the like which has a problem in stability and economy. The polyphosphoric acid compound such as pyrophosphoric acid used in the present invention is excellent in stability as compared with CoA, and since a cheap synthetic product is commercially available, it is economically very advantageous.

The enhancing effect of bioluminescence obtained by the present invention far exceeds the enhancing effect of CoA. Specifically, when compared with the detection sensitivity of luciferase, the conventional method using CoA has a limit of about 10 ⁻²² M, whereas in a preferred embodiment of the present invention, the detection sensitivity is further 5 times higher. It is clear that the enhancing effect according to the present invention is excellent. Further, comparing the effect of extending the light emission time, the conventional method using CoA is comparable to the conventional method and can be used instead of CoA. INDUSTRIAL APPLICABILITY The present invention makes it possible to enhance the luminescence intensity in a bioluminescence reaction with a stable component that is inexpensive and easy to handle, and to maintain luminescence with a stable intensity for a long time. It is expected that the present invention will greatly advance the practical application of bioluminescence in various fields.

According to the present invention, not only the luminescence intensity in the bioluminescence reaction is enhanced but also the luminescence intensity is less likely to be attenuated. Therefore, when applied to various measuring methods, a remarkable improvement in sensitivity can be expected. That is, conventionally, only the initial luminescence amount was quantified, so that the detection sensitivity was limited. However, if the present invention is utilized, stable and strong light emission can be obtained over a long period of time, and a very high sensitivity can be realized by integrating the total amount of light emission. Further, according to the present invention, strong light emission continues for a long time, so that sufficient sensitivity can be maintained without strict control of the reaction time. Due to this effect, the method for measuring bioluminescence, which conventionally required a special device, can be traced by a general luminometer, and as a result, the bioluminescence reaction can be popularized. Next, the present invention will be described in more detail based on examples.

[0041]

【Example】

1. Effect of adding polyphosphoric acid compound in the middle of luminescence reaction (without SH compound) An additional test was conducted on the effect of adding an inorganic polyphosphate such as pyrophosphoric acid in the middle of luminescence reaction in the prior art. . In the luciferin-luciferase reaction, it has been reported that the amount of luminescence increases when pyrophosphate is added during the luminescence reaction, but these reports indicate that luciferin and luciferase prepared in the 1950s are partially purified. It was the result of using. Therefore, first, a confirmation experiment was conducted to determine whether or not this phenomenon was reproduced even when highly purified luciferase and luciferin were used. The reagents used in the experiment are shown below.

Luciferin: Boehringer Mannheim From Photinus pyralis, Cat. No. 634409 Luciferin: Boehringer Mannheim D-(-)-Liciferin (Photinus pyralis Luciferin), Cat.
No.411400 ・ ATP: manufactured by Boehringer Mannheim 100 μl of a luciferase solution having the composition shown below and 100 μl of a substrate solution were mixed at 25 ° C., and 1
Pyrophosphate was added to a final concentration of 5 μM-50 mM at 20 seconds. Luminescence intensity was measured every 15 seconds with a luminescence reader (BLR-201: manufactured by Aloka). No change in pH was observed due to the addition of a pyrophosphoric acid aqueous solution or the like. Luciferase solution: 640ng / ml Luciferase in buffe
r A buffer A: 20 mM Tricine (pH 7.75), 1 mg / ml BS
A, 8 mM MgSO4, 0.13 mM EDTA substrate solution: 470 μM luciferin, 530 μM ATP,
10 µM-100 mM pyrophosphate in buffer A The results are shown in Fig. 1. It was confirmed that the amount of luminescence increased when 5 μM-0.5 mM pyrophosphoric acid was added. It was also found that the amount of luminescence decreases conversely when 5 mM or more is added.

2. Regarding the timing of adding the polyphosphoric acid compound From the result of 1), it was confirmed that strong phosphorescence temporarily occurs when pyrophosphoric acid or the like is added after the reaction in the presence of ATP.
However, when these phosphate compounds are added before the addition of ATP, the initial luminescence amount is drastically reduced even if the reaction is started by adding ATP (WDMcElroy et al. (1955) Arch. Bioche
m.Biophys., 46, 399-416), it was thought that the increase in luminescence due to pyrophosphate, etc. occurred only during the reaction and could not be used for amplification of luminescence including initial luminescence.
Therefore, the timing of adding the polyphosphoric acid compound to the reaction system was changed and the effect on the luminescence reaction was observed. The timing of adding the polyphosphate compound is (1) adding pyrophosphate to the luciferase solution, (2) adding to the substrate solution, and (3) preparing a pyrophosphate solution separately from these solutions and adding this. We examined three types: yes. As a result, it was confirmed that the amount of luminescence can be increased and the inhibitory effect of the polyphosphate compound can be avoided except for the case (1) where luciferase and pyrophosphate contact each other before the initiation of the luminescence reaction. Therefore, it became clear that the polyphosphoric acid compound should be used at the following timing A or B. Timing A: Add "luciferin + polyphosphate compound" to "solution of luciferase solution added with ATP in advance". Timing B: Add "luciferin + ATP + polyphosphate compound solution" to "luciferase solution" to start the reaction.

3. Effects of combining a polyphosphoric acid compound and an SH compound In the results 1 and 2, although the initial luminescence amount was increased by the polyphosphoric acid compound, the luminescence intensity rapidly decreased after the strong initial luminescence (FIG. 1). Therefore, the decay of this emission is S
It was investigated whether it could be suppressed by the addition of the H compound.
The operation is as follows. Luciferase solution containing 33.3 mM DTT and 33.3 mM DTT and 0.2 mM
Each of 10 substrate solutions containing potassium pyrophosphate (final concentration of potassium pyrophosphate in the reaction solution is 0.1 mM)
0 μl was mixed and the luminescence reaction at 25 ° C. was followed by the same luminometer as in 1. The results are shown in FIGS. When the luminescence enhancing component is not contained, the initial luminescence intensity is low and the luminescence intensity is almost instantaneously decreased, whereas when potassium pyrophosphate is added, the initial luminescence intensity is enhanced (Fig. 3). Alternatively, when DTT was added, the effect of maintaining the emission intensity was confirmed (Fig. 4). And the result according to the present invention of combining potassium pyrophosphate and DTT (FIG. 5)
In Example 1, it was confirmed that the initial emission intensity was enhanced and the emission intensity was maintained at a high level.

4. Optimal Concentration of Polyphosphate Compound in the Present Invention The optimal concentration of pyrophosphoric acid in the present invention was determined. Adjust the DTT concentration of the luciferase solution and substrate solution to 33.3 mM
And the pyrophosphate concentration of the substrate solution is 0.02-22 mM
(The final concentration of pyrophosphate in the reaction solution is 0.01-11.
The bioluminescence reaction was performed under the same conditions as in 3 except that the concentration was changed to mM), and the effect of the pyrophosphate concentration on the luminescence intensity was investigated. The results are shown in Fig. 6. It was found that the initial amount of luminescence increased most when the final concentration of pyrophosphoric acid in the reaction solution was about 10 ^-4 M.

5. Optimal concentration of SH compound in the present invention The optimal concentration of SH compound in the present invention was determined. Set the DTT concentrations of the luciferase solution and the substrate solution to 4.2-33.
SH which gives bioluminescence reaction under the same conditions as 3 except that the concentration of pyrophosphate is 0.2 mM (the final concentration of pyrophosphate in the reaction solution is 0.1 mM) is 3 mM.
The effect of compound concentration was investigated. The results are shown in Fig. 7. It was found that the emission intensity was maintained at a high level for a long time when DTT having a final concentration of about 8-33 mM was present in the reaction solution.

6. Effect of polyphosphoric acid compounds such as potassium pyrophosphate and tripolyphosphoric acid Pyrophosphoric acid was used as the polyphosphoric acid compound in the experiments so far. In this example, the effects of pyrophosphate and tripolyphosphate were examined by replacing pyrophosphate in 3 with various polyphosphate compounds. As a result, when the final concentration of 0.1 mM potassium pyrophosphate was added to the reaction solution, it was possible to obtain 1.3 times as much luminescence amount as when 0.1 mM pyrophosphate was added. Also, 0.
When 1 mM tripolyphosphoric acid was added, it was possible to obtain 1.7 times as much luminescence amount as when pyrophosphoric acid was added. From these results, it was confirmed that not only pyrophosphoric acid but also polyphosphoric acid compounds such as tripolyphosphoric acid and salts thereof are useful for enhancing the emission intensity.

7. Detection of Luciferase Activity The application of the bioluminescence measuring method according to the present invention to the detection of luciferase activity was tried. By the same procedure as 3 except that the DTT concentration of the luciferase solution and the substrate solution is set to 16.6 mM, and the pyrophosphate concentration of the substrate solution is set to 0.2 mM (the final concentration of pyrophosphate in the reaction solution is 0.1 mM). The emission intensity was measured. As a comparative control, an additive-free section containing no enhancer and 1 mM CoA + 33.3 mMD, which is the conventional enhancement technology.
The results were compared by setting a section to which TT (both final concentrations) was applied. The results are shown in Table 1 (pyrophosphate is abbreviated as PPi in the table) and FIG. The luminescence intensity obtained by the present invention was 30 times as high as that without addition, and was 7 times as strong as that of the known enhancer CoA + DTT. Further, as shown in FIG. 8, it was confirmed that the luminescence enhancement technique of the present invention not only simply enhances, but also maintains a high degree of quantification. Since the enhancing effect was quantitative, the luciferase activity could be quantified with high sensitivity and accuracy.

[0049]

[Table 1]

8. Detection of ATP The application of the bioluminescence measuring method according to the present invention to the detection of ATP activity was tried. DTT of luciferase solution and substrate solution
The concentration of the substrate solution was 16.6 mM, the concentration of potassium pyrophosphate was 0.2 mM (the final concentration of pyrophosphate in the reaction solution was 0.1 mM), and the ATP concentration was 0.0001-10.
The emission intensity was measured by the same operation except that the concentration was 0 nM. As a comparative control, a non-added section containing no enhancer was provided to compare the results. The results are shown in Fig. 9. According to the present invention, it was confirmed that ATP can be quantified in a wide range from low concentration to high concentration. Especially in the high concentration range, even higher concentration is included in the measurement range as compared with the conventional case of no addition, and it is clear that the present invention contributes to the expansion of the measurement range. Further, in the low concentration range, the slope of the standard curve of the present invention is large, resulting in improvement in sensitivity. Furthermore, in this experiment, not just the enhancement of the emission intensity,
Since the luminescence intensity is enhanced while maintaining a high degree of quantification, it is clear that the present invention is useful for measurement of ATP by bioluminescence reaction.

[Brief description of drawings]

FIG. 1 is a graph showing changes in luminescence intensity when pyrophosphoric acid is added after the initiation of luminescence reaction. The vertical axis is the emission intensity (KCPM),
The horizontal axis represents time (seconds).

FIG. 2 is a graph showing a change in luminescence intensity when a bioluminescence reaction is started under a condition without a luminescence enhancing component. The vertical axis represents emission intensity (KCPM) and the horizontal axis represents time (seconds).

FIG. 3 is a graph showing changes in luminescence intensity when a bioluminescence reaction is started in the presence of pyrophosphate. The vertical axis shows the emission intensity (KCP
M), the horizontal axis represents time (seconds).

FIG. 4 is a graph showing changes in luminescence intensity when a bioluminescence reaction is started in the presence of DTT. The vertical axis is the emission intensity (KCPM),
The horizontal axis represents time (seconds).

FIG. 5 is a graph showing changes in luminescence intensity when a bioluminescence reaction is initiated in the presence of both pyrophosphate and DTT. The vertical axis represents emission intensity (KCPM) and the horizontal axis represents time (seconds).

FIG. 6 is a graph showing the effect of pyrophosphate concentration on the emission intensity. The vertical axis represents emission intensity (KCPM) and the horizontal axis represents time (seconds).

FIG. 7 is a graph showing the influence of DTT concentration on the emission intensity. The vertical axis represents emission intensity (KCPM) and the horizontal axis represents time (seconds).

FIG. 8 is a graph showing changes in luminescence intensity with changes in luciferase concentration in the present invention. The vertical axis shows the emission intensity (KCP
M), the horizontal axis shows the luciferase concentration (amol / tube).

FIG. 9 is a graph showing changes in emission intensity with changes in ATP concentration in the present invention. The vertical axis represents emission intensity (KCPM) and the horizontal axis represents ATP concentration (nmol / tube).

Claims (24)

[Reference number] P-000304 [Claims] 1. A method for measuring bioluminescence generated by allowing luciferase to act on a luciferase substrate, which comprises performing a bioluminescence reaction in the presence of a polyphosphate compound or a salt thereof and a sulfhydryl compound. 2. The method for measuring bioluminescence according to claim 1, wherein the luciferase is derived from firefly, and the firefly luciferase substrate or its derivative is used as the luciferase substrate, and adenosine triphosphate is added as the substrate. 3. The method for measuring bioluminescence according to claim 2, wherein the firefly luciferase is obtained by gene recombination or is made thermostable by substituting a part of amino acids. 4. The method for measuring bioluminescence according to claim 1, wherein the polyphosphate compound is present at 10 ⁻⁶ −10 ⁻² M. 5. The method for measuring bioluminescence according to claim 4, wherein the polyphosphate compound is present at 10 ⁻⁵ −10 ⁻³ M. 6. The method for measuring bioluminescence according to claim 1, wherein the polyphosphate compound is an inorganic polyphosphate compound. 7. The method for measuring bioluminescence according to claim 6, wherein the inorganic polyphosphoric acid compound is selected from the group consisting of tripolyphosphoric acid, metaphosphoric acid, hexametaphosphoric acid, pyrophosphoric acid, and salts thereof. 8. The method for measuring bioluminescence according to claim 1, wherein the polyphosphate compound is an organic polyphosphate compound. 9. The organic polyphosphate compound is cytidine 5'-
Triphosphate (CTP), cytidine 5'-2 phosphate (CD
P), deoxycytidine 5′-3 phosphate (dCTP),
Deoxycytidine 5'-2 phosphate (dCDP), dideoxycytidine 5'-3 phosphate (ddCTP), cytidine 5'-3 phosphate or cytidine 5'-2 phosphate periodate, cytidine 5'- The method for measuring bioluminescence according to claim 8, which is selected from the group consisting of an etheno derivative of triphosphate or cytidine 5'-2 phosphate, and salts thereof. 10. The method for measuring bioluminescence according to claim 1, wherein the polyphosphate compound is not contacted with luciferase until the bioluminescence reaction is started. 11. The method for measuring bioluminescence according to claim 1, wherein the polyphosphate compound is added to the luciferase together with the luciferase substrate and adenosine triphosphate. 12. A sulfhydryl compound is selected from the group consisting of dithiothreitol, dithioerythritol, β-mercaptoethanol, 2-mercaptopropanol, 3-mercaptopropanol, 2,3-dithiopropanol, glutathione, and coenzyme A. The method for measuring bioluminescence according to claim 1, which is selected. 13. The method for measuring bioluminescence according to claim 12, wherein the sulfhydryl compound is dithiothreitol. 14. A sulfhydryl compound is added to 0.1-200.
The method for measuring bioluminescence according to claim 1, wherein mM is present. 15. The method for measuring bioluminescence according to claim 14, wherein the sulfhydryl compound is present at 8-100 mM. 16. The method for measuring bioluminescence according to claim 1, wherein the luciferase is a labeling enzyme. 17. The method for measuring bioluminescence according to claim 16, wherein the substance to be labeled is selected from the group consisting of immunologically active substances, nucleic acids, and specific binding ligands. 18. A luciferase, a luciferase substrate,
Reagent for bioluminescence measurement containing polyphosphoric acid compound or salt thereof, and sulfhydryl compound 19. A reagent for measuring the activity of firefly luciferase by a bioluminescence reaction containing a luciferase substrate, a polyphosphate compound or a salt thereof, and a sulfhydryl compound. 20. A reagent for measuring adenosine triphosphate by bioluminescence, which comprises a firefly luciferase, a luciferase substrate, a polyphosphate compound or a salt thereof, and a sulfhydryl compound. 21. A method for enhancing bioluminescence produced by acting luciferase on a luciferase substrate, which comprises performing a bioluminescence reaction in the presence of a polyphosphate compound or a salt thereof and a sulfhydryl compound. 22. A luciferase, comprising a polyphosphate compound and a sulfhydryl compound.
And bioluminescence enhancers by luciferase substrates 23. A method for carrying out a bioluminescence reaction by allowing a luciferase to act on a luciferase substrate in the presence of a polyphosphoric acid compound or a salt thereof and a sulfhydryl compound, the method comprising contacting each component substantially at the same time to generate luminescence. Method for conducting bioluminescence reaction characterized by enhancing and prolonging luminescence time 24. A bioluminescent reaction composition comprising a polyphosphate compound or a salt thereof, a sulfhydryl compound, a luciferase substrate, and a luciferase.