JP2011043447A - Emission enhancement method of peroxidase chemiluminescent reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of enhancing emission intensity and emission durability of a chemiluminescent reaction using peroxidase (hereafter referred to as 'POD') originated in basidiomycete having a defect of inferior emission durability in comparison with a chemiluminescent reaction using POD originated in horseradish. <P>SOLUTION: One kind or two or more kinds of chemiluminescence enhancers selected from an EDTA analog, a hydrazine derivative or an acene-based polycyclic aromatic hydrocarbon are added into a final reaction solution of the chemiluminescent reaction. Hereby, the emission intensity and the emission durability of the chemiluminescent reaction can be enhanced significantly in the chemiluminescent reaction using POD originated in basidiomycete. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for enhancing luminescence intensity and enhancing luminescence persistence in a chemiluminescence reaction using a basidiomycete-derived peroxidase.

  Horseradish-derived peroxidase (hereinafter referred to as “POD”) is a solid phase enzyme immunoassay (ELISA), for example, a non-competitive or competitive method, and a Western blotting method, as an analytical reagent using the enzyme activity as an index. It is widely used in various enzyme immunoassay methods such as (see, for example, Patent Document 1). However, since POD derived from horseradish takes a long time to cultivate horseradish as a raw material, it is produced by a method of destroying the plant body and purifying POD from a wide variety of contaminated components. It was difficult to say that its production efficiency was high, and mass production was difficult. In addition, in recent years, there has been a growing concern about the supply of horseradish as a raw material for POD due to poor cultivation efficiency and conversion to grains for bioethanol, which are in high demand. Needs are great.

  Further, POD derived from horseradish has a problem that many isozymes exist. Most of the PODs derived from horseradish that are widely used at present are restricted to be distributed at a constant price within a low production efficiency, and thus are mostly a mixture of many isozymes. However, when performing various measurements using such POD, for example, enzyme immunoassay, the content of various isozymes having different reaction characteristics varies from production lot to production lot of POD. There is a serious problem that it is difficult to obtain a stable measurement result.

  POD derived from microorganisms is a POD expected to overcome the problems of POD derived from horseradish. Microorganisms can be cultivated in a large amount in a short time, and POD derived from microorganisms can be purified with much less labor than when purifying from a plant body. In addition, it is easy to artificially increase the expression level in a microbial host by using a gene recombination technique. If gene recombination technology is used, only the target POD can be expressed in a large amount. Therefore, it is easy to avoid the problem of isozyme contamination, and at the same time, it is relatively easy to modify and improve the POD. Easy. Therefore, it can be said that POD derived from microorganisms is a promising enzyme that can replace POD derived from horseradish.

  The present inventors have already prepared PODs derived from microorganisms such as Arthromyces genus and basidiomycete Coprinus genus as enzymes that can be substituted for horseradish-derived POD (for example, Patent Document 2, Non-Patent Documents 1 and 2). Whether it is used in the free state or labeled as a POD-labeled antibody, it has a reactivity equivalent to or higher than that of POD derived from horseradish in a chemiluminescent reaction using luminol as a substrate. I have confirmed that.

  However, PODs derived from microorganisms including PODs derived from the genus Coprinus show a high emission intensity immediately after the start of the chemiluminescence reaction under the conditions of the conventional chemiluminescence reaction, but have a drawback of poor sustainability. . Therefore, it is difficult to measure the amount of luminescence in a stable state, and there is a problem to be solved for improving the luminescence persistence in order to replace POD derived from microorganisms with POD derived from horseradish and use it for enzyme immunoassay and the like. It is left.

  Various chemiluminescence enhancers with luminescence intensity enhancement effect and luminescence persistence effect are added to compensate for weak luminescence intensity when measuring POD derived from horseradish using luminol as a substrate. How to do is known. Examples of chemiluminescence enhancers that exhibit luminescence intensity enhancement effect and luminescence persistence effect against POD derived from horseradish include, for example, p-iodophenol, p-bromophenol, p-chlorophenol, p-phenylphenol, 2- Chloro-4-phenylphenol, 2,4-dichlorophenol, 4- (cyanomethylthio) phenol, 4-cyanomethylthio-2-chlorophenol, phenolindole, p-hydroxycinnamic acid, 4- [4 '-(2 '-Methyl) thiazolyl] phenol, 4- (4'-thiazolyl) phenol, 4- (2'-thienyl) phenol, 4- [4'-(2 '-(3'-pyridyl)) thiazole] phenol, phenothiazine -N-propyl sulfonate, phenolindophenol, 4-hydroxycinnamic acid, -Phenol derivatives such as hydroxycinnamic acid, 6-hydroxybenzothiazole, 4- (4-hydroxyphenyl) thiazole, 4- (4-hydroxyphenyl) -2-methyl-thiazole, 4- (4'-hydroxyphenyl) -2 '-(3'-pyridyl) thiazole, firefly luciferin, thiazole derivatives such as dehydroluciferin, naphthol derivatives such as 2-naphthol, 1,6-dibromo-2-naphthol, 3- (10-phenothiazyl) propylsulfonic acid Examples thereof include salts, p-hydroxyphenylpropionate and diethylaniline, N, N, N ′, N′-tetramethylbenzidine and the like (for example, see Non-Patent Documents 3 to 4 and Patent Documents 3 to 6). However, these chemiluminescence enhancers are peculiar to POD derived from horseradish, and do not show a light emission intensity enhancing effect or a light emission sustaining effect with respect to POD derived from microorganisms.

  On the other hand, as a chemiluminescence enhancer for POD derived from microorganisms, hydrazobenzene, indigo, 3-methyl-2-benzothiazoline hydrazone hydrochloride, α-naphthoquinone, phenylhydrazine are used for detection or quantification of trace amounts of hydrogen peroxide. Chemiluminescence measurement methods using 8-hydroxyquinoline, phenolphthaline, L-tryptophan, N, N-dimethylindoaniline, calcein, indigo, carmine and resorcin are known (for example, see Patent Document 7). Further, as a method for enhancing the enzymatic reaction of microorganism-derived POD for the purpose of bleaching dyes in paper or paper pulp, etc., 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate), 4- Amino-4'-methoxystilbene, 4,4'-diaminostilbene-2,2'-disulfonic acid, 4,4'-dihydroxybenzophenone, N-benzylidene-4-biphenylamine, 10-methylphenothiazine, 10-phenothiazine- Propionic acid, 10-phenylphenothiazine, 10-methylphenoxazine, benzidine, 3,3′-dimethylbenzidine, 6-amino-3-methyl-2-benzothiazolinone azine, 6-hydroxy-2-naphthoic acid, 6-bromo-2-naphthol, 7-amino-2-naphthalenesulfonic acid, 1,5-di A method of adding minonaphthalene, 10-phenothiazinepropionic acid, 10-ethylphenothiazine-4-carboxylic acid, 10-methylphenoxazine and 10-phenoxazinepropionic acid is known (see, for example, Patent Documents 8 to 10). .

  As described above, methods for enhancing the enzymatic reaction of POD derived from microorganisms are known for the detection or quantification of trace amounts of hydrogen peroxide, bleaching of dyes in paper or paper pulp, or prevention of dye transfer, etc. In various enzyme immunoassay methods such as solid phase enzyme immunoassay (ELISA), for example, non-competitive method or competitive method or Western blotting method, a method for enhancing the chemiluminescent reaction of microorganism-derived POD is not known.

  That is, in an enzyme immunoassay method using a chemiluminescence reaction using POD derived from a microorganism as an index, development of a method for enhancing the luminescence intensity of the chemiluminescence reaction and increasing the reaction persistence is desired.

JP 2000-88850 A JP 61-104784 A Japanese National Patent Publication No.59-500262 JP 59-171839 A JP-A-2-291299 JP-A-8-38195 Japanese Patent No. 3005238 Japanese National Patent Publication No. 8-503371 Japanese National Patent Publication No. 8-506209 Japanese National Patent Publication No. 10-500728

Kim et al. Analytical Biochemistry 199, 1-6 (1991) Kjalke et al. Biochim Biophys Acta. 1992 Apr 17; 1120 (3): 248-56. Gray et al. Methods in Enzymology 133, 331-353 (1986) Akio Tsuji. Clinical examination 38, 162-166 (1994)

  An object of the present invention is to provide a method for maintaining a high luminescence intensity for a long time from the start of a chemiluminescence reaction in a chemiluminescence reaction using a POD derived from a microorganism.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that, in a system that chemiluminescence is generated using a chemiluminescent substance and an oxidizing agent, from EDTA analogs, hydrazine derivatives, and acene polycyclic aromatic hydrocarbons. Knowing that one or more selected compounds can improve the reactivity of basidiomycete-derived POD, the present invention has been completed. That is, the present invention relates to the following.
1) In chemiluminescence reaction using basidiomycete-derived peroxidase, one or more chemiluminescence enhancers selected from EDTA analogs, hydrazine derivatives or acene polycyclic aromatic hydrocarbons are used. A method for enhancing luminescence of a peroxidase chemiluminescence reaction.
2) EDTA analog is Mn (II) -EDTA, N, N-bis (2-hydroxyethyl) glycine (Bicine), 1,6-hexamethylenediamine-N, N, N ′, N′-tetraacetic acid (HDTA), 1,3-diamino-2-hydroxypropane-N, N, N ′, N′-tetraacetic acid (DPTA-OH), triethylenetetramine-N, N, N ′, N ″, N ′ ″, N ″ ′-hexaacetic acid (TTHA), diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA), O ′, O′-bis (2-aminoethyl) The method for enhancing luminescence of peroxidase chemiluminescence reaction according to 1) above, which is either ethylene glycol-N, N, N ′, N′-tetraacetic acid (GEDTA) or calcein blue.
3) The method for enhancing luminescence of the peroxidase chemiluminescence reaction according to 1) above, wherein the hydrazine derivative is any one of dansyl hydrazine, iproniazide phosphate, and 1-phenyl-3-thiosemicarbazide.
4) The method for enhancing luminescence of the peroxidase chemiluminescence reaction according to 1) above, wherein the acene polycyclic aromatic hydrocarbon is either anthracene or naphthacene.

  According to the present invention, the luminescence intensity and luminescence persistence of a chemiluminescence reaction using basidiomycete-derived POD can be significantly enhanced, and a highly sensitive and stable measurement method can be provided.

It is the figure which showed the influence which calcein blue density | concentration at the time of using calcein blue as a chemiluminescence enhancer has on the first time emission intensity. It is the figure which showed the influence which the calcein blue density | concentration at the time of using Mn (II) -EDTA as a chemiluminescence enhancer has on the first time luminescence intensity.

(Basidiomycete-derived POD)
Examples of the POD used in the present invention include basidiomycete-derived POD. Examples of the microorganism belonging to the basidiomycete include the genus Coprinus, the genus Uredinales, the genus Auriculariales, the genus Agaricales, and the like. Among them, POD derived from the genus Coprinus has excellent enzyme chemical properties (optimal pH, pH stability, optimal temperature, temperature stability) that are equivalent to or higher than those derived from horseradish. Moreover, in the color reaction using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate, the specific activity (U / mg) is 9 more than POD derived from horseradish. It is preferable because it is about 10 times higher. Examples of microorganisms belonging to the genus Coprinus include Coprinus cinereus (NBRC30114), Coprinus macrorhizus (ATCC20120), Coprinellus disseminatus, Coprinus comatus (ATCC12640), Coprinus clastophyllus, Coprinus alkalinus, Coprinus amphibius, Coprinus micaceus, Coprinus micaceus, Coprius micaceus, Colut Examples include trisporus, Coprinus sclerotiger, Coprinus domesticus, Coprinus stercorarius, Coprinus radiatus. In addition, NBRC indicates the National Institute for Product Evaluation and Technology Biological Resource Department, and ATCC indicates the American Type Culture Collection.

  Coprinus genus-derived POD may be naturally occurring, may have improved heat resistance, improved substrate specificity, or may be artificially introduced with one or more other mutations, such as a chimeric protein. Also good. Moreover, you may use commercially available Coprinus genus POD. A plurality of PODs containing isozymes can be used together as long as there is no problem in measurement, but it is preferable to use a single purified product for the stability of measurement.

  In addition, even in the case of microorganisms that do not belong to the genus Coprinus, for example, PODs derived from microorganisms closely related to the genus Coprinus or PODs derived from the genus Coprinus are similar in amino acid sequence. POD or the like having the same reactivity can be used.

  The basidiomycete-derived POD used in the present invention can be used in a free state or a state in which various antibodies are labeled in the final reaction solution described later. As a method for labeling (crosslinking) an antibody to POD, various known labeling methods can be used. For example, generally known methods include glutaraldehyde method, periodic acid method, maleimide method, pyridyl Examples thereof include a disulfide method, an isocyanate crosslinking method, and a benzoquinone crosslinking method. In particular, the maleimide method is suitable in terms of the presence or absence of polymer formation, the maintenance of the activity of antigens, antibodies and enzymes, and the efficiency of labeling.

(Chemiluminescence enhancer)
As a chemiluminescence enhancer of microorganism-derived POD, one or more chemiluminescence enhancers selected from (a) EDTA analogs, (b) hydrazine derivatives or (c) acene polycyclic aromatic hydrocarbons Can be mentioned.

(A) As an EDTA analog, any organic compound that forms a cyclic structure containing a metal ion can be used, and a tricoordinate chelating agent and a tetracoordinate chelating agent are used. For example, these chelating agents include ethylenediaminetetraacetic acid (EDTA), EDTA · 2Na, EDTA · 3Na, EDTA · 4Na, EDTA · 2K, EDTA · 3K, EDTA · 2ammonium, Ca (II) -EDTA, Co ( II) -EDTA, Cu (II) -EDTA, Fe (III) -EDTA, Mg (II) -EDTA, Mn (II) -EDTA, Zn (II) -EDTA, O, O′-bis (2-amino) Phenyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid (BAPTA), N, N-bis (2-hydroxyethyl) glycine (Bicine), trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (CyDTA), diethylenetriamine-N, N, N ′, N′-pentaacetic acid (DTPA), N- ( -Hydroxyethyl) ethylenediamine-N, N ', N'-triacetic acid (EDTA-OH), O', O'-bis (2-aminoethyl) ethylene glycol-N, N, N ', N'-tetraacetic acid (GEDTA), 1,6-hexamethylenediamine-N, N, N ′, N′-tetraacetic acid (HDTA), 1,3-diamino-2-hydroxypropane-N, N, N ′, N′-four Acetic acid (DPTA-OH), 1,2-diaminopropane-N, N, N ′, N′-tetraacetic acid (Methyl-EDTA), N- (hydroxyethyl) iminodiacetic acid (HIDA), nitrilotriacetic acid (NTA) ), Nitrilotris (methylenephosphonic acid) · 3Na (NTPO · 3Na), triethylenetetramine-N, N, N ′, N ″, N ′ ″, N ′ ″-hexaacetic acid (TTHA), nitrilotri Propio Acid (NTP), calcein blue, and the like. Among these, Mn (II) -EDTA, Bicine, HDTA, DPTA-OH, TTHA, and calcein blue are preferable because they exhibit particularly high chemiluminescence persistence.

(B) As a hydrazine derivative, 1,1-diphenylhydrazine hydrochloride, 3-methyl-2-benzothiazolinone hydrazine hydrochloride monohydrate, phenylhydrazine, 2-hydrazinobenzothiazole, dansylhydrazine, iproniadi Dolinate, 2,4-dinitrophenylhydrazine hydrochloride, 4-hydrazinobenzoic acid, 1-phenyl-3-thiosemicarbazide, 4-phenyl-3-thiosemicarbazide, 1-acetyl-2-phenylhydrazine, 4-hydra Examples include dinobenzenesulfonamide hydrochloride, hydrazinoethanol, 1-hydrazinophthalazine hydrochloride, 2-hydrazinopyridine, and 2-hydrazinoquinoline. Among these, dansyl hydrazine, iproniazide phosphate, and 1-phenyl-3-thiosemicarbazide are preferable because they exhibit particularly high chemiluminescence persistence.

(C) Examples of the acene-based polycyclic aromatic hydrocarbon include naphthalene, anthracene, naphthacene, pentacene, and hexacene. Of these, anthracene and naphthacene are preferable because they exhibit particularly high chemiluminescence persistence.

(Oxidant)
Examples of the oxidizing agent used in the present invention include inorganic peroxides such as hydrogen peroxide, perborate and hypochlorite, and organic peroxides such as peracetic acid and perpropionic acid. Etc. From the viewpoint of ease of handling, it is preferable to use hydrogen peroxide.

  What is necessary is just to set content of an oxidizing agent suitably according to the kind of chemiluminescent substance to be used, density | concentration, or the measuring method to apply. In order to maintain the luminescence intensity at a high value, the concentration of the oxidizing agent in the final reaction solution is preferably in the range of 0.001 to 100 mM, more preferably in the range of 0.01 to 10 mM, and still more preferably. Is in the range of 0.05 to 2 mM.

(Chemiluminescent substance)
As the chemiluminescent substance used in the present invention, various known chemiluminescent substances can be used. For example, 2,3-dihydro-1,4-phthalazinedione compounds can be used. Specific examples of the chemiluminescent substance include, for example, luminol, isoluminol, N-ethylisoluminol, N- (4-aminobutyl) -N-ethylisoluminol hemisuccimide, N- (6-aminohexyl). ) -N-ethylisoluminol and the like, or metal salts thereof. As the metal salt, an alkali metal salt, an alkaline earth metal salt, or the like can be used. Examples of the alkali metal salt include sodium salt and potassium salt, and examples of the alkaline earth metal salt include calcium salt and magnesium salt. In particular, luminol or a luminol metal salt is preferable because of its excellent stability and emission quantum yield.

  The content of the chemiluminescent substance may be appropriately set depending on the type and concentration of the oxidizing agent used, the measurement method to be applied, etc., but the initial emission intensity is maintained at a high value in order to maintain the emission intensity at a high value. Therefore, the concentration of the chemiluminescent substance in the final reaction solution is preferably in the range of 0.001 to 100 mM, more preferably in the range of 0.01 to 10 mM, and still more preferably in the range of 0.1 to 5 mM. It is a range.

(Buffer solution)
The buffer solution used in the present invention is not particularly limited as long as it is a buffer solution that is generally used and has an excellent buffer action. For example, acetate buffer solution, citrate buffer solution, phosphate buffer solution, Tris buffer Solution, Good buffer solution, borate buffer solution, glycine buffer solution, pyrophosphate buffer solution, ammonium buffer solution, and carbonate buffer solution can be used.

  The pH of the buffer solution may be appropriately set depending on the type and concentration of the chemiluminescent substance or oxidizing agent to be used, the measurement method to be applied, or the like. It is preferable to make it the range of 0.0-12.0, More preferably, it is the range of 7.0-11.0, More preferably, it is the range of 7.5-10.0.

(Final reaction solution)
The chemiluminescent reaction is performed in a final reaction solution containing the POD, chemiluminescent enhancer, chemiluminescent substance, oxidizing agent and buffer solution. In addition, evaluation of the light emission amount of this invention can be performed by the relative value or integrated value of the light emission amount immediately after the reaction start, or the light emission amount for every measurement time.

  The final reaction solution using the chemiluminescence enhancer of the present invention exhibits excellent effects as a measurement method and measurement kit using basidiomycete-derived POD. Therefore, as an immunoassay method and an immunoassay kit, it is particularly preferable to use an enzyme immunoassay method and an enzyme immunoassay kit, and it is most preferable to use a heterogeneous enzyme immunoassay kit using an insoluble carrier.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Search for enhancer (EDTA analog) using POD-labeled mouse IgG antibody]
Using a POD-labeled antibody (POD-IgG), a search was performed for EDTA analogs among chemiluminescence enhancers.

1. POD labeling to anti-mouse IgG antibody POD labeling to anti-mouse IgG antibody was performed as follows.

1) Preparation of IgG-SH Solution replacement of goat anti-mouse antibody (IgG) with 0.1 M sodium phosphate buffer (pH 6.4) containing 0.15 M NaCl and 10 mM EDTA results in 1 to 10 mg / ml It was prepared as follows. 500 mM cysteamine (manufactured by Sigma; product code M9768-25G) (6 mg of cysteamine dissolved in 0.1 ml of 0.1 M sodium phosphate buffer (pH 6.4) containing 0.15 M NaCl and 10 mM EDTA) A 1/10 (v / v) amount of the antibody solution was added and incubated at 37 ° C. for 1.5 hours. Excess cysteamine was dialyzed or gel filtration column Sephadex G-25 (manufactured by GE; product code 17-0033-01, equilibration solution: 0.1 M sodium phosphate buffer (pH 6. containing 0.15 M NaCl and 10 mM EDTA). 8), eluent: same as the equilibration liquid, column size: 10 ml, elution by gravity).

2) Preparation of maleimidated POD 0.1M sodium phosphate buffer (pH 7.) containing 0.15 M NaCl and 10 mM EDTA so that the commercially available basidiomycete-derived POD (Roche) was 10 to 30 mg / ml. 4) dissolved. 100 mM SMCC (Pierce; product code 22360) (3.34 mg dissolved in 0.1 ml DMSO) was prepared and immediately 5 times the molar concentration of POD was added. After incubating at room temperature for 30 minutes, according to the above conditions, the gel filtration column Sephadex G-25 was used to remove excess SMCC. The enzyme concentration was determined from the measured absorbance at 403 nm using ε403 nm = 8.33 × 10 ⁴ l / (mol · cm).

3) Labeling (crosslinking) of antibody with maleimidated POD and purification of labeled antibody The maleimidated POD and SH group-reduced antibody (solvent is 0.1 M sodium phosphate buffer (pH 6. containing 0.15 M NaCl and 10 mM EDTA). 8)) was mixed so that the molar ratio of enzyme: antibody was 10: 1. After incubation at 4 ° C. for 6 hours or more (overnight), the reaction solution after labeling (crosslinking) was equilibrated with 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl, Superdex 200 10 / 300GL (manufactured by GE; product code 17-5175-01, elution: 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl, column size: about 24 ml, flow rate: 0.5 ml / min The enzyme that was not bound to the antibody was removed, and a basidiomycete-derived POD-labeled anti-mouse IgG (POD-IgG) was obtained. Hereinafter, POD-IgG was used as the microorganism-derived POD.

2. Search for Enhancer (EDTA Analog) Using POD-IgG An EDTA analog that significantly enhances the chemiluminescence of basidiomycete-derived POD-IgG was searched.
1.875 mM luminol (manufactured by Sigma) and 100 mM, pH 9.5 Tris-HCl buffer (hereinafter referred to as “screening reagent 1”) containing 12.5 ng / ml POD-IgG and 0.133 mM hydrogen peroxide ( 100 mM, pH 9.5 Tris-HCl buffer solution (hereinafter referred to as “screening reagent 2”) containing Wako Pure Chemical Industries, Ltd.) was prepared.

  Next, as an EDTA analog, calcein blue is appropriately dissolved in dimethyl sulfoxide (DMSO), and Mn (II) -EDTA is appropriately dissolved in ion-exchanged water, so that calcein blue is 0.01-1. A DMSO solution containing a concentration of 0 mM or an aqueous solution containing Mn (II) -EDTA in a concentration range of 0.1 to 5.0 mM was prepared. The DMSO solution containing the EDTA analog and 10 μl of aqueous solution are dispensed into a 96-well microtiter plate, 160 μl of screening reagent 1 is added, and then 30 μl of screening reagent 2 is added to start the chemiluminescence reaction. did. The reaction temperature was 30 ° C. As a control group, DMSO containing no EDTA analog and 10 μl of ion-exchanged water were used, and a chemiluminescence reaction was performed as in the experimental group.

  The amount of luminescence was measured using MicroLumatPlus LB 96V (manufactured by Berthold) as the average amount of luminescence accumulated for 1 second at 0 minutes (first time), 5 minutes, 10 minutes, and 15 minutes from the start of the reaction. In order to confirm the chemiluminescence enhancing action of each EDTA analog, a relative value with the initial luminescence amount of the control group as 100% was calculated as the initial luminescence intensity, and the luminescence amount in each reaction time of the control group was 100 respectively. The relative value as% was calculated as the relative light emission intensity. The initial emission intensity at each EDTA analog concentration is shown in FIGS. 1-2, and the relative emission intensity is shown in Tables 1-2.

  As shown in FIGS. 1 and 2 and Tables 1 and 2, when subjected to chemiluminescence reaction using basidiomycete-derived POD, as the reaction time elapses, the emission intensity decreases rapidly, whereas chemiluminescence It was confirmed that when EDTA analogs calcein blue and Mn (II) -EDTA were added as enhancers, the emission intensity and emission persistence were uniformly enhanced. In particular, it was confirmed that calcein blue showed an excellent enhancing action at an addition concentration of 0.1 to 1.0 mM, and Mn (II) -EDTA at an addition concentration of 1.0 to 5.0 mM.

  Next, the enhancing action of various EDTA analogs Bicine, HDTA, DPTA-OH, TTHA, DTPA, GEDTA, 8-hydroxyquinoline and Cu (II) -EDTA was confirmed in the same manner as in the above method. Calcein blue and 8-hydroxyquinoline were dissolved in dimethyl sulfoxide (DMSO), and the other EDTA analogs were dissolved in ion-exchanged water. Table 3 shows the relative light emission intensities. The results show the concentration having the strongest enhancing action for various EDTA analogs after confirming the influence of the EDTA analog concentration on the chemiluminescence reaction.

  As shown in Table 3, calcein blue, Mn (II) -EDTA, Bicine, HDTA, DPTA-OH, TTHA, DTPA and GEDTA among EDTA analogues are excellent in chemiluminescence reaction using basidiomycete-derived POD. It was confirmed to show a potentiating action. On the other hand, 8-hydroxyquinoline and Cu (II) -EDTA could not confirm the enhancing action.

  As described above, it was suggested that the chemiluminescence reaction using basidiomycete-derived POD is enhanced by selecting an appropriate EDTA analog and adjusting the concentration appropriately.

[Search for potentiators (hydrazine derivatives) using POD-labeled mouse IgG antibodies]
Hydrazine derivatives that significantly enhance the chemiluminescence of basidiomycete-derived POD-IgG were searched.

  Using a commercially available hydrazine derivative, the enhancement effect on the chemiluminescence reaction of the hydrazine derivative was confirmed in the same manner as in the method described in Example 1. Dimethyl sulfoxide (DMSO) was used as the solvent for the hydrazine derivative. The results are shown in Table 4.

  As shown in Table 3, among the hydrazine derivatives, dansyl hydrazine, iproniazidophosphate and 1-phenyl-3-thiosemicarbazide exhibit an excellent enhancing action in the chemiluminescence reaction using basidiomycete-derived POD. Was confirmed. In particular, it was confirmed that iproniazidphosphate exhibits an extremely excellent enhancing action. On the other hand, with 1,1-diphenylhydrazine hydrochloride, the enhancing action could not be confirmed.

[Search for enhancer (acene polycyclic aromatic hydrocarbon) using POD-labeled mouse IgG antibody]
Acene polycyclic aromatic hydrocarbons that significantly enhance the chemiluminescence of basidiomycete-derived POD-IgG were searched.

  Using a commercially available acene-based polycyclic aromatic hydrocarbon, in the same manner as in the method described in Example 1, the enhancing action of the acene-based polycyclic aromatic hydrocarbon on the chemiluminescence reaction was confirmed. Note that DMSO was used as the solvent for the acene polycyclic aromatic hydrocarbon. The results are shown in Table 5.

  As shown in Table 5, it was confirmed that anthracene and naphthacene, which are acene polycyclic aromatic hydrocarbons, show an excellent enhancing action in the chemiluminescence reaction using basidiomycete-derived POD. In particular, it was confirmed that naphthacene exhibits an extremely excellent enhancing action.

Claims (4)

In a chemiluminescence reaction using a basidiomycete-derived peroxidase, one or more chemiluminescence enhancers selected from EDTA analogs, hydrazine derivatives or acene polycyclic aromatic hydrocarbons are used. Luminescence enhancement method of chemiluminescence reaction. EDTA analogs include Mn (II) -EDTA, N, N-bis (2-hydroxyethyl) glycine (Bicine), 1,6-hexamethylenediamine-N, N, N ′, N′-tetraacetic acid (HDTA). ), 1,3-diamino-2-hydroxypropane-N, N, N ′, N′-tetraacetic acid (DPTA-OH), triethylenetetramine-N, N, N ′, N ″, N ′ ″ , N ′ ″-hexaacetic acid (TTHA), diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA), O ′, O′-bis (2-aminoethyl) ethylene glycol The method for enhancing luminescence of a peroxidase chemiluminescence reaction according to claim 1, which is either -N, N, N ', N'-tetraacetic acid (GEDTA) or calcein blue. The method for enhancing luminescence of a peroxidase chemiluminescence reaction according to claim 1, wherein the hydrazine derivative is any one of dansyl hydrazine, iproniazidophosphate, or 1-phenyl-3-thiosemicarbazide. The method for enhancing light emission of a peroxidase chemiluminescence reaction according to claim 1, wherein the acene polycyclic aromatic hydrocarbon is either anthracene or naphthacene.

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