JPH07311197A - Chemiluminescence enhancing method - Google Patents

Abstract

PURPOSE:To determine the presence or the quantity of a catalyst or 2,3- dihydro-1,4-phthalazinedione in a reaction liquid by effecting chemiluminescence method under presence of a compound represented by a specified formula thereby achieving sufficient background emission suppressing effect and signal emission enhancing effect. CONSTITUTION:An emission reaction is effected under presence of any one or more kind of compound selected from the group consisting of four-position substituted phenol derivatives. (In the formula, X represents a hydrogen atom or a halogen atom, Y represents thiophene having or not having a substituent bonded at position 2, 3, 4 or 5 with phenol. Alternatively, Y represents pyrrole having or not having a substituent bonded at position 1 with phenol, or represents a benzene having a substituent at position 4 and bonded at position 1 with phenol where the substituent is a methyl group, a halogen group or a carboxyl group).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for reducing background luminescence and enhancing signal luminescence in luminescence analysis such as chemiluminescent enzyme-labeled immunoassay (CLEIA).

[0002]

2. Description of the Related Art Enzyme-linked immunosorbent assay (EIA) is used to measure or quantify the presence of trace amounts of substances in the living body.
In the recent EIA, it is mainstream to use a fluorescent substrate for the detection of the labeling enzyme (FEIA), but the measurement of a fluorescent substance is not easy to perform a highly sensitive measurement due to the influence of excitation light for exciting the substance. Absent.

For this reason, CLEIA using a luminescent substrate capable of more sensitive measurement was developed.

[0004]

The enzymes used in CLEIA mainly include alkaline phosphatase and peroxidase. In the case of a luminescent substrate used for alkaline phosphatase, the phosphate group of the luminescent substrate containing a dioxetane structure is hydrolyzed. It utilizes the light emission when it decomposes and becomes unstable and the substrate decomposes. However, at the optimum pH of alkaline phosphatase, non-enzymatic hydrolysis of the phosphoric acid ester of these luminescent substrates is likely to occur, and reduction of background luminescence resulting from this is a problem.

On the other hand, in the case of peroxidase, when luminol and an oxidizing agent such as hydrogen peroxide are reacted alone, short-time luminescence is observed up to several fentomoles as the absolute amount of enzyme, but when the amount is less than that. Measurement is difficult because it is hidden by background light emission. For this task, T
horpe et al. report that the addition of compounds such as phenol derivatives and hydroxybenzothiazole to the above system can remarkably enhance the luminescence and can maintain the luminescence for a long time (Methods in Enzymology 133, p331). -
353, 1986).

[0006] Such a series of chemiluminescence enhancers (enhancers) has the effect of reducing background luminescence generated when an oxidant and luminol are mixed, and at the same time enhancing luminescence by a catalyst such as peroxidase.
As a result, peroxidase can be measured up to several tens of attomoles. Therefore, in CLEIA using peroxidase as a label, the use of this enhancement reaction made it possible to measure with higher sensitivity.

However, in CLEIA or the like for the purpose of measuring a very small amount of a component in a living body, there remains a problem that the measurement sensitivity is insufficient even if a conventional enhancer is used.

[0008]

[Means for Solving the Problems] There are two approaches for solving such problems. One is the enhancement of chemiluminescence, that is, the search for enhancers that enhance the signal emission, and the other is the development of a technique for suppressing background emission to a low level. The enhancer reported so far has the property of suppressing background emission and enhancing signal emission, but the effect of reducing background emission is insufficient even in the practical use of p-indophenol, and It is difficult to say that the degree of luminescence enhancement is sufficient.

As a result of intensive studies to solve these problems, the present inventors have found an enhancer having a sufficient background light emission suppressing effect and a sufficient signal light emission enhancing effect, and completed the present invention. I arrived.

That is, the present invention is based on 2,3 having chemiluminescence.
-Forming a reaction solution containing dihydro-1,4-phthalazinone, an oxidizing agent and heme or peroxidase, causing a luminescence reaction by utilizing the catalytic action of heme or peroxidase, and measuring the reaction to measure the reaction in the reaction solution. Catalyst or a few
-A chemiluminescence method for measuring the presence or abundance of dihydro-1,4-phthalazinone, wherein the luminescence reaction is any one or more compounds selected from the group of 4-substituted phenol derivatives represented by the following chemical formula: The method for enhancing chemiluminescence is characterized in that it is carried out in the presence of The present invention will be described in detail below.

[0011]

[Chemical 2]

(In the formula, X represents a hydrogen atom or a halogen atom, Y represents (1) thiophene having no substituent or a substituent, and the bonding position with the phenol is 2, 3, 4 or 5). Position, (2) means an unsubstituted or substituted pyrrole, and the binding site to the phenol is at the 1-position, or (3) means benzene having a substituent at the 4-position. The binding site with the phenyl is the 1-position, the substituent is a methyl group,
In the enhancer represented by the above chemical formula, X means a hydrogen atom or a halogen atom, but among the halogen atoms, the chlorine element is excellent in the light emission enhancing effect and the background suppressing effect. And is preferable. In the case where Y is an unsubstituted or substituted thiophene, the binding site to the phenol may be the 2, 3, 4 or 5 position of the thiophene skeleton, but the 3 or 4 position is preferred. Examples of the substituent include a methyl group and a chlorine atom, but those without a substituent are preferable. When Y has a pyrrole skeleton, the binding site to the phenol is preferably the 1-position of the pyrrole skeleton. Further, the pyrrole skeleton is preferably unsubstituted. When Y is 4-position-substituted benzene, the binding site with phenol is preferably the 1-position of 4-position-substituted benzene. Examples of the substituent at the 4-position include a methyl group, a carboxyl group and a halogen atom, and the halogen atom is preferably a chlorine atom or a bromine atom.

Chemiluminescent light in the reaction system of the present invention
Examples of 3-dihydro-1,4-phthalazione include luminol and isoluminol, and luminol is particularly preferable. Examples of the oxidizing agent include hydrogen peroxide and salts having hydrogen peroxide in the form of crystal water, such as perboric acid and its salts, percarbonic acid and its salts, and the like, and hydrogen peroxide is preferred. Examples of the oxidation catalyst include plant- or microorganism-derived peroxidase, microperoxidase (microPOD), heme and the like, and horseradish peroxidase (HRP) is particularly preferable.

Chemiluminescent 2,3-dihydro-1,4-
The concentration conditions of each component in the luminescence reaction by phthalazinone, an oxidizing agent and heme or peroxidase include:
Although the effect of the enhancer is recognized in the range of 0.005 mM to 1 mM, 0.05 mM to 0.2 mM is the most effective in suppressing the background and enhancing chemiluminescence. Here, in the reaction solution of the present invention, an amine-based or borate-based buffer solution, preferably Tris / hydrochloric acid buffer solution, boric acid / NaOH buffer solution, is used to adjust the pH to 7.5 to 10, particularly preferably to a pH of around 8.5. It is effective to achieve the above effect.

The concentration of the oxidizing agent can be in the range of 1 μM to 5 mM, preferably 0.5 mM to 2 mM. Chemiluminescent 2,3-dihydro-1,4-such as luminol
The concentration of phthalazin can be in the range of 1 μM to 2 mM, but 0.1 mM to 1 mM is preferable.

The amount of luminescence can be measured with a commercially available or self-made luminometer, but when the enhancer of the present invention is used, luminescence continues for a long time, so that a reaction solution is formed and waited for several seconds to several minutes. After that, it is preferable to adopt a method of integrating the amount of light emission for several tens of seconds to several minutes.

[0017]

According to the method of the present invention, it is possible to obtain a higher effect of enhancing chemiluminescence and a higher effect of suppressing background luminescence, as compared with the conventionally known measuring method using an enhancer. Therefore, in a reaction solution containing chemiluminescent 2,3-dihydro-1,4-phthalazinone, an oxidizing agent and heme or peroxidase, the presence of the catalyst or 2,3-dihydro-1,4-phthalazione in the reaction solution Alternatively, the abundance can be measured with higher sensitivity.

The present invention is particularly suitable for measuring (quantifying) the amount of peroxidase present. Therefore, CLEI using peroxidase-labeled antibody or antigen
When applied to A, it is possible to measure a substance that can be recognized by an antibody (immunoreactive). As a result, it is possible to measure important physiologically active substances, microbial antigens, antimicrobial antibodies, cancer-related markers and the like in vivo.

[0019]

EXAMPLES Examples will be described below to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Enhancer (4- (3-Thienyl
) Synthesis of phenol) 3 g of p-aminophenol was dissolved in 25 ml of thiophene and 10 ml of trifluoroacetic acid and ice-cooled.
4 g of n-Butylnitrite was added dropwise and kept in ice for 1 hour.
Then, the reaction solution was heated and refluxed for 1.0 hour.

100 ml of ethyl acetate was added to the reaction solution, and the organic phase was washed twice with water, twice with NaHCO3 saturated water and twice with NaCl saturated water.
After washing twice, it was dried over MgSO4, concentrated, and purified by silica gel column chromatography (elution solvent; hexane: ethyl acetate = 3: 1).

The target fraction was concentrated and then recrystallized from ethanol / cyclohexane. Since recrystallization was a mixture of 4- (3-Thienyl) phenol and 4- (2-Thienyl) phenol, reverse phase preparative thin-layer chromatography (RP-8F: Merck Ar) was used.
The product was separated and purified by t15388) (developing solvent; H2 O: methanol = 1: 2).

Example 2 Enhancer (4- (1-Pyrroly
l) Synthesis of phenol) 2.18 g of p-aminophenol and 2.64 g of 2,5
-Dimethosytetrahydrofurane and 10 ml of glacial acetic acid were added and the mixture was refluxed for 1 hour and then poured into saturated NaHCO3 water to obtain a precipitate. After filtering the precipitate, the residue was dissolved in ethanol, treated with activated carbon, and then purified with a silica gel column (elution solvent; hexane: ethyl acetate = 4: 1). The target fraction was concentrated and then recrystallized from isopropanol / cyclohexane.

Example 3 Enhancer (4- (4-Tolyl)
Synthesis of phenol) 3 g of p-aminophenol was added to 25 ml of toluene and 1
It was dissolved in 0 ml of trifluoroacetic acid and ice-cooled.
n-Butylnitraite (g) was added dropwise, and the mixture was kept in ice for 1 hour.
Then, the reaction solution was heated and reflaked for 1.5 hours.
100 ml of ethyl acetate was added to the reaction solution, the organic phase was washed twice with water, twice with NaHCO3 saturated water and twice with NaCl saturated water, dried over MgSO4, concentrated, and purified by silica gel column chromatography ( Elution solvent; hexane: ethyl acetate =
4: 1), the target fraction was concentrated and then recrystallized from cyclohexane.

Example 4 Chemiluminescence Measurement The following reagents were used in this example. (1) Luminol sodium salt: Luminol sodium HG (manufactured by Wako Pure Chemical Industries, Ltd.) (2) Hydrogen peroxide: 30% hydrogen peroxide solution (manufactured by Wako Pure Chemical Industries, Ltd.) (3) Peroxidase: horseradish Peroxidase (IC) (manufactured by Toyobo Co., Ltd.) The chemiluminescence reaction in this example was carried out by RIA tube (3
No., manufactured by Eiken Co., Ltd.), and the amount of luminescence was measured using a luminescence reader BLR-301 (manufactured by Aloka). The measurement conditions are as follows. (1) Range: x10 (Kcpm) (2) Sensitivity: x1 (3) Time Const. : 0.1 (4) Wait time: 60 sec. (5) Integ. time: 60 sec. A reaction solution having the following composition was prepared, 0.2 ml was dispensed into a tube, kept at 37 ° C. for 4 minutes, set in a luminescence reader, and the background luminescence amount was measured under the above measurement conditions. . Then an absolute amount of 250 attomole horseradish peroxidase solution (10 μl) was added,
The amount of light emission for 1 minute was measured.

When no enhancer was added as a negative control and when 4-Iodophenol, which is known as an enhancer as a positive control, was added, the background emission and signal emission were also measured. In addition, 4 carboxy4'hydroxybiphen
yl and 4 Bromo4'hydroxyphenyl
Is an enhancer having the structure of the present invention.

Composition of reaction solution (1) 0.1 M boric acid / NaOH buffer solution (pH 8.
5) (2) 1.0 mM hydrogen peroxide (3) 0.5 mM luminol-Na salt (4) 0.1 mM CyDTA * (5) 0.05 mM to 1 mM of various enhancers
(Including those synthesized in Examples 1 to 3) * CyTA: trans-1,2-Diaminocyclohexane-N, N, N ', N'-tetr
The results of aacetic acid are shown in Table 1. According to Table 1, in the novel enhancer described in the present invention as compared with the conventionally known 4-Iodophenol, the background is reduced but the amount of light emission is increased, and the S / N ratio is significantly increased (10 It can be seen that it will be improved from double to 57 times. Of the five enhancers of the present invention, 4-
(3-Thienyl) phenol, 4- (1-Py)
rrollyl) phenol and 4- (4-Toly)
l) The three enhancers of phenol are especially S / N
The improvement in the ratio is remarkable.

[0028]

[Table 1]

Example 5 Comparison of Detection Limits of Horseradish Peroxidase A horseradish peroxidase dilution solution (1 mg / ml gelatin, 0.1 M boric acid-
Dissolve in NaOH, 0.1 mM CyDTA, pH 8.5) and use the same diluent to prepare 10-6, 10-7, 10-8 (1 for each).
ng / ml, 100 pg / ml, corresponding to 10 pg / ml). Luminescent solution containing enhancer 200μ
10 μl of the horseradish peroxidase solution for 1
1 was added, the amount of luminescence was measured, and the S / N ratio at each concentration was determined. The measurement conditions and the like were the same as in Example 4.

The results are shown in Table 2. According to Table 2, the three enhancers of the present invention- (4- (3-Thienyl) p
henol, 4- (1-Pyrrolyl) pheno
1 and 4- (4-Tolyl) phenol), HRP can be detected to a concentration lower than that of 4-Iodophenol by one digit or more.

[0031]

[Table 2]

Example 6 TSH CLEIA A monoclonal antibody against TSH (human thyroid hormone) was digested with pepsin to form F (ab) '2, which was then purified by gel filtration and hydrophobic chromatography and reduced with dithiothreitol. Made into a Fab. The Fab-fragment was purified by gel filtration, and SMCC (Succ
inimidyl4- (N-maleimidomethyl) cyclohexane-1-carboxy
3) Add horseradish peroxidase modified in late)
After reacting at 7 ° C for 1 hour, the conjugate fraction was separated by gel filtration. The collected conjugate is UV2
After measuring the absorption at 80 nm, it was stored at 4 ° C.

Twelve magnetic beads (diameter 1.4 mm) on which an anti-TSH F (ab) 2 -conjugated antibody was immobilized were placed in a reaction cup, and TSH zero serum or a known degree (4.81 μm) was placed in this.
(IU / ml) serum (100 μl) was added, and the mixture was reacted at 37 ° C. for 5 minutes while stirring, followed by B / F separation and washing once (washing solution composition: 0.1 M NaCl, 50 mM Tris buffer, 0.5% Tween 20). After pH 8.5), 100 μl of the peroxidase-labeled conjugate (diluted 200 times with a diluting solution) prepared by the above-mentioned method was added, and further reacted at 37 ° C. for 10 minutes. Next, after B / F separation and washing five times, a luminescence detector (BLR manufactured by Aloka Co., Ltd.
-301) and set it to the above-mentioned luminescent reagent (enhancer-
4-Pyrrolylphenol (as a control 4-Iodopheno
l)) was added thereto, and after waiting for 1 minute, the luminescence amount for 1 minute was integrated, and the lower limit of detection was determined from the values of zero serum and positive serum.

Table 3 shows the results when the same operation was performed three times.
Shown in. According to Table 3, the enhancer (4-
(1-Pyrrolyl) phenol) to CLEIA
It can be seen that, when used in, the lower limit of detection can be measured as compared with 4-Iodophenol.

[0035]

[Table 3]

Claims (1)

[Claims] 1. 2,3-Dihydro-having chemiluminescent property
A reaction solution containing 1,4-phthalazinone, an oxidant and heme or peroxidase is formed, a luminescent reaction is caused by utilizing the catalytic action of heme or peroxidase, and the reaction is measured to measure the catalyst or 2 in the reaction solution. , 3-dihydro-
In the chemiluminescence method for measuring the presence or abundance of 1,4-phthalazione, the luminescence reaction is performed in the presence of any one or more compounds selected from the 4-position substituted phenol derivative group represented by the following chemical formula. The method for enhancing chemiluminescence, which is characterized in that [Chemical 1] (In the formula, X is a hydrogen atom or a halogen atom, and Y is
(1) means thiophene which is unsubstituted or has a substituent,
The bonding position with the phenol is 2, 3, 4 or 5 position,
(2) means pyrrole which is unsubstituted or has a substituent,
The binding site with the phenol is at the 1-position, or (3) means benzene having a substituent at the 4-position, the binding site with the phenol is at the 1-position, and the substituent is a methyl group, halogen or Indicates that it is a carboxyl group)