JPH0749311A - Composition and highly sensitive emission analysis using the same - Google Patents

Abstract

PURPOSE:To enable the realization of a composition suitable for emission analysis highly stable with high emission performance by arranging a composition composing a three-dimensional structure hydrophilic compound having a void inside and a chemical luminescence intensifier as selected from luminol, isoluminol and a derivative thereof. CONSTITUTION:A hydrophilic compound 2 has a proper void 1 inside a three- dimensional structure and encloses other compounds therein. alpha, beta or gammacyclodexitrine, 18-crown-6,15-crown-5, paracyclophane and the like are utilized preferably. A chemical luminescence intensifier is provided to enhance the quantity of light in an emission reaction of luminol, isoluminol and a derivative thereof. 2-hydroxy-9-fluorenone, 2-phenyl-6-hydroxybenzoxazol. N,N,N',N'- tetramethylbenzine and the like are utilized preferably. A composition can be produced by mixing the hydrophilic compound and the chemical luminescence intensifier in water and heating the mixture as required.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition suitable for use in a highly sensitive emission analysis method.

More specifically, in the fields of clinical inspection, food inspection, environmental analysis, animal and plant inspection, manufacturing process control inspection, etc.
It is preferably used when performing chemiluminescence measurement using an enzyme reaction, an antigen-antibody reaction, a nucleic acid hybrid reaction, or the like.

[0003]

2. Description of the Related Art Luminol, isoluminol or their derivatives [hereinafter referred to as chemiluminescent DPD (abbreviated as chemiluminescent DPD (2,3-dihydro-1,4-phthalazinedione)] by peroxidase is used as a luminescent reaction in an immunoassay. , Elastase analysis, glucose analysis, oxidant analysis, etc.

It is known that it is effective to add the following chemiluminescence enhancer to the reaction system in order to improve the luminescence intensity of the above-mentioned luminescence reaction.

6-Hydroxybenzothiazole (JP-A-59-500252) Certain phenols (JP-A-59-171839) Certain aromatic amines (JP-A-59-171839) The chemiluminescence enhancer is often used by dissolving it in an organic solvent such as DMSO and then dispersing it in water or a buffer solution, and crystals are precipitated during long-term storage, resulting in a decrease in luminescence intensity. Had. Further, since it is used in coexistence with a buffering agent and other chemicals, it has a drawback that it is susceptible to chemical changes.

[0006]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned drawbacks of the prior art, and provides a composition suitable for high-sensitivity emission analysis which is excellent in stable emission performance, and a high sensitivity using the same. It is intended to provide an emission analysis method.

[0007]

In order to achieve the above object, the present invention has the following constitution.

That is, the present invention provides a composition comprising a hydrophilic compound having a three-dimensional structure having pores therein and a chemiluminescence enhancer of a compound selected from luminol, isoluminol and derivatives thereof, and the presence of the composition. The present invention relates to high-sensitivity emission analysis, which is characterized in that

The hydrophilic compound in the present invention is a compound having pores of an appropriate size inside the three-dimensional structure, in which other compounds are included. An example of the structure of the hydrophilic compound is shown in FIGS. 1 and 2. Specific examples thereof include cyclodextrin, crown compounds and cyclophane. Among them, cyclodextrin is preferably used because of its high water solubility.

Further, specific compounds include, as cyclodextrins, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and 2,6-di-
O-methyl-α-cyclodextrin, 2,6-di-O
-Methyl-β-cyclodextrin, 2,3,6-tri-O-methyl-β-cyclodextrin, 6-O-α-
D-glucosyl-α-cyclodextrin, 6-O-α
-D-glucosyl-β-cyclodextrin, 6-O-
α-maltosyl-α-cyclodextrin, 6-O-α
-Maltosyl-β-cyclodextrin and 2,3,3
As crown compounds such as 6-tri-O- (2-hydroxypropyl) -β-cyclodextrin, 18-crown-6, 15-crown-5, dibenzo-14-crown-4, dibenzo-18-crown-6. , Dibenzo-24-crown-8, dibenzo-15-crown-
5, dicyclohexyl-18-crown-6, dibenzo-30-crown-10 and cryptand [2.2.
2] and the like, examples of cyclophane include paracyclophane.

The chemiluminescence enhancer used in the present invention is not particularly limited as long as it increases the amount of light by the luminescence reaction of luminol, isoluminol and its derivatives.
Phenols and aromatic amines are preferably used. Specific compounds include phenols 2
-Hydroxy-9-fluorenone, 4-hydroxy-3
-[-(4-hydroxyphenyl) -1-oxo-2-
Propenyl] -2H-1-benzopyran-2-one, 6
-Hydroxybenzoxazole, 2-fluoromethyl-6-hydroxybenzoxazole, 2-chloromethyl-6-hydroxybenzoxazole, 2-bromomethyl-6-hydroxybenzoxazole, 2-methoxycarbonyl-6-hydroxybenzoxazole, 2
-Ethoxycarbonyl-6-hydroxybenzoxazole, 2-propoxycarbonyl-6-hydroxybenzoxazole, 2-phenyl-6-hydroxybenzoxazole, 2- (2-methylphenyl) -6-hydroxybenzoxazole, 2- (3 -Methylphenyl) -6-hydroxybenzoxazole, 2- (4-
Methylphenyl) -6-hydroxybenzoxazole, 2- (2-fluorophenyl) -6-hydroxybenzoxazole, 2- (2-chlorophenyl) -6-
Hydroxybenzoxazole, 2- (2-bromophenyl) -6-hydroxybenzoxazole, 2- (2
-Iodophenyl) -6-hydroxybenzoxazole, 2- (2,4-dichlorophenyl) -6-hydroxybenzoxazole, 2- (2,5-dichlorophenyl) -6-hydroxybenzoxazole, p-iodophenyl, p- As aromatic amines such as phenylphenol, p-hydroxycinnamic acid and 6-hydroxybenzothiazole, N, N, N ', N'-tetramethylbenzidine, 1-aminopyrene, 6-aminochrysene, 4-aminophenyl Examples thereof include ether, 2-aminofluorene, 3-aminofluoranthrene, 4-methoxyaniline, 3-ethylaniline and 4-ethylaniline.

The composition of the present invention can be prepared by mixing a hydrophilic compound and a chemiluminescence enhancer in water and heating the mixture if necessary. More specifically, the hydrophilic compound is preferably used in an amount of 1 to 100 times the mol of the chemiluminescence enhancer, and the reaction temperature in preparing the composition is preferably in the range of 0 to 50 ° C. Also, the composition can be made in about 3 to 48 hours.

The peroxidase used in the present invention is
Although not particularly limited, plant peroxidase such as horseradish peroxidase is preferable.
Examples of peroxidase derivatives include peroxidase antibody conjugates, peroxidase antigen conjugates, peroxidase streptavidin conjugates and biotin-conjugated peroxidases. The antibody used here is not particularly limited,
For example, in addition to intact IgG and IgM, Fab and F
Partial structures such as (ab) ₂ can also be preferably used.

The antigen is not particularly limited, and low molecular weight haptens such as fluorescein and steroid hormones and high molecular weight substances such as polypeptides, proteins and polysaccharides can be used.

In the case of the nucleic acid hybrid reaction, DNA and RNA having 10 or more bases are preferably used and may be linear or circular.

Hydrogen peroxide is preferably used as the oxide, but perborate and hypochlorite can also be used.

Chemiluminescent DP used in the practice of the present invention
D is luminol, isoluminol, N- (4-aminobutyl) -N-ethylisoluminol, or an antigen, antibody, nucleic acid or the like bound thereto, and luminol is particularly preferable.

The composition of the present invention can be used in a method for measuring a substance utilizing a luminescence reaction consisting of peroxidase, oxide and chemiluminescent DPD, and is particularly preferably used for measurement by an enzyme immunoassay and a DNA hybrid reaction.

[0019]

EXAMPLES The present invention will be specifically described below with reference to examples.

The following reagents and devices were used in the examples.

Reagent Tris [hydroxymethyl] aminomethane (Trisma bas
e) and tris [hydroxymethyl] aminomethane hydrochloride (Trisma HCl) are available from Sigma Chemical Co.
Manufactured by luminol (5-amino-2,3-dihydro-
1,4-phthalazinedione) and Tween 20 are manufactured by Tokyo Kasei Co., Ltd., and horseradish peroxidase (HR
P) is Boehringer Mannheim
Gmbh), powdered PBS manufactured by Nissui Pharmaceutical Co., Ltd., bovine serum albumin (BSA) manufactured by Seikagaku Corporation, and γ-
Cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin and 2,3,6-tri-O- (2-
Hydroxypropyl) -β-cyclodextrin was manufactured by Wako Pure Chemical Industries, Ltd., disodium hydrogen phosphate was manufactured by Kishida Co., Ltd., and citric acid was manufactured by Nacalai Tesque Co., Ltd.

Breast cancer associated antigen (CA15-3), anti-CA1
5-3 antibody (mouse monoclonal antibody) pancreatic cancer-related antigen (CA19-9) and anti-CA19-9 antibody (mouse monoclonal antibody) are available from Saint Core (Malvern, USA).
The liver cancer-related antigen (AFP) and anti-AFP antibody (mouse monoclonal antibody) manufactured by Dako Co., Ltd. were used. Two
-Phenyl-6-hydroxybenzoxazole is 4-
Aminoresonol hydrochloride and methyl orthobenzoate were condensed and cyclized, and then recrystallized from acetone / water were used. The luminol sodium salt used was obtained by reacting luminol in sodium hydroxide and recrystallizing the product with water. As the hydrogen peroxide solution, pharmacopoeia oxidol was used.

Anti-CA1 labeled with horseradish peroxidase
5-3 antibody, horseradish peroxidase-labeled anti-CA1
9-9 antibody and horseradish peroxidase labeled anti-A
The FP antibody is a maleimide hinge method [Yoshitake.S.at al:
J. Biochem., 92, 1413-1424 (1982)], and separated and purified by Pharmaciasha FPLC using a hydroxyapatite column (Mitsui Toatsu, HCA-column, φ7.6 mm × L100 mm).

Analytical device Luminometer LB95 manufactured by Berthold Co.
0 or Lumileizer manufactured by Toray Industries, Inc.

Example 1 HRP labeled anti-CA15-using luminol and composition
Luminescence Assay of 3 Antibody 500 ml of ultrapure water 649 mg (0.5 mmol) of ultrapure water was placed in a 1-liter Erlenmeyer flask and stirred for 5 hours. 3.7 mg (0.0175 mmol) of 2-phenyl-6-hydroxybenzoxazole was added to the solution, and the mixture was stirred at room temperature for one day to dissolve the crystals completely.

Into the clathrate solution, 100 mg (1 mmol) of luminol sodium salt, 4.36 g of Trismabase,
2.21 g Trisma HCl and "Tween 20" 15
After adding 0 μl and stirring for several hours, it was used as a reagent for luminescence.

On the other hand, HRP-labeled anti-CA15-3 antibody was added to a phosphate buffer containing 0.25% bovine serum albumin (P
BS), 10ng / ml, 2ng / ml, 0ng / ml
(PBS containing 0.25% bovine serum albumin) was prepared, and each solution was used as a standard CA15-3 solution.

Standard CA15-for plastic cuvettes
50 μl of 3 solutions, 200 μl of reagent for luminescence, 100 μl of 0.9 mM hydrogen peroxide / disodium hydrogen phosphate / citrate buffer (10 mM, pH 4.0) were added, and a luminescence reaction was performed to measure luminescence intensity after 3 minutes. did. The results are shown in Table 1.

Examples 2 and 3 Instead of the γ-dextrin of Example 1, 2,6-di-O was used.
-Methyl-β-cyclodextrin (Example 2), 2,
3,6-tri-O- (2-hydroxypropyl) -β-
The emission intensity was measured in the same manner as in Example 1 except that cyclodextrin (Example 3) was used, and the results are shown in Table 1.

[0030]

[Table 1] Example 4 CA19-9 Antibody Luminescence Assay Using the Composition The CA19-9 antigen solution was diluted with phosphate buffer (PBS) containing 0.25% bovine serum albumin to 300 U /
ml, 200U / ml, 100U / ml, 50U / ml, 25U
/ Ml, 0 U / ml (PBS containing 0.25% bovine serum albumin) to prepare a solution having a concentration of CA19
-9 solution.

20 μl of standard CA19-9 solution of each concentration
Dispense each into a plastic cuvette and add 150 μl of CA19-9 antibody-immobilized magnetic microbeads to 37 ° C.
And reacted for 10 minutes. Magnetic microbeads are attracted with a magnet, the solution is discarded, and then P containing 0.03% Tween 20 is added.
Washed with BS.

After washing, peroxidase-labeled anti-CA19
300 μl of −9 antibody was added and reacted at 37 ° C. for 15 minutes. After completion of the reaction, the plate was washed 3 times with PBS containing 0.03% Tween 20.

After washing, 200 μ of the reagent for luminescence of Example 1
l, 0.9 mM hydrogen peroxide / disodium hydrogen phosphate
100 μl of citrate buffer (10 mM, pH 4.0) was added, and the luminescence intensity after 5 minutes was measured. The results are shown in Table 2 and FIG.

Example 5 AFP antigen solution in place of the CA19-9 antigen solution of Example 4, 300 U / ml and 25 U / ml standard CA19-.
350 U / ml and 10 U / ml standard AFP solution instead of 9 solution, CA19-9 antibody-immobilized magnetic microbeads and peroxidase labeled anti-CA19-9 antibody instead of AFP antibody-immobilized magnetic microbeads and peroxidase labeled anti-AFP The luminescence intensity was measured by the same method as in Example 4 except that the antibody was used, and the results are shown in Table 2 and FIG.

[0035]

[Table 2]

[0036]

EFFECT OF THE INVENTION By using the composition of the present invention for chemiluminescence analysis, highly sensitive and rapid measurement of a substance becomes possible.

[Brief description of drawings]

FIG. 1 is a drawing showing a structure of a hydrophilic compound of the present invention.

FIG. 2 is a drawing showing a structure of a hydrophilic compound of the present invention.

FIG. 3 shows emission intensities (relative values) obtained in Examples 4 and 5.

[Explanation of symbols]

 1: Void 2: Hydrophilic compound 3: Result of Example 4 4: Result of Example 5

Claims (9)

[Claims] 1. A composition comprising a hydrophilic compound having a three-dimensional structure having internal pores, and a chemiluminescence enhancer selected from luminol, isoluminol and derivatives thereof. 2. The hydrophilic compound is selected from cyclodextrin, crown compounds and cyclophane.
The composition as described. 3. The hydrophilic compound is α-cyclodextrin,
β-cyclodextrin, γ-cyclodextrin,
2,6-di-O-methyl-α-cyclodextrin,
2,6-di-O-methyl-β-cyclodextrin,
2,3,6-tri-O-methyl-β-cyclodextrin, 6-O-α-D-glucosyl-α-cyclodextrin, 6-O-α-D-glucosyl-β-cyclodextrin, 6-O A selected from -α-maltosyl-α-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin and 2,3,6-tri-O- (2-hydroxypropyl) -β-cyclodextrin. 1
The composition as described. 4. The composition according to claim 1, wherein the hydrophilic compound is γ-cyclodextrin. 5. The composition according to claim 1, wherein the chemiluminescence enhancer is selected from phenols and aromatic amines. 6. The chemiluminescence enhancer is 2-hydroxy-9-.
Fluorenone, And the composition according to claim 1, which is selected from the oxazole derivatives represented by the general formula [1]. [Chemical 2] [In the formula, R represents hydrogen or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms or a phenyl group. ] 7. The composition according to claim 1, wherein the chemiluminescence enhancer is 2-phenyl-6-hydroxybenzoxazole. 8. (a) Peroxidase or peroxidase derivative, (b) oxide and (c) luminol,
A chemiluminescent reaction resulting from the reaction of isorminol or a derivative thereof is used to detect or measure a substance, and the luminescent reaction is carried out in the presence of the composition according to claim 1. Luminescence analysis method. 9. The high-sensitivity emission analysis method according to claim 8, which is carried out in the presence of the composition according to any one of claims 2 to 7.