JP2002350352A - Chemiluminescent enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new chemiluminescent enhancers superior to know in chemiluminescent effect. SOLUTION: The chemiluminescent enhancers are composed of compounds containing anion exchange groups treated with polycarboxylic acids or their salts. For example, polyacrylic acid is preferred as the polycarboxylic acid and trimethylbenzylammonium-poly(styrene-divinylbenzen) and agarose gel having trimethylammonium groups are preferred as the compound containing anion exchange groups.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence enhancer used when a chemiluminescent substrate emits chemiluminescence under the action of an enzyme.

[0002]

2. Description of the Related Art Chemiluminescence measurement using a chemiluminescence reaction in which an enzyme such as alkaline phosphatase is caused to act on a chemiluminescent substrate such as 1,2-dioxetane to cause chemiluminescence is carried out in a sample to be measured. The presence or concentration of
It can be measured with high sensitivity, and viruses such as HIV and HCV,
It is widely used to measure other trace components in the body.

As a chemiluminescence enhancer for enhancing chemiluminescence in the above chemiluminescence reaction, a water solution containing a quaternary ammonium group such as poly (vinylbenzyl (benzyldimethylammonium) chloride (BDMQ) or polyvinylbenzyltributylammonium chloride (TBQ) is used. (JP-A-3-53897 and JP-A-4-124185) By using these chemiluminescence enhancers, the chemiluminescence intensity is increased about 20 to 30 times.

[0004]

Although known chemiluminescence enhancers are effective, if a chemiluminescence enhancer having a better chemiluminescence enhancement effect is provided, the chemiluminescence required for obtaining the same chemiluminescence intensity can be obtained. It is needless to say that the amount of the luminescent substrate, the test material, other constituent reagents, and the like can be reduced, or the time required for the measurement can be shortened.

Accordingly, an object of the present invention is to provide a novel chemiluminescence enhancer having a better chemiluminescence enhancement effect than known chemiluminescence enhancers.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a substance containing an anion-exchange group treated with a polycarboxylic acid or a salt thereof exhibits a high chemiluminescence enhancing effect, and developed the present invention. completed.

That is, the present invention provides a chemiluminescence enhancer comprising an anion exchange group-containing substance treated with a polycarboxylic acid or a salt thereof.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As described above, the chemiluminescence enhancer of the present invention is obtained by treating a substance containing an anion exchange group with a polycarboxylic acid or a salt thereof. Here, “polycarboxylic acid” means a compound having a plurality of carboxyl groups in one molecule. Preferred examples of the polycarboxylic acid include a polymer of an unsaturated carboxylic acid or a salt thereof, a copolymer containing an unsaturated carboxylic acid or a salt thereof,
Aliphatic polycarboxylic acids obtained by substituting at least 2 carboxyl groups or salts thereof with up to 20 alkanes or hydroxyalkanes can be mentioned.

Here, a preferred example of the unsaturated carboxylic acid is a vinyl carboxylic acid having a vinyl group. The vinyl carboxylic acid means a carboxylic acid having a vinyl group, and as a preferred example, the following general formula
Those represented by [I] can be mentioned, and particularly preferred examples include acrylic acid and methacrylic acid.

[0010]

Embedded image (Wherein, in the formula [I], R is hydrogen or an alkyl group having 1 to 6 carbon atoms)

Preferred examples of the unsaturated carboxylic acids include unsaturated aliphatic polycarboxylic acids in which a carboxyl group is substituted by an alkene having 2 to 20 carbon atoms. A preferred example of the unsaturated aliphatic polycarboxylic acid is maleic acid.

The above-mentioned unsaturated carboxylic acids or salts thereof can be polymerized singly or copolymerized by combining them to obtain the polycarboxylic acids or salts thereof used in the present invention. Alternatively, one or more unsaturated carboxylic acids or salts thereof may be copolymerized with other copolymerizable monomers to obtain the polycarboxylic acids or salts thereof used in the present invention. In this case, since what is necessary in the present invention is that the polycarboxylic acid or a salt thereof has two or more carboxyl groups or a salt thereof, other copolymerizable monomers are not limited at all. Examples thereof include vinyl compounds such as styrene, vinylpyrrolidone, and alkyl esters of vinylcarboxylic acids represented by the general formula [I] (the alkyl portion preferably has 1 to 6 carbon atoms).

When a polymer or copolymer of an unsaturated carboxylic acid or a salt thereof is used as a polycarboxylic acid, the average molecular weight (number average molecular weight) of the polymer or copolymer is not particularly limited, but may be 2,000,000 to 2,000,000. Degree is preferable, and about 5000 to 200,000 is more preferable. Further, the number of carboxyl groups or salts thereof in one molecule is not particularly limited, but is preferably about 20,000, more preferably about 50,000.

Preferred examples of the polycarboxylic acid include aliphatic polycarboxylic acids such as citric acid in which an alkane or hydroxyalkane having 2 to 20 carbon atoms is substituted with two or more carboxyl groups or salts thereof. Can be mentioned.

Preferred examples of the polycarboxylic acid salt that can be used in the present invention include alkali metal salts such as sodium and potassium.

Preferred specific examples of the above-mentioned polycarboxylic acids include polyacrylic acid, polysodium methacrylate, poly (acrylic-maleic acid), poly (1-vinylpyrrolidone-acrylic acid), and poly (methylvinyl-maleic). Acid), but is not limited thereto.

The anion exchange group-containing substance used in the chemiluminescence enhancer of the present invention includes, for example, polymers having tertiary or quaternary ammonium groups, latex particles having tertiary or quaternary ammonium groups, tertiary or quaternary ammonium groups. Liposomes having a quaternary ammonium group, cationic amino acid polymers, cationic amino acid copolymers, and the like can be used.
Examples of the polymer having a tertiary or quaternary ammonium group include, for example, an anion exchanger having a tertiary or quaternary ammonium group. The polymer is not particularly limited, but a polymer having a quaternary ammonium group is preferable. Anion exchangers having a quaternary ammonium group are well known and various ones are commercially available. In the present invention, these commercially available anion exchangers can be preferably used. Examples of the tertiary ammonium group include an ammonium group formed from a diethylaminoethyl group, a dimethylaminoethyl group, or the like. Examples of the quaternary ammonium group include a diethyl- (2-hydroxypropyl) aminoethyl group, a triethylaminoethyl group, and a trimethylaminoethyl group. Examples of the polymer include polystyrene, polystyrene copolymer, agarose gel, cross-linked dextran gel, and cellulose. Furthermore, examples of the amino acid polymer include polylysine and polyarginine. Examples of the amino acid copolymer include copolymers of amino acids such as lysine and arginine.

Although the shape of the anion exchanger is not particularly limited, the anion exchanger is in the form of a powder or an emulsion in order to sufficiently bring the polycarboxylic acid into contact with the anion exchanger. Is particularly preferred.

Examples of preferred anion exchangers include trimethylbenzylammonium-poly (styrene-divinylbenzene) (trade name "AG1 Resins", commercially available from Bio-Rad), dimethylethanolbenzylammonium-poly (styrene-divinylbenzene) ) Product name "AG2 Resin
s ", commercially available from Bio-Rad), agarose gel with trimethylammonium groups (trade name" Q Sepharose ", Ph
armacia Biotech), cross-linked dextran gel having diethyl (2-hydroxypropyl) aminoethyl group (trade name “QAE Sephadex”, Pharmacia Bi
otech) and cellulose having a triethylaminoethyl group (TEAE cellulose), but are not limited thereto.

The chemiluminescence enhancer of the present invention is, for example, one obtained by treating the above anion exchanger with the above polycarboxylic acid or a salt thereof. Here, “treatment” means contacting a molecule of a polycarboxylic acid or a salt thereof with an anion exchanger molecule, which is a solution of a polycarboxylic acid or a salt thereof or a liquid polycarboxylic acid or a salt thereof. Can be easily performed by contacting with an anion exchanger. When a solution of a polycarboxylic acid or a salt thereof or a liquid polycarboxylic acid or a salt thereof is brought into contact with an anion exchanger, this is preferably carried out by immersing the anion exchanger in the solution or the liquid and immersing the mixture in the solution or liquid. The reaction can be carried out at room temperature for at least 30 minutes, preferably for about 1 to 5 hours, by standing or stirring. Alternatively, when the polycarboxylic acid or a salt thereof is a polymer or copolymer of an unsaturated polycarboxylic acid or a salt thereof, by performing the polymerization or copolymerization in the presence of an anion exchanger, The polymer or copolymer of the generated unsaturated polycarboxylic acid or a salt thereof can be brought into contact with an anion exchanger, and the above-mentioned "treatment" can be performed by such a method. A polycarboxylic acid-treated anion exchanger obtained by such a method is also commercially available, and such a commercially available product can also be preferably used. For example, an ion delay resin (trade name “AG11 Resins”, commercially available from Bio-Rad) obtained by polymerizing acrylic acid with trimethylbenzylammonium-poly (styrene-divinylbenzene) can be preferably used.

By the above treatment, the carboxyl group of the polycarboxylic acid or a salt thereof or the salt thereof is combined with the anion exchanger.
It is presumed that the quaternary or quaternary ammonium group ion-bonds to form an ion pair, and this ion pair is presumed to exhibit a chemiluminescence enhancing effect.

The above chemiluminescence enhancers can be used alone or in combination.

The chemiluminescence reaction itself to which the chemiluminescence enhancer of the present invention is applied is known. Preferred examples of the enzyme for performing the chemiluminescence reaction include acid phosphatase, alkaline phosphatase and galactosidase. These enzymes can be purified from animals or plants by known methods, and are also commercially available. Commercial products can also be preferably used. These enzymes may be free or bound to other substances such as antigens, antibodies or haptens.

As the substrate for the chemiluminescence reaction, any chemiluminescence substrate used in conventional chemiluminescence measurement can be used. A preferred group of chemiluminescent substrates include 1,2-dioxetane derivatives represented by the following general formula [II].

[0025]

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(Wherein, in the formula [II], R ¹ is a hydrogen atom or a halogen atom; R ² is an alkyl group having 1 to 6 carbon atoms or a cycloalkyl group having 3 to 6 carbon atoms; Ar ¹ is a phenylene group or a naphthylene A group; R ³ is -OPO ₃ ^2-・ 2M ⁺ (where M represents a sodium atom, a potassium atom or NH ₄ ) or a galactosyl group.

In the above general formula [II], R ¹ is a hydrogen atom or a halogen atom such as chlorine, bromine or iodine atom. R ² has 1 to 1 carbon atoms such as a methyl, ethyl, propyl, butyl, pentyl or hexyl group.
A linear or branched alkyl group having 6 carbon atoms; or a cycloalkyl group having 3 to 6 carbon atoms such as a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group. Ar ¹ is an aromatic hydrocarbon group such as a phenylene group or a naphthylene group.

As a specific example of the chemiluminescent substrate represented by the general formula [II], 3- [4-methoxyspiro (1,2-dioxetane-3,
2'-tricyclo [3,3,1,1 ^3,7 ] decane) -4-yl] phenyl phosphate disodium salt (hereinafter referred to as “AMPPD”), 7- [4-
Methoxyspiro (1,2-dioxetane-3,2'-tricyclo [3,
3,1,1 ^3,7 ] decane) -4-yl] naphthyl-2-yl phosphate disodium salt, 3- [4-methoxyspiro (1,2-dioxetane-
3,2′-Tricyclo [3,3,1,1 ^3,7 ] decane) -4-yl] phenyl β-D-galactopyranose, 3- [4-methoxyspiro [1,
2-dioxetane-3,2 '-(5'-chloro) tricyclo [3,3,1,1
^3,7 ] decane) -4-yl] phenyl phosphate disodium salt, 3
- [4-methoxy-spiro [1,2-dioxetane-3,2 '- (5'-chloro) tricyclo [3,3,1,1 ^3,7] decane) -4-yl] phenyl β
-D-galactopyranose and the like.

Of these, AMPPD is preferably used as a chemiluminescent substrate, since it is widely known and practically used in the field of clinical test drugs.

As the chemiluminescent substrate, the following general formula [III]
The 1,2-dioxetane derivative represented by

[0031]

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(However, in the formula [III], R ⁴ , R ⁵ and R
⁶ is independently an alkyl group having 1 to 6 carbon atoms or a cycloalkyl group having 3 to 6 carbon atoms; Ar ² is any one of the following formulas (A) to (C). )

[0033]

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(However, R ⁷ is -OPO ₃ ^2- · 2M ⁺ (however,
M represents a sodium atom, a potassium atom or NH ₄ ) or a galactosyl group; R ⁸ represents a hydrogen atom, an alkyl group or a substituted alkyl group having 1 to 20 carbon atoms, a cycloalkyl group or a substituted cycloalkyl group having 3 to 20 carbon atoms. , 5
20 is a 20-membered aryl, an alkoxyalkoxyl group having 1 to 20 carbon atoms, an alkoxyalkoxyalkoxyl group having 3 to 20 carbon atoms, an aryloxy group having 6 to 20 carbon atoms or an aralkyloxy group having 7 to 20 carbon atoms; Oxygen or sulfur atom),

[0035]

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(However, R⁷Is defined above in formula (A)
W means C-R⁹(However, R ⁹Is hydrogen atom, charcoal
An alkyl group having 1 to 20 or a substituted alkyl group,
A cycloalkyl group or a substituted cycloalkyl having a prime number of 3 to 20;
Alkyl group, 5-20 membered aryl, 1-20 carbon atom
Alkoxy alkoxyl group, alkoxy having 3 to 20 carbon atoms
Alkoxyalkoxyl group, aryl having 6 to 20 carbon atoms
An oxy group or an aralkyloxy group having 7 to 20 carbon atoms)
Represents a nitrogen atom; X represents an oxygen atom or a sulfur atom)

[0037]

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(Wherein, R ⁷ has the same meaning as in formula (A); Y represents an oxygen atom, a sulfur atom or NR ¹⁰ ; Z is a hydrogen atom, an alkyl group having 1 to 20 carbon atoms or a substituted alkyl group) A cycloalkyl group having 3 to 20 carbon atoms or a substituted cycloalkyl group, a 5- to 20-membered aryl, -OR ¹¹ , -SR ¹² or a general formula (D)

[0039]

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(Provided that R ¹⁰ is a hydrogen atom and has 1 to 1 carbon atoms)
20 alkyl groups or substituted alkyl groups, having 3 to 3 carbon atoms
20 cycloalkyl groups or substituted cycloalkyl groups, 5 to 20 membered aryls, hydroxyl groups, 1 carbon atoms
R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each independently represent a hydrogen atom, a carbon atom of 1 to 20 alkoxyl groups, an alkoxyalkoxyl group having 2 to 20 carbon atoms or an alkoxyalkoxy alkoxyl group having 3 to 20 carbon atoms; To 20 alkyl groups or substituted alkyl groups, cycloalkyl groups or substituted cycloalkyl groups having 3 to 20 carbon atoms,
Represents a 20-membered aryl (provided that (i) R ¹⁰ and R ¹¹ , (ii) R ¹⁰ and R ¹² , (iii) R ¹⁰ and R ¹³ ,
Or (iv) R ¹³ and R ¹⁴ may be taken together to form a ring, and this ring may contain two or more heterocycles)).

In the 1,2-dioxetane derivative represented by the general formula [II], the alkyl group having 1 to 6 carbon atoms is a straight-chain or branched methyl, ethyl, propyl, butyl, pentyl or hexyl group. And the cycloalkyl group having 3 to 6 carbon atoms may be a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.

In the general formulas (A), (B), (C) and (D), the alkyl group having 1 to 20 carbon atoms or the substituted alkyl group is a straight-chain or branched alkyl group having 1 to 20 carbon atoms.
20 unsubstituted alkyl groups, ie, methyl, ethyl,
Propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,
A heptadecyl, octadecyl, nonadecyl or eicosanyl group, or a linear or branched alkyl group having 1 to 20 carbon atoms, having 1 to 5 hydroxyl groups and 1 to 5 carbon atoms having 1 to 5 carbon atoms; 20 alkoxyl groups, 1 or more and 5 or less C2-20 alkoxyalkoxyl groups, 1 or more and 5 or less C3-20 alkoxyalkoxyalkoxyl groups, and / or 1 or more and 5 or less It may be a substituted alkyl group substituted by a 5-20 membered aryl group. Here, carbon number 1-2
0 alkoxyl group, C2-20 alkoxyalkoxyl group and C3-20 alkoxyalkoxyalkoxyl group are methoxy, ethoxy, propoxy,
It may be a linear or branched alkoxy-containing group, such as butoxy, pentoxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy. The 5- to 20-membered aryl group substituted on the alkyl group is an aromatic hydrocarbon group having 6 to 20 carbon atoms such as phenyl and naphthyl; or nitrogen, oxygen and furyl, thienyl or pyridyl. It may be a 5-20 membered heteroaryl group containing 1-5 heteroatoms selected from the group consisting of sulfur.

In the above formulas (A), (B), (C) and (D), the cycloalkyl group having 3 to 20 carbon atoms or the substituted cycloalkyl group is an unsubstituted cycloalkyl group having 3 to 20 carbon atoms. I.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, cyclotridecyl, cyclotetradecyl, cyclopentadecyl, cyclohexadecyl, cycloheptadecyl, cyclo An octadecyl, cyclononadecyl or cycloeicosanyl group, or a cycloalkyl group having 3 to 20 carbon atoms is a hydroxyl group having 1 to 5 carbon atoms, an alkoxyl group having 1 to 5 carbon atoms having 1 to 5 carbon atoms, 1 to 5 carbon atoms having 2 to 20 carbon atoms Jobs Shi alkoxyl group, 1
From 3 to 20 alkoxyalkoxyalkoxyl groups, and / or from 1 to 5 carbon atoms
It may be a substituted alkyl group substituted by a 20-membered aryl group. As the description of these substituents, the description of the above substituted alkyl group can be applied as it is.

In the general formulas (A), (B), (C) and (D), an alkoxyl group having 1 to 20 carbon atoms, an alkoxyalkoxyl group having 2 to 20 carbon atoms, and 3 to 2 carbon atoms
For the alkoxyalkoxyalkoxy group and aryl group of 0, the description of these substituents on the alkyl group can be applied as it is.

In the general formulas (A), (B), (C) and (D), the aryloxy group having 6 to 20 carbon atoms may be, for example, phenoxy, naphthoxy, etc.
The aralkyloxy group having 7 to 20 carbon atoms may be, for example, benzyloxy, phenethyloxy and the like.

When Ar ² in the general formula [II] is represented by the general formula (C), Y in the general formula (C) is preferably oxygen, and R ¹³ and R ^{14 in} Z are Preferably cooperating 3
A 7-membered ring is formed, and more preferably, Z represents a group represented by the following formula.

[0047]

Embedded image

When Ar ² in the general formula [II] is represented by the general formula (C), Y may also preferably be NR ¹⁰ ;
Preferably may be -OR ^{11, R 10} and ^{R 11} preferably forms a 3-membered to 7-membered ring. In this case, more preferably, R ¹⁰ and R ¹¹ form a group represented by the following formula.

[0049]

Embedded image

The above-mentioned chemiluminescent substrates can be used alone or in combination.

The concentration of the chemiluminescence enhancer of the present invention in the chemiluminescence reaction solution is not particularly limited, and it can be used in a dissolvable concentration range, but is usually about 1 to 200 mg / ml.
Preferably, it is about 10 to 100 mg / ml. The concentration of the chemiluminescent substrate in the chemiluminescent reaction solution is not particularly limited, and it can be used in a concentration range in which the chemiluminescent substrate can be dissolved, but is usually about 0.2 to 2 mg / ml.

The chemiluminescence measurement itself can be performed in exactly the same manner as the conventional chemiluminescence measurement, except that the above-mentioned chemiluminescence enhancer of the present invention is coexisted in the reaction system. The method of causing the chemiluminescence enhancer to coexist in the reaction system is not limited at all, for example, adding a chemiluminescence enhancer to the substrate solution,
This substrate solution may be mixed with the enzyme solution, or the chemiluminescence enhancer may be added to the reaction system separately from the substrate. The chemiluminescence measurement is preferably performed at room temperature to about 40 ° C., more preferably at the optimal temperature of the enzyme used. The reaction time is not limited, and is usually 1 minute to 60 minutes, preferably 3 minutes to 20 minutes. The reaction medium can be performed in a buffer suitable for the enzyme reaction, such as a phosphate buffer, as in conventional chemiluminescence measurements.
H is preferably at or near the optimum pH of the enzyme used. Chemiluminescence from the reaction product can be measured using a well-known and commercially available photocounter or luminometer, similarly to the conventional chemiluminescence measurement.

The chemiluminescence measurement of the present invention can be applied to any field to which conventional chemiluminescence measurement is applied. Among these fields, enzyme immunoassay is an important field. Enzyme immunoassays themselves are well known in the art. In the enzyme immunoassay, the enzyme is used as a label or a marker bound to an antigen, an antibody, a hapten or the like. Detected or quantified. The use of chemiluminescence measurements in enzyme immunoassays is well known in the art. For example, in a sandwich ELISA, which is a typical example of enzyme immunoassay, a first antibody is immobilized on a solid phase, and an antigen to be quantified, which is specific to the first antibody, is reacted with the first antibody. After washing, an enzyme-labeled second antibody specific to the antigen is reacted with the antigen captured on the solid phase. After washing, the captured second antibody, ie, the captured enzyme, is quantified using chemiluminescence measurements. In addition, the above-mentioned sandwich ELIS
A is only an example, and the enzyme immunoassay is not limited to the above example.

In the enzyme immunoassay, the target substance to be detected or quantified is not limited, and examples thereof include antigens such as hormones, enzymes, proteins, cytokines, bacterial cells and viruses, and antibodies against these antigens. Can be. The sample subjected to the enzyme immunoassay is also not limited at all, for example, blood, plasma,
Examples include body fluids such as serum, urine and tissue fluids, and foods and beverages.

In the immunoassay, the antibody may be a polyclonal antibody or a monoclonal antibody, and the latter is preferred from the viewpoint of specificity. Monoclonal antibodies
It can be produced by the method of Kohler and Milstein (Nature, 256, 495 (1975)), and the produced antibody can be purified by a conventional method. The purified antibody can be used as it is or after being fragmented into an antigen-binding fragment such as a Fab fragment or F (ab ') ₂ fragment.

[0056]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Reference Example 1 Preparation of conjugate bonded to ferrite particles Ferrite particle liquid having a concentration of 0.04% to which HBs antigen was bonded; 1 m
l to anti-HBs antibody binding alkaline phosphatase solution; 0.1ml
And left at room temperature for 30 minutes. After that, collect the ferrite particles and remove the excess liquid, and then add 10 mM Tris / HCl pH
1 ml of 7.2, 0.01% Triton X-100 (registered trademark) (hereinafter referred to as “washing solution”) was added. After repeating this washing operation 5 times, finally, 0.5 ml of 0.1 M diethanolamine / HCl p
Ferrite particles were suspended in H10, 1 mM magnesium chloride (hereinafter, referred to as “substrate buffer”), and used as HBs particle-conjugate.

Example 1, Comparative Example 1 Comparison of Luminescence Enhancing Effect between Quaternary Ammonium Water-Soluble Polymer and AG11 Quaternary ammonium water-soluble polymer chemiluminescence enhancer (hereinafter, the chemiluminescence enhancer is sometimes referred to as “enhancer”. ) BDMQ (polyvinylbenzyl (benzyldimethylammonium) chloride) 0.5mg / ml, Chemiluminescent substrate AMPP
5 μl of the HBs particle conjugate of Reference Example 1 was added to 50 μl of a substrate solution containing 1 mg / ml of D, and the luminescence count for 20 minutes was measured with a luminometer (Comparative Example 1). Also, about 50 μl of AG1
1 Resins (trade name, listed above) was mixed with AMPPD at a final concentration of 1 mg / ml, and 5 μl of HBs particle conjugate was added, and the luminescence count was measured (Example 1). Separately, the same measurement was performed for AMPPD alone without an enhancer as a control (control).

Table 1 shows the counts after 5, 10, 15 and 20 minutes. Counting with BDMQ added substrate solution (Comparative Example 1)
Was increased about 27-fold after 5 minutes as compared to the case of AMPPD alone (control), whereas the substrate solution containing AG11 (Example 1) was about 300-fold higher.
It was doubled, showing a count of more than 10 times that of BDMQ.

[0060]

[Table 1] Table 1 Note) "Control" indicates the count for only AMPPD without enhancer.

Example 2 Polyacrylic Acid Treatment of AG1 Powder and Effect of Enhancing Chemiluminescence AG1 Resins (trade name, listed above) were placed in a mortar and ground until the shape of the beads disappeared. Ground powder
AG1 was placed in a test tube of about 20 mg, mixed with 4% polyacrylic acid (average molecular weight 90,000), and allowed to stand at room temperature for 2 hours.
Thereafter, the plate was washed with water and the substrate buffer until the final pH was the same as that of the substrate buffer, and finally suspended at a concentration of 200 mg / ml in the substrate buffer. Take 5 μl aliquot from here and make a final concentration of 1 mg / ml.
Mix with AMPPD and add HBs particle conjugate of Reference Example 1 to 2
Five minutes after the addition of μl, the luminescence count was measured with a luminometer.

Table 2 shows the results. The count of AG1 powdered and treated with polyacrylic acid (Example 2) was increased by about 180 times.

[0063]

[Table 2] Table 2 *: "Control" is without AG1

Example 3 Treatment of AG1 Powder with Various Polycarboxylic Acids and Effect of Enhancing Chemiluminescence As in Example 2, AG1 Resins (trade name, listed above) was placed in a mortar and ground until the shape of the beads disappeared. Take about 20 mg of the ground powdered AG1 in a test tube, and add 4
% Of sodium polymethacrylate, poly (acrylic acid-maleic acid), poly (1-vinylpyrrolidone-acrylic acid), and poly (methylvinyl-maleic acid), and left at room temperature for 2 hours. Thereafter, the plate was washed with water and the substrate buffer until the final pH was the same as that of the substrate buffer, and finally suspended at a concentration of 200 mg / ml in the substrate buffer. 5μl from here
And mixed with AMPPD at a final concentration of 1 mg / ml.
The luminescence count 5 minutes after adding 2 μl of the Bs particle conjugate was measured with a luminometer.

Table 3 shows the results. It can be seen that the emission count is about 35 to 95 times higher than the control using any of the polycarboxylic acids.

[0066]

[Table 3] Table 3 *: "Control" is without AG1

Example 4, Comparative Example 2 Polyacrylic acid treatment of other anion exchangers and the effect of enhancing chemiluminescence Q-Sepharose (registered trademark, supra) was selected as a representative anion exchanger other than AG1. 2 ml of gel equivalent to about 0.5 ml
Was reacted with a 4% acrylic acid polymer solution at room temperature for 2 hours. Thereafter, washing was carried out with water and the substrate buffer until the final pH was the same as that of the substrate buffer, and finally a suspension of about 50% was obtained with the substrate buffer. An excess of the substrate buffer was removed by aliquoting 50 μl from this, mixed with AMPPD at a final concentration of 1 mg / ml, and 2 μl of the HBs particle-conjugate of Reference Example 1 was added. Was measured.

Table 4 shows the results. Even though Q-Sepharose (registered trademark) is not treated with polyacrylic acid (Comparative Example 2), it exhibits about 40 times the luminescence count when not treated.
The polyacrylic acid-treated Q-Sepharose (Example 4) showed a count increase of about 130 times when not added.

[0069]

[Table 4] Table 4 *: Control is without Q-Sepharose

Example 5, Comparative Example 3 Comparison of (α-fetoprotein) AFP Standard Curve AFP antigen 0, 0
20 μl of 10, 100, 800, 2000 ng / ml was added and reacted at 37 ° C. for 10 minutes. Thereafter, the magnet was brought close to the outside of the test tube containing the reaction solution to collect the ferrite particles, and the excess solution was removed by a suction operation, and the ferrite particles were washed three times with 0.7 ml of a washing solution. Next, add 0.25 ml of
Add AFP antibody-bound alkaline phosphatase and add
After reacting for 1 minute, the ferrite particles were magnetically collected and washed in the same manner as above to obtain ferrite particles that had been completed up to the immune reaction. In the test tube containing the ferrite particles, the substrate solution added with BDMQ used in Example 1 (Comparative Example 3) or AMPPD
Only 200 μl of the substrate solution (control) was added, and the luminescence count for 5 minutes was measured with a luminometer. When using the substrate solution to which AG1 has been added, add 40 μl of the substrate buffer to the ferrite particles that have been subjected to the immune reaction, suspend them sufficiently, and take 2 μl from the suspension (1/20 of the total particle amount). 4 μl of the substrate solution (Example 5) to which the acrylic acid polymer-treated AG1 used in Example 2 was added, and the luminescence count for 5 minutes was measured with a luminometer.

Table 5 shows the results. The AG1-added substrate showed higher counts than the BDMQ-added substrate in the high AFP concentration range, although the ferrite particles used for the luminescence reaction were used only in other twentieth amount. Also, comparing the ratio of the counts of 10 ng / ml and 2000 ng / ml after subtracting the zero blank with each substrate, 214 times without enhancer, 88 times with BDMQ substrate, AG1
The substrate was 187-fold, significantly improving the linearity of the standard curve (the curve plotting the known AFP concentration on the horizontal axis and the luminescence count on the vertical axis) compared to the conventional BDMQ substrate.

[0072]

[Table 5] Table 5 1): No enhancer 2): Relative value with the amount of ferrite particles used as control set to 1

[0073]

As described above, according to the present invention, a novel chemiluminescence enhancer having a higher chemiluminescence enhancement effect than the conventional chemiluminescence enhancer is provided. When the chemiluminescence enhancer of the present invention is used, the linearity of the standard curve is improved, and more accurate quantification becomes possible.

Continued on the front page (72) Inventor Akio Hikita 2-62-5 Nihonbashihamacho, Chuo-ku, Tokyo F-Termio within Fujirebio Inc. 2G054 AA06 AA07 CA20 CA21 CA23 CA28 CE02 EA01 4B063 QA01 QQ30 QR10 QR57 QR66 QR66 QS24 QS36 QX02

Claims (8)

[Claims] 1. A chemiluminescence enhancer comprising an anion exchange group-containing substance treated with a polycarboxylic acid or a salt thereof. 2. The polycarboxylic acid is a polymer of an unsaturated carboxylic acid or a salt thereof, a copolymer containing an unsaturated carboxylic acid or a salt thereof, or two or more alkane or hydroxyalkane having 2 to 20 carbon atoms. The chemiluminescence enhancer according to claim 1, wherein the carboxyl group or a salt thereof is a substituted aliphatic polycarboxylic acid. 3. The chemiluminescence enhancer according to claim 2, wherein the polycarboxylic acid is a polymer of vinyl carboxylic acid or a salt thereof or a copolymer containing vinyl carboxylic acid or a salt thereof. 4. The chemiluminescence enhancer according to claim 3, wherein the vinyl carboxylic acid is acrylic acid or methacrylic acid or a salt thereof. 5. The anion exchange group-containing substance is a polymer having a tertiary or quaternary ammonium group, a latex particle having a tertiary or quaternary ammonium group, a liposome having a tertiary or quaternary ammonium group, a cationic The chemiluminescence enhancer according to any one of claims 1 to 4, which is an amino acid polymer or a cationic amino acid copolymer. 6. The chemiluminescence enhancer according to claim 5, wherein the polymer having a tertiary or quaternary ammonium group is an anion exchanger having a tertiary or quaternary ammonium group. 7. The chemiluminescence enhancer according to claim 6, wherein the polymer is polystyrene, polystyrene copolymer, agarose gel, cross-linked dextran gel or cellulose. 8. The method according to claim 1, wherein the treatment comprises bringing a solution of a polycarboxylic acid or a salt thereof or a liquid polycarboxylic acid or a salt thereof into contact with the anion exchange group-containing substance. The chemiluminescence enhancer according to the above.