JP2002022740A - Biochemical reaction accelerator, clinical diagnostic and clinical examination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biochemical reaction accelerator finishing a biochemical reaction system such as an antigen-antibody reaction, an enzyme-substrate reaction, and a DNA-DNA reaction for a clinical examination purpose in a short time so as to allow highly sensitive measurement when it is simply added into the reaction. SOLUTION: This biochemical reaction accelerator consists of a polymer prepared by polymerizing a monomer composition including a specific-structure phosphorylcholine analogue containing monomer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biochemical reaction accelerator which promotes a biochemical reaction when measuring an object to be measured in a sample in the field of clinical examination, a clinical test agent using the same, and Related to clinical test methods.

[0002]

2. Description of the Related Art In the field of clinical examination, an examination by agglutination of latex such as polystyrene is performed as an examination method for measuring the amount of antigen with high sensitivity. In the latex agglutination method, an antigen (or antibody) is physically or chemically bound to the latex surface, and the turbidity and particle size change when the latex is agglutinated by an immune reaction with an antibody (or antigen) to be detected. This is a method of detecting the distribution and the like and evaluating the amount of the substance to be measured.

As another method, an enzyme immunoassay has been used. For example, a sandwich assay, which is a type of enzyme immunoassay, employs two types of antibodies (Ab1, Ab2) having different epitopes on an analyte (test substance, Ag).
It is a method using. First, Ab1 is physically or chemically immobilized on the surface of a solid phase made of a synthetic polymer or the like, and Ag is added thereto and bonded. Then, labeled Ab2
(Ab2 is labeled as Ab2 ^* ), and then washed to remove free Ab2 ^* .
This is a method for measuring the activity of Ab2 ^* bound to a solid phase.
Usually, enzymes, fluorescent substances, luminescent substances, radioactive substances and the like are used for these labels.

As another method, a hybridization method of a gene-related substance such as DNA or RNA is used. This method comprises reacting a solid-phase on which a DNA having a sequence complementary to a gene-related substance such as a DNA to be measured is immobilized with a sample containing the DNA to be measured, This is a method of removing the DNA other than the DNA to be measured by washing, and finally measuring the activity of the labeling substance bound to the solid phase. For these labels, fluorescent substances, enzymes, luminescent substances, radioactive substances and the like are usually used.

[0005] In the field of clinical examinations, when measuring an object to be measured using the above-mentioned latex agglutination method, enzyme immunoassay method, hybridization method or the like, it is required to perform the measurement with high sensitivity in a short time. Conventionally, when measuring a sample using the latex agglutination method, sandwich measurement method, and hybridization method, the purpose is to suppress the non-specific adsorption of each measurement target substance or labeling substance to a container or the like. It is widely known to add proteins such as bovine serum albumin (hereinafter abbreviated as BSA) and casein. However, although these proteins suppress nonspecific adsorption, they cannot promote antigen-antibody reaction, enzyme-substrate reaction, DNA-DNA reaction and the like. That is, these reactions could not be completed in a short time.

On the other hand, a phosphorylcholine group-containing polymer is
Due to the phospholipid-like structure derived from biological membranes, it has excellent biocompatibility such as blood compatibility, deactivation of complement, non-adsorption of biological substances, and excellent properties such as antifouling property and moisturizing property. It is known that the polymer has such a function, and research and development on synthesis of a polymer for the purpose of developing a bio-related material utilizing each function and its use are actively performed.

[0007] Among them, JP-A-7-5177, JP-A-7-83923, and JP-A-10-114800
Japanese Patent Application Laid-Open Publication No. H11-157, discloses a technique that can perform a clinical test with high accuracy by utilizing the fact that a phosphorylcholine group-containing polymer can suppress the adsorption of a protein to a container or the like. However, phosphorylcholine group-containing polymers can be used as antigen-
It is known that the reaction can be completed in a short time and highly sensitive measurement can be performed by adding the compound to a biochemical reaction system such as an antibody reaction, an enzyme-substrate reaction, or a DNA-DNA reaction. I didn't.

[0008]

SUMMARY OF THE INVENTION A first object of the present invention is to add a compound to a biochemical reaction system such as an antigen-antibody reaction, an enzyme-substrate reaction, or a DNA-DNA reaction for clinical examination. It is therefore an object of the present invention to provide a biochemical reaction accelerator which allows the reaction to be completed in a short time and enables highly sensitive measurement. A second object of the present invention is to provide a clinical test agent containing a biochemical reaction accelerator, which promotes a biochemical reaction for clinical tests. Further, a third object of the present invention is to provide a clinical test method using the above-mentioned clinical test drug.

[0009]

Means for Solving the Problems As a result of intensive studies in view of the above problems, the present inventors polymerized a monomer composition containing a specific phosphorylcholine analog-containing monomer, and obtained the resulting polymer. The inventors of the present invention have found that the use of the combination for the biochemical reaction in a clinical test promotes the biochemical reaction, thereby completing the present invention. That is, the present invention provides the following [1] to [5]
It is as follows.

[1] The following equation [1]

[0011]

Embedded image

Wherein X represents a divalent organic residue; Y represents an alkyleneoxy group having 1 to 6 carbon atoms;
Is a hydrogen atom or R ⁵ —O— (C ＝ O) — (provided that R ⁵
Represents an alkyl group having 1 to 10 carbon atoms or a hydroxyalkyl group having 1 to 10 carbon atoms). R ¹ represents a hydrogen atom or a methyl group, and R ² , R ³ and R ⁴ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group. m represents 0 or 1. n is an integer of 1 to 4. A biochemical reaction accelerator comprising a polymer obtained by polymerizing a monomer composition containing a phosphorylcholine-like group-containing monomer represented by｝.

[2] A monomer composition containing a phosphorylcholine-like group-containing monomer is composed of 20 to 100 mol% of a phosphorylcholine-like group-containing monomer of component A and 0 to 80 mol% of a hydrophobic monomer of component B. The biochemical reaction accelerator according to the above [1], which is a monomer composition comprising:

[3] The phosphorylcholine analog-containing monomer of the component A is 2-((meth) acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, and the hydrophobic monomer of the component B is Is the following equation [2]

[0015]

Embedded image

Wherein R ⁶ represents a hydrogen atom or a methyl group, and L ¹ represents —C ₆ H ₄ —, —C ₆ H ₁₀ —,
(C = O) -O-, -O-,-(C = O) NH-, -O
- (C = O) - indicates and -O- (C = O) group selected from -O-, L ² is a hydrogen _{atom, - (CH 2) g -L} 3 and - ((CH _{2) p} -O) shows a hydrophobic functional group selected from _h -L ^3. (G and h are integers from 1 to 24, and p is 3 to 5
L ³ represents a hydrogen atom, a methyl group, —C ₆ H ₅
And a functional group selected from —OC ₆ H ₅ ).} The biochemical reaction accelerator according to [1] or [2], which is a hydrophobic monomer represented by {}.

[4] A clinical test agent comprising the biochemical reaction accelerator according to any one of [1] to [3]. [5] A clinical test method using the clinical test agent according to the above [4] for a biochemical reaction to measure a bio-related substance.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The biochemical reaction accelerator of the present invention comprises
Specific phosphorylcholine analog-containing monomers (hereinafter referred to as PC
(Hereinafter abbreviated as monomer) as a main component is a polymer obtained by polymerizing a monomer composition containing the polymer (hereinafter, referred to as a PC polymer). The monomer composition containing a PC monomer may contain only the PC monomer or may contain other polymerizable monomers. Furthermore, the above-mentioned PC polymer specifically contains 20 to 100 mol% of a PC monomer as an A component and B as a component.
A polymer obtained by polymerizing a monomer composition comprising a hydrophobic monomer in an amount of 0 to 80 mol% as a component can be preferably exemplified. More preferably, PC monomer 4 is used as the A component.
0 to 80 mol%, hydrophobic monomer 20 to 60 as B component
mol% of the monomer composition.

When the content of the hydrophobic monomer of the component B is more than 80 mol%, a phosphorylcholine-like group (hereinafter abbreviated as PC group) is used.
It is not preferable because it is difficult to exert the effect of (1).
Further, the PC polymer contains 20 to 100 mol% of a PC monomer as the component A and a hydrophobic monomer 0 as the component B.
-40 mol% and hydrophilic monomer 0-7 as C component
A polymer obtained by polymerizing 0 mol% of the monomer composition can be preferably exemplified. More preferably, it is a monomer composition comprising 40 to 80 mol% of a PC monomer as the component A, 10 to 30 mol% of a hydrophobic monomer as the component B, and 10 to 60 mol% of a hydrophilic monomer as the component C. If the amount of the PC monomer of the component A is less than 20 mol%, it is difficult to exert the effect of the PC group, which is not preferable. Here, the PC monomer is represented by the following formula [1]

[0020]

Embedded image

Wherein X represents a divalent organic residue; Y represents an alkyleneoxy group having 1 to 6 carbon atoms;
Is a hydrogen atom or R ⁵ —O— (C ＝ O) — (provided that
R ⁵ is an alkyl group having 1 to 10 carbon atoms or 1 to 1 carbon atoms.
0 represents a hydroxyalkyl group). Also, R ¹
Represents a hydrogen atom or a methyl group, and R ² , R ³ and R ⁴
Are the same or different groups and each represent a hydrogen atom,
And represents an alkyl group or a hydroxyalkyl group.
m represents 0 or 1. n is an integer of 1 to 4. ｝

Examples of the divalent organic residue X in the formula [1], for _{example, -C 6 H 4 -, -} C 6 H 10 -, - (C = O)
-O -, - O -, - CH 2 -O -, - (C = O) NH
-, -O- (C = O)-, -O- (C = O) -O-,-
_{C 6 H 4 -O -, -} C 6 H 4 -CH 2 -O -, - C 6 H 4 -
And a group such as (C = O) -O-.

Y in the formula [1] is an alkyleneoxy group having 1 to 6 carbon atoms, for example, a group such as a methyloxy group, an ethyloxy group, a propyloxy group, a butyloxy group, a pentyloxy group and a hexyloxy group. No.

Z in the formula [1] is a hydrogen atom or R ⁵
It represents a -O- (C = O)-group. However, R ⁵ has 1 carbon atom
Represents an alkyl group having 10 to 10 or a hydroxyalkyl group having 1 to 10 carbon atoms. Here, examples of the alkyl group having 1 to 10 carbon atoms include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group,
Examples include an octyl group, a nonyl group, and a decyl group.

Examples of the hydroxyalkyl group having 1 to 10 carbon atoms include a hydroxymethyl group, a 2-hydroxyethyl group, a 3-hydroxypropyl group, a 2-hydroxypropyl group, a 4-hydroxybutyl group and a 2-hydroxybutyl group. Butyl group, 5-hydroxypentyl group, 2-hydroxypentyl group, 6-hydroxyhexyl group, 2-hydroxyhexyl group, 7-hydroxyheptyl group, 2-
Hydroxyheptyl group, 8-hydroxyoctyl group, 2
-Hydroxyoctyl group, 9-hydroxynonyl group, 2
-Hydroxynonyl group, 10-hydroxydecyl group, 2
-Hydroxydecyl group and the like.

As the PC monomer, specifically, for example,
2-((meth) acryloyloxy) ethyl-2'-
(Trimethylammonio) ethyl phosphate, 3-
((Meth) acryloyloxy) propyl-2 '-(trimethylammonio) ethyl phosphate, 4-((meth) acryloyloxy) butyl-2'-(trimethylammonio) ethyl phosphate, 5-((meth) acryloyloxy ) Pentyl-2 '-(trimethylammonio) ethyl phosphate, 6-((meth) acryloyloxy) hexyl-2'-(trimethylammonio)
Ethyl phosphate,

2-((meth) acryloyloxy) ethyl-2 '-(triethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'
-(Tripropylammonio) ethyl phosphate, 2
-((Meth) acryloyloxy) ethyl-2 '-(tributylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'-(tricyclohexylammonio) ethyl phosphate, 2-((meth) Acryloyloxy) ethyl-2 ′-(triphenylammonio) ethyl phosphate,

2-((meth) acryloyloxy) ethyl-2 '-(trimethanolammonio) ethyl phosphate, 2-((meth) acryloyloxy) propyl-2'-(trimethylammonio) ethylphosphate, ((Meth) acryloyloxy) butyl-2 ′
-(Trimethylammonio) ethyl phosphate, 2-
((Meth) acryloyloxy) pentyl-2 ′ (trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) hexyl-2 ′-(trimethylammonio) ethylphosphate,

2- (vinyloxy) ethyl-2 '-(trimethylammonio) ethylphosphate, 2- (allyloxy) ethyl-2'-(trimethylammonio) ethylphosphate, 2- (p-vinylbenzyloxy)
Ethyl-2 ′-(trimethylammonio) ethyl phosphate, 2- (p-vinylbenzoyloxy) ethyl-
2 ′-(trimethylammonio) ethyl phosphate,
2- (styryloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (p-vinylbenzyl) ethyl-2'-(trimethylammonio) ethyl phosphate, 2- (vinyloxycarbonyl) ethyl-2 '-(Trimethylammonio) ethyl phosphate,

2- (allyloxycarbonyl) ethyl-
2 ′-(trimethylammonio) ethyl phosphate,
2- (acryloylamino) ethyl-2 '-(trimethylammonio) ethylphosphate, 2- (vinylcarbonylamino) ethyl-2'-(trimethylammonio) ethylphosphate, ethyl- (2'-trimethylammonioethylphosphoryl) Ethyl) fumarate, butyl- (2′-trimethylammonioethyl phosphorylethyl) fumarate, hydroxyethyl- (2′-trimethylammonioethyl phosphorylethyl) fumarate,

Ethyl- (2'-trimethylammonioethylphosphorylethyl) malate, butyl- (2'-trimethylammonioethylphosphorylethyl) malate, hydroxyethyl- (2'-trimethylammonioethylphosphorylethyl) malate, etc. Can be mentioned.

Of these, 2-((meth) acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate is preferable, and 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate is preferred. It may be referred to as 2- (methacryloyloxy) ethyl phosphorylcholine. Hereinafter, MPC is more preferable in terms of availability and the like.

When polymerizing the PC monomer, the above-mentioned PC
One of the monomers can be used alone, or two or more can be used as a mixture. The PC monomer can be produced by a known method. For example, it can be produced according to a known method disclosed in JP-A-54-63025, JP-A-58-155491, and the like. The hydrophobic monomer of the B component copolymerized with the PC monomer has the formula [2]

[0034]

Embedded image

Wherein R ⁶ represents a hydrogen atom or a methyl group, and L ¹ represents —C ₆ H ₄ —, —C ₆ H ₁₀ —,
(C = O) -O-, -O-,-(C = O) NH-, -O
- (C = O) - and -O- (C = O) represents a group selected from the group consisting of -O-, L ² is a hydrogen atom, - (C
H _{2) g} -L ³ and - indicates a ((CH _{2) p} -O) hydrophobic functional group selected from _h -L ^3. (G and h are an integer of 1 to 24, p is an integer of 3 to 5, L ³ is a hydrogen atom,
It represents a functional group selected from a methyl group, -C ₆ H ₅ and -O-C ₆ H _5. ) Represented by｝.

Specifically, for example, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth)
Linear or branched alkyl (meth) such as acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, and stearyl (meth) acrylate
Acrylate; cyclic alkyl (meth) acrylate such as cyclohexyl (meth) acrylate; aromatic ring (meth) acrylate such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate; polyalkylene glycol (meth) such as polypropylene glycol (meth) acrylate ) Acrylates; styrene-based monomers such as styrene, methylstyrene and chloromethylstyrene; vinyl ether-based monomers such as methyl vinyl ether and butyl vinyl ether; vinyl ester-based monomers such as vinyl acetate and vinyl propionate; . These one
Species or two or more species are used.

The hydrophilic monomer of the component C used in the present invention includes:
For example, a hydroxy group, a carboxyl group, a phosphone group,
It is a monomer having a hydrophilic group selected from the group consisting of a sulfone group, an amide group, an amino group, a dialkylamino group, a trialkylamino group, a trialkylphosphonium base and a polyoxyethylene group. Furthermore, specifically, for example, 2-hydroxyethyl (meth) acrylate,
Hydroxyl-containing (meth) acrylates such as 2-hydroxybutyl (meth) acrylate and 4-hydroxybutyl (meth) acrylate; carboxylic acids such as acrylic acid and methacrylic acid; styrenesulfonic acid, (meth) acryloyloxyphosphonic acid, Ionic group-containing monomers such as hydroxy-3- (meth) acryloxypropyltrimethylammonium chloride; nitrogen-containing monomers such as (meth) acrylamide, aminoethyl methacrylate, dimethylaminoethyl (meth) acrylate; polyethylene glycol ( (Meth) acrylate and the like. One or more of these are used.

The PC polymer is a monomer composition of the above-mentioned A component PC monomer and the B component hydrophobic monomer, or the aforementioned A component PC monomer and the B component hydrophobic monomer. And C
What is necessary is just to polymerize the monomer composition with the hydrophilic monomer of the component, and it can be produced by ordinary radical copolymerization.

The molecular weight of the PC polymer of the present invention is preferably in the range of 5,000 to 5,000,000 and more preferably in the range of 100,000 to 2,000,000 by weight. If the molecular weight of the polymer is less than 5,000, it is difficult to sufficiently promote the biochemical reaction. If the molecular weight of the polymer is more than 5,000,000, the viscosity of the aqueous solution of the PC polymer becomes too high. This is not preferable because it may inhibit biochemical reactions.

The biochemical reaction in the present invention is not particularly limited, but refers to various reactions used in clinical tests and the like, and specifically, for example, a reaction between proteins such as an antigen-antibody reaction and an antibody-antibody reaction; Reaction of an enzyme with its specific substrate, such as an enzyme-substrate reaction; DNA-DNA reaction, DNA-R
Examples include reactions between gene-related substances such as NA reaction, DNA-enzyme reaction, and RNA-enzyme reaction, and reactions between gene-related substances and proteins.

Examples of immunoreactive substances such as antigens and antibodies include immunoglobulins, plasma proteins, hormone-related substances, tumor-related substances, virus-related substances, antibodies to drugs, antibodies to enzymes, and the like. Antibodies to substances are also included. Examples of the plasma protein include C-reactive protein (CRP), an apoprotein-related substance, rheumatoid factor (RF), and transferrin. Examples of hormone-related substances include thyroid stimulating hormone (TSH) and triiodothyronine (T
3), thyroxine (T4), thyroxine binding protein (TBG) and the like.

Examples of the tumor-related substance include carcinoembryonic antigen (CEA), β2-microglobulin, α-fetoprotein (AFP) and the like. Examples of virus-related substances include HBs antigen, HBs antibody, HBe
Antigen, HBe antibody, mumps, herpes, measles, rubella,
Anti-AIDS antibodies and the like. Antibodies to drugs include, for example, antibodies such as phenobarbital, acetaminophenone, salicylic acid, and cyclosporin.
Specific examples of the enzyme include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, peroxidase, glucoamylase, glucose oxidase, and lysozyme. Examples of the fluorescent substance include fluorescein isothiocyanate, 4-chloro-7-nitrobenzenezofurazan,
-Fluoro-7-nitrobenzenezofurazan, sulforhodamine 101 acid chloride and the like.

Examples of the luminescent substance include 10-methyl-9- [4- {2- (succinimidyloxycarbonyl) ethyl} phenyloxycarbonyl] acridinium fluorosulfate.

As a method of accelerating the biochemical reaction by the PC polymer of the present invention, a method of adding the PC polymer to the biochemical reaction system in an amount of 0.0001 to 10% by weight is used. When the concentration of the PC polymer is lower than 0.0001% by weight, the effect of accelerating the biochemical reaction cannot be sufficiently exhibited. When the concentration is higher than 10% by weight, the viscosity of the aqueous solution becomes too high. It is not preferable because biochemical reactions may be inhibited.

When the PC polymer of the present invention is used as a biochemical reaction accelerator, P is added to the biochemical reaction system.
It is only necessary to add the C polymer. When adding the PC polymer, one of the reaction between the protein and the protein, the reaction between the enzyme and its specific substrate, the reaction between gene-related substances or the reaction between the gene-related substance and the protein,
Alternatively, the PC polymer may be added to both before the biochemical reaction, or may be added later to the biochemically reacting system. Further, the state of the PC polymer to be added is
The solution state may be a powder state, and a preferred example is a solution state that quickly becomes uniform with the reaction system.

The biochemical reaction used in the clinical test and the like in the present invention refers to the biochemical reaction described above, and is not particularly limited. The clinical test agent of the present invention is the antigen described above.
Antibody reaction, antibody-antibody reaction, enzyme-substrate reaction, DNA-
DNA reaction, DNA-RNA reaction, DNA-enzyme reaction,
Serum, using biochemical reactions such as RNA-enzyme reaction,
When measuring proteins, enzymes, gene-related substances, etc., which are measurement objects (test objects) in specimens such as plasma, urine, tears, tissues, etc., the aforementioned proteins, enzymes, gene-related substances, etc. This is a measurement reagent containing a biochemical reaction accelerator. The mixing ratio of the protein, enzyme, gene-related substance and the biochemical reaction promoter is not particularly limited by the characteristics of the object to be measured in the above-mentioned reaction.
The weight / weight ratio of the polymer / protein, enzyme or gene-related substance is 1.0 × 10 ^{−12 to} 1.0 × 10 ¹⁸ , particularly preferably 1.0 × 10 ⁻⁹ to 1.0 × 10 ^18. It is ¹⁵ .
Wherein the weight ratio is difficult to sufficiently exhibit the effect in promoting 1.0 × 10 ^-12 smaller than biochemical reactions, and larger than 1.0 × 10 ^18, PC in the reaction system It is not preferable because the polymer concentration is high and the viscosity is high, which may hinder the biochemical reaction.

The clinical test method of the present invention specifically includes
This is a method of measuring a biological substance in the biochemical reaction system using the biochemical reaction accelerator. Examples of the biologically-related substance include the aforementioned antigens, proteins such as immunologically active substances such as antibodies, gene-related substances such as nucleic acid-related substances such as DNA and RNA, and substrates to be used for enzymes and enzyme reactions. .

The biochemical reaction accelerator of the present invention includes a surfactant, a solvent, and a solvent within a range that does not impair the effects of the present invention.
Other components such as preservatives may be added.

[0049]

The biochemical reaction accelerator of the present invention is a polymer containing a constituent component based on a specific phosphorylcholine analog-containing monomer, and promotes a biochemical reaction when a clinical test is carried out. Therefore, measurement can be performed with high sensitivity in a short time. In addition, the clinical test agent of the present invention,
It is a test reagent containing the above-mentioned biochemical reaction accelerator, and is a clinical test reagent suitable for an automatic analyzer because it can be measured in a short time with high sensitivity. Further, the clinical test method of the present invention,
This is a method using the above-mentioned clinical test agent, which facilitates the reaction, so that the measurement can be easily performed with high sensitivity in a short time.

[0050]

The present invention will be described in more detail with reference to the following examples. Synthesis Example 1 MPC 35.7 g, butyl methacrylate (BMA)
4.3 g was dissolved in 160 g of ethanol, placed in a four-necked flask, and blown with nitrogen for 30 minutes.
0.82 g of azobisisobutyronitrile (hereinafter abbreviated as AIBN) was added thereto, and a polymerization reaction was carried out for 8 hours. The polymerization solution was added dropwise to 3 L of diethyl ether while stirring, and the precipitated precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 29.6 g of a powder. The molecular weight was analyzed by gel permeation chromatography (GPC). The analysis conditions were 20 mM phosphate buffer (pH 7.
4) was used as an eluent, polyethylene glycol was used as a standard substance, and UV (210 nm) and the refractive index were detected.
The molecular weight evaluated by GPC was 15 weight average molecular weight.
It was 3,000. This is designated as polymer 1 (P-1). Table 1 shows the results. Polymer 1 (P-1); pol
y (MPC0.8-co-BMA0.2), weight average molecular weight 153,000.

Synthetic Examples 2 and 3 Polymers having the following copolymer composition were obtained in the same manner as in Synthetic Example 1 except that the type, composition ratio, and solvent type of the monomers used in Synthetic Example 1 were changed. Synthesis Example 2; Polymer 2 (P-2); poly (MPC
0.3-co-BMA 0.7), weight average molecular weight 13
It was 0000. The polymerization conditions were MPC;
4.1 g, BMA; 15.9 g, ethanol; 120
g, AIBN: Synthesis Example 1 except for changing to 0.35 g
Reacted according to Synthesis Example 3; Polymer 3 (P-3);
poly (MPC) and the weight average molecular weight was 529,000. The reaction was carried out in the same manner as in Synthesis Example 1 except that the polymerization conditions were changed to 50.0 g for MPC, 100 g for ethanol, and 0.24 g for AIBN. Table 1 shows the results.

[0052]

[Table 1]

Example 1: Effect of promoting antigen-antibody reaction-1 10 μg / mL goat anti- (mouse antibody) antibody {manufactured by Wako Pure Chemical Industries, Ltd.} solution (100 mM phosphate buffer, pH
7.5) was added to a polystyrene 96-well titer plate (Immunoplate F96, Maxisorp, manufactured by Nunc) at 100 μL / well, incubated at 4 ° C. overnight, and allowed to physically adsorb. Washing was performed four times with a buffer solution (hereinafter abbreviated as PBS). Then
PBS containing 1% bovine serum albumin (hereinafter abbreviated as BSA) was added at 300 μL / well, and the mixture was incubated at 4 ° C. overnight. Then, the BSA solution was removed to prepare an antibody-adsorbed solid phase. Next, the polymer 1 was added to the antibody-adsorbed solid phase.
0 μg / mL containing 1.0% by weight of (P-1), 0.1 μ
g / mL and 0.4 μg / mL of mouse antibody {Wako Pure Chemical Industries, Ltd.} solution (PBS) was added at 100 μL / well and incubated at 25 ° C. for 15 minutes to obtain antigen-
An antibody reaction was performed and the cells were washed four times with PBS. Next, a peroxidase-labeled anti- (mouse antibody) antibody {manufactured by Wako Pure Chemical Industries, Ltd.} was added to a solution containing PBS containing 1% BSA for 1000 times.
Dilute 0-fold and add at 100 μL / well, 25 ° C, 2
Incubated for 4 hours and washed 4 times with PBS. Next, after each well was colored at room temperature for 10 minutes with a peroxidase color development kit T (manufactured by Sumitomo Bakelite Co., Ltd.), 450 nm of each well was measured using SPECTRA MAX250 (microplate reader, manufactured by Molecular Devices). The absorbance was measured. The number of measurements (n) was 8, and the average value, standard deviation, and CV value (%) {(average value / standard deviation) × 100} were shown as the results. Table 2 shows the results.

Examples 2 and 3: Effect of promoting antigen-antibody reaction-2, 3 In place of polymer 1 (P-1) used in Example 1, polymer 2 (P-2; The same operation as in Example 1 was performed except that 2) and Polymer 3 (P-3; Example 3) were used. Table 2 shows the results. Comparative Example 1 Instead of the polymer 1 (P-1) used in Example 1, BS was used.
The same operation as in Example 1 was performed except that A was used. Table 2 shows the results.

[0055]

[Table 2]

Example 4 Effect of Promoting Antigen-Enzyme-Labeled Antibody Reaction-1 The antibody-adsorbed solid phase prepared in Example 1 contained 1% by weight of BSA at 0 μg / mL, 0.1 μg / mL, and 0.4 μg / mL.
Mouse antibody (Wako Pure Chemical Industries, Ltd.) solution (PBS) was added at 100 μL / well, incubated at 25 ° C. for 2 hours, and washed four times with PBS. Then
Peroxidase labeled anti- (mouse antibody) antibody containing 1.0% by weight of polymer 1 (P-1) {Wako Pure Chemical Industries, Ltd.
Make the solution 10000-fold diluted with PBS, and add 100 μL
Per well, and incubated at 25 ° C. for 15 minutes to perform an antigen-enzyme-labeled antibody reaction, followed by washing four times with PBS.
Next, after each well was colored at room temperature for 5 minutes with a peroxidase color developing kit T (manufactured by Sumitomo Bakelite Co., Ltd.), using SPECTRA MAX250 (microplate reader, manufactured by Molecular Devices),
The absorbance at 450 nm of each well was measured. The number of measurements (n) was 8, and the average value, standard deviation, and CV value (%) were shown as results. Table 3 shows the results.

Examples 5 and 6: Promoting effect of antigen-enzyme-labeled antibody reaction-2, 3, Polymer 2 (P-2; Example 5) in place of polymer 1 (P-1) used in Example 4, and , Polymer 3 (P-
3; The same operation as in Example 4 was performed except that Example 6) was used. Table 3 shows the results. Comparative Example 2 Instead of polymer 1 (P-1) used in Example 4, BS
The same operation as in Example 4 was performed except that A was used. Table 3 shows the results.

[0058]

[Table 3]

Example 7: Hybridization (DN
(A-DNA Reaction) Acceleration Effect-1 The names, sequences and terminal modifications of the oligo DNAs used (Espec Oligo Service Co., Ltd.) are shown in Table 4.

[0060]

[Table 4]

Adenine is abbreviated as A, guanine as G, cytosine as C, and thymine as T.

250 pmol (= picomolar: 10 ^-12 )
/ ML PUC-amino solution ｛50 mM phosphate buffer containing 1 mM EDTA, pH 8.5 (hereinafter EDTA
-NaPB) was added to 96-well DNA-BIND (trademark, manufactured by Corning Coaster) at 100 μL / well, and incubated at 37 ° C. for 1 hour to chemically form P-BIND.
After adsorbing UC-amino, washing was performed four times with PBS. Then EDTA- with 3% BSA
After adding NaPB at 300 µL / well and incubating at 37 ° C for 1 hour, the BSA solution was removed and PUC-
An amino binding solid phase was prepared. Next, PUC-am
1.0% by weight of polymer 1 (P-1) in the ino-bound solid phase
Containing 0 fmol (femtomole: 10 ^-15 ) / mL, 1
PU of each concentration of 0 fmol / mL and 40 fmol / mL
C-biotin solution (7 containing 750 mM NaCl)
5 mM sodium citrate) was added at 100 μL / well and incubated at 55 ° C. for 15 minutes to perform hybridization (DNA-DNA reaction), and 0.1% SD
The plate was washed four times with 30 mM sodium citrate aqueous solution containing S and 300 mM NaCl. Next, a solution of peroxidase-labeled-avidin (manufactured by Sigma) was added to 3% BS.
Diluted 10000-fold with EDTA-NaPB containing A,
The solution was added at 100 μL / well, incubated at 37 ° C. for 1 hour, and washed four times with PBS. Next, each well was colored at room temperature for 10 minutes with a peroxidase color developing kit T (manufactured by Sumitomo Bakelite Co., Ltd.), and then SPECT.
Using RA MAX250 (microplate reader, manufactured by Molecular Devices), 450 n of each well
m was measured. The number of measurements (n) was 8, and the average value, standard deviation, and CV value (%) were shown as results. Table 5 shows the results.

Examples 8 and 9: Promoting effect of hybridization (DNA-DNA reaction) -2,3 Polymer 2 (P-2; Example) instead of polymer 1 (P-1) used in Example 7 8) and polymer 3 (P-
3 The operation was carried out in the same manner as in Example 7, except that Example 9) was used. Table 5 shows the results. Comparative Example 3 Example 7 was repeated, except that casein {manufactured by Wako Pure Chemical Industries, Ltd.} was used instead of the polymer 1 (P-1) used in Example 7.
The same operation was performed. Table 5 shows the results.

[0064]

[Table 5]

Example 10: Promoting effect of avidin-biotin reaction-1-1 PUF-amino binding solid phase prepared in Example 7, containing 1.0% by weight of casein, 0 fmol / mL, 10 fmo
PUC-bi at each concentration of 1 / mL and 40 fmol / mL
otin solution (75 mM sodium citrate containing 750 mM NaCl) at 100 μL / well and add
C., 1 hour, 0.1% by weight SDS, 3%
The plate was washed four times with a 30 mM sodium citrate aqueous solution containing 00 mM NaCl. Then, the peroxidase-labeled-avidin solution was added to E containing 1% of polymer 1 (P-1).
Dilute 10,000 times with DTA-NaPB, and add 100 μL
Per well and incubated at 37 ° C. for 15 minutes to perform an avidin-biotin reaction, followed by washing four times with PBS.
Next, after each well was colored at room temperature for 10 minutes with a peroxidase color development kit T (manufactured by Sumitomo Bakelite Co., Ltd.), 450 nm of each well was measured using SPECTRA MAX250 (microplate reader, manufactured by Molecular Devices). The absorbance was measured. The number of measurements (n) was 8, and the average value, standard deviation, and CV value (%) were shown as results. Table 6 shows the results.

Examples 11 and 12: Promoting effect of avidin-biotin reaction-2,3 Polymer 2 (P-2; Example 11) and polymer 3 in place of polymer 1 (P-1) used in Example 10 (P
-3: The same operation as in Example 10 was performed except that Example 12) was used. Table 6 shows the results. Comparative Example 4 Instead of the polymer 1 (P-1) used in Example 10, B
The same operation as in Example 10 was performed except that SA was used.
Table 6 shows the results.

[0067]

[Table 6]

Example 13: Effect of promoting enzyme-substrate reaction
1 0 μg / m containing 0.1% by weight of polymer 1 (P-1)
L, 0.01 μg / mL and 0.1 μg / mL peroxidase (Wako Pure Chemical Industries, Ltd.) solution (PBS) 9
A 6-well titer plate (ELISA plate S, manufactured by Sumitomo Bakelite Co., Ltd.) was added at 100 μL / well. 2,2'-azino- as a substrate for peroxidase
1 Component AB containing di (3-ethyl-benzothiazoline-6-sulfonate) (ABTS)
TS Microwell Peroxidase S
ubstrate (Kirkegaard & Per
lyLaboratories) at 100 μL / well, and incubated at 25 ° C. for 10 minutes.
A substrate reaction was performed. Then, 1% by weight SDS solution was added to 10%.
The reaction was stopped by adding 0 μL / well. Then SP
The resulting absorbance at 405 nm was measured using ECTRA MAX250. Table 7 shows the results.

Examples 14 and 15: Promoting effect of enzyme-substrate reaction-2, 3, Polymer 2 (P-2; Example 14) and polymer 3 in place of polymer 1 (P-1) used in Example 13 (P
-3: The same operation as in Example 13 was performed except that Example 15) was used. Table 7 shows the results. Comparative Example 5 Instead of the polymer 1 (P-1) used in Example 13, B
The same operation as in Example 13 was performed except that SA was used.
Table 7 shows the results.

[0070]

[Table 7]

From the above results, Comparative Examples 1 to 5
In the case of Examples 1 to 15, it can be seen that the measured value is high. That is, it is understood that each reaction is completed in a short time, and a highly sensitive analysis can be performed in a short time.

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Claims (5)

[Claims] (1) The following formula [1] ｛Wherein, X represents a divalent organic residue, Y represents an alkyleneoxy group having 1 to 6 carbon atoms, and Z represents a hydrogen atom or R ⁵ —O— (C ＝ O) — (provided that R ⁵ has 1 to 1 carbon atoms
10 alkyl groups or hydroxyalkyl groups having 1 to 10 carbon atoms). R ¹ represents a hydrogen atom or a methyl group, and R ² , R ³ and R ⁴ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group. m represents 0 or 1. n
Is an integer of 1 to 4. A biochemical reaction accelerator comprising a polymer obtained by polymerizing a monomer composition containing a phosphorylcholine-like group-containing monomer represented by｝. 2. A monomer composition containing a phosphorylcholine-like group-containing monomer, comprising 20 to 100 mol% of a phosphorylcholine-like group-containing monomer of component A and 0 to 80 mol% of a hydrophobic monomer of component B. The biochemical reaction accelerator according to claim 1, which is a monomer composition comprising: 3. A single component containing a phosphorylcholine-like group of component A
When the body is 2-((meth) acryloyloxy) ethyl-
With 2 '-(trimethylammonio) ethyl phosphate
And the hydrophobic monomer of the component B is represented by the following formula [2]:｛Wherein, R⁶Represents a hydrogen atom or a methyl group
Then L¹Is -C₆H_Four-, -C₆H_Ten-,-(C = O)-
O-, -O-,-(C = O) NH-, -O- (C = O)
-And -O- (C = O) -O-
Then L^TwoIs a hydrogen atom,-(CH_Two)_g-L^ThreeAnd-
((CH_Two)_p-O)_h-L^ThreeA hydrophobic functional group selected from
Show. (G and h are integers of 1 to 24, and p is 3 to 5
Indicates an integer and L ^ThreeIs a hydrogen atom, a methyl group, -C₆H_FiveYou
And -OC₆H_FiveRepresents a functional group selected from )｝
3. The method according to claim 1, wherein the monomer is a hydrophobic monomer.
Reaction accelerator. A clinical test agent comprising the biochemical reaction promoter according to any one of claims 1 to 3. 5. A clinical test method comprising using the clinical test agent according to claim 4 for a biochemical reaction to measure a biological substance.