JP2010169604A - Agent for preventing non-specific adsorption and coating method of article - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for preventing non-specific adsorption, preventing non-specific adsorption of a protein, a protein-loaded latex, etc. to an article, and also to provide a coating method of an article using the agent for preventing non-specific adsorption. <P>SOLUTION: The agent for preventing non-specific adsorption contains a copolymer having: a repeating unit (A) having acrylamide represented by a formula (1) (wherein R<SP>1</SP>is hydrogen atom or methyl group; and R<SP>2</SP>is at least one chosen from hydrogen atom, methyl group, hydroxymethyl group and hydroxyethyl group), or an acrylamide derivative having an N-substituent; and a repeating unit (B) represented by formula (2) (wherein R<SP>3</SP>is hydrogen atom or methyl group; and Z is 4-12C organic group having at least one aldo group or keto group). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to, for example, a nonspecific adsorption inhibitor for preventing nonspecific adsorption of various proteins used in clinical diagnostics, clinical diagnostic devices, biochips, etc., and coating of articles using the nonspecific adsorption inhibitor Regarding the method.

  In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. Even in diagnostic drugs using solid phases such as polystyrene plates and magnetic particles, a method that uses enzyme color development as a detection method is being switched to a method that uses fluorescence or chemiluminescence for higher sensitivity in order to improve sensitivity. However, in reality, sufficient sensitivity has not been obtained. The reason for this is that in the diagnosis of detecting a specific substance in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. are adsorbed non-specifically to solid phases, instruments and containers, As a result, it can be mentioned that noise increases and hinders high sensitivity. Therefore, in immunodiagnostic measurement, albumin is usually used to reduce the decrease in sensitivity due to non-specific adsorption to substances other than the substance that specifically binds to the solid phase surface or instrument / container used for the immune reaction. By using biologically derived substances such as casein and gelatin as non-specific adsorption inhibitors, non-specific adsorption is suppressed and noise is reduced.

  However, even when a non-specific adsorption inhibitor according to the conventional method is added, non-specific adsorption still remains. Further, when a non-specific adsorption inhibitor derived from a living body is used, there is a problem of biological contamination represented by BSE. For this reason, development of a high-performance nonspecific adsorption inhibitor by chemical synthesis is desired.

  As non-specific adsorption inhibitors by chemical synthesis, polymers having polyoxyethylene are proposed in JP-A-10-153599 and JP-A-11-352127, and a specific methacrylic copolymer is proposed in Japanese Patent No. 3434891. However, these nonspecific adsorption preventing effects were insufficient.

JP-A-10-153599 JP-A-11-352127 Japanese Patent No. 3434891

  The present invention relates to a non-specific adsorption inhibitor capable of preventing non-specific adsorption of proteins and protein-supported latex to solid phase surfaces, articles such as instruments and containers used in immunodiagnostic measurement, and the like. Methods for coating agents and articles are provided.

  In order to solve this problem, the present inventors have found that a copolymer having a specific composition has a high non-specific adsorption preventing effect, and completed the present invention.

The non-specific adsorption inhibitor according to one aspect of the present invention is
A copolymer having a repeating unit (A) represented by the following general formula (1) and a repeating unit (B) represented by the following general formula (2) is contained.

・・・・・（１）
（式中、Ｒ^１は、水素原子またはメチル基を示し、Ｒ^２は、水素原子、メチル基、ヒドロキシメチル基、およびヒドロキシエチル基から選ばれる少なくとも１種を示す。）

(1)
(In the formula, R ¹ represents a hydrogen atom or a methyl group, and R ² represents at least one selected from a hydrogen atom, a methyl group, a hydroxymethyl group, and a hydroxyethyl group.)

・・・・・（２）
（式中、Ｒ^３は水素原子またはメチル基を示し、Ｚは少なくとも１個のアルド基またはケト基を有する炭素数４〜１２の有機基を示す。）

(2)
(In the formula, R ³ represents a hydrogen atom or a methyl group, and Z represents an organic group having 4 to 12 carbon atoms having at least one aldo group or keto group.)

  In the non-specific adsorption inhibitor, in the general formula (2), the organic group represented by Z may have 5 to 9 carbon atoms.

  In the nonspecific adsorption inhibitor, in the general formula (2), the organic group represented by Z can be represented by the following general formula (3).

・・・・・（３）
（式中、Ｘは酸素原子またはＮＨを示し、Ｙは少なくとも１個のアルド基またはケト基を有する炭素数３〜１０の有機基を示す。）

(3)
(In the formula, X represents an oxygen atom or NH, and Y represents an organic group having 3 to 10 carbon atoms having at least one aldo group or keto group.)

  A method for coating an article according to another embodiment of the present invention includes a step of bringing the article into contact with a solution containing the non-specific adsorption inhibitor.

  According to the nonspecific adsorption inhibitor, it contains a copolymer having the repeating unit (A) represented by the general formula (1) and the repeating unit (B) represented by the general formula (2). By doing so, it has a high non-specific adsorption preventing effect.

  Hereinafter, a non-specific adsorption inhibitor according to an embodiment of the present invention, and a method for coating an article using the non-specific adsorption inhibitor and an article will be specifically described.

1. Non-specific adsorption inhibitor 1.1. Non-specific adsorption inhibitor The non-specific adsorption inhibitor according to this embodiment includes a repeating unit (A) represented by the following general formula (1) and a repeating unit (B) represented by the following general formula (2). ).

・・・・・（１）
（式中、Ｒ^１は、水素原子またはメチル基を示し、Ｒ^２は、水素原子、メチル基、ヒドロキシメチル基、およびヒドロキシエチル基から選ばれる少なくとも１種を示す。）

(1)
(In the formula, R ¹ represents a hydrogen atom or a methyl group, and R ² represents at least one selected from a hydrogen atom, a methyl group, a hydroxymethyl group, and a hydroxyethyl group.)

・・・・・（２）
（式中、Ｒ^３は水素原子またはメチル基を示し、Ｚは少なくとも１個のアルド基またはケト基を有する炭素数４〜１２の有機基を示す。）

(2)
(In the formula, R ³ represents a hydrogen atom or a methyl group, and Z represents an organic group having 4 to 12 carbon atoms having at least one aldo group or keto group.)

  In the nonspecific adsorption inhibitor according to this embodiment, in the general formula (2), the organic group represented by Z may have 5 to 9 carbon atoms.

  In the nonspecific adsorption inhibitor according to this embodiment, in the general formula (2), the organic group represented by Z can be represented by the following general formula (3).

・・・・・（３）
（式中、Ｘは酸素原子またはＮＨを示し、Ｙは少なくとも１個のアルド基またはケト基を有する炭素数３〜１０（好ましくは４〜８）の有機基を示す。）

(3)
(In the formula, X represents an oxygen atom or NH, and Y represents an organic group having 3 to 10 (preferably 4 to 8) carbon atoms having at least one aldo group or keto group.)

  The non-specific adsorption inhibitor according to the present embodiment may contain the above copolymer in a part thereof, or may be composed of only the above copolymer.

1.2. Physical Properties and Use of Nonspecific Adsorption Agent In the nonspecific adsorption inhibitor according to this embodiment, the copolymer has a mass average molecular weight of usually 1,000 to 1,000,000, preferably 5,000 to 500,000, more preferably 10,000 to 100,000. In addition, the molecular weight distribution of the copolymer typically has a mass average molecular weight / number average molecular weight of 1.5 to 8. When the weight average molecular weight of the copolymer is less than the above range, the nonspecific adsorption preventing effect may be insufficient. On the other hand, when the weight number average molecular weight of the copolymer is larger than the above range, The viscosity may increase and handling may be difficult.

  The copolymer contained in the nonspecific adsorption inhibitor according to this embodiment is water-soluble. In the present invention, “water-soluble” means that when a copolymer is added to and mixed with water so as to have a polymer solid content of 1% at 25 ° C., it is visually or translucently dissolved. Say.

  The nonspecific adsorption inhibitor according to this embodiment has a high nonspecific adsorption preventing effect. Specifically, the non-specific adsorption inhibitor is considered to act as follows.

  In the non-specific adsorption inhibitor according to this embodiment, the non-specific adsorption inhibitor can be adsorbed on the wall surface of a container / apparatus by the hydrophobic bond of the repeating unit (B) of the copolymer, and By making the wall surface hydrophilic by the repeating unit (A), nonspecific adsorption of protein, lipid, protein-carrying latex and the like can be prevented.

  In the non-specific adsorption inhibitor according to this embodiment, the repeating unit (B) of the copolymer interacts with the protein, and the repeating unit (A) has a dispersing action on water. The conformation can be changed to prevent hydrophobicity, and the protein can be solubilized in an aqueous solvent.

  In the non-specific adsorption inhibitor according to the present embodiment, in particular, the repeating unit (A) and the repeating unit (B) are balanced by including the repeating unit (A) and the repeating unit (B) at a ratio described later. Containing copolymers. For this reason, since adsorption speed is high, it can prevent effectively non-specific adsorption | suction causative substances, such as protein, adsorb | sucking to wall surfaces, such as a container and an instrument.

  The non-specific adsorption inhibitor according to the present embodiment includes, for example, a method of coating an article such as a container / equipment, a method of adding to a diluent of a diagnostic agent, a reaction solvent, or a preservative, a cleaning agent of an automatic diagnostic machine, and a rinse It has the effect of strongly preventing non-specific adsorption of proteins and protein-carrying latex by the method of incorporating into the automatic diagnosis process as a liquid. That is, the article coating method according to an embodiment of the present invention includes a step of bringing the article into contact with a solution containing the non-specific adsorption inhibitor according to the present embodiment.

  Moreover, the nonspecific adsorption inhibitor according to the present embodiment can suppress the signal of a nonspecific sample by using it as a diluent for an immunodiagnostic drug.

  Furthermore, the nonspecific adsorption inhibitor according to the present embodiment includes, for example, a labeled antibody, a labeled antigen, an enzyme, a primary antibody, a primary antigen stabilizer, and a protein stabilizer contained in a plasma preparation used as a clinical diagnostic agent. When added to a protein solution as a stabilizer such as an enzyme used for cleaning contact lenses, it has the effect of maintaining the protein activity for a long period of time.

1.3. Repeating unit (A)
In the copolymer, the repeating unit (A) represented by the general formula (1) can contribute to the development of a high nonspecific adsorption preventing effect.

In the general formula (1), R ¹ represents a hydrogen atom or a methyl group, and R ² represents at least one selected from a hydrogen atom, a methyl group, a hydroxymethyl group, and a hydroxyethyl group. The repeating unit (A) has the following general formula (1): R ¹ = R ² = hydrogen atom, R ¹ = R ² = methyl group, R ¹ = hydrogen atom and R ² = hydroxymethyl group, or R ¹ = preferably having a hydrogen atom and ^{R 2} = hydroxyethyl ^groups, those of ^R 1 = R ² = methyl group is more preferable.

1.4. Repeating unit (B)
In the copolymer, the repeating unit (B) represented by the general formula (2) contributes to the development of a higher non-specific adsorption preventing effect by shifting the hydrophilic / hydrophobic balance of the copolymer to the hydrophobic side. can do.

In the general formula (2), R ³ represents a hydrogen atom or a methyl group, preferably a hydrogen atom. Z represents an organic group having 4 or more carbon atoms having at least one aldo group or keto group, and preferably 5 carbon atoms having one aldo group or keto group and one ester group or amide group. To 9 organic groups (for example, in the above general formula (2), the organic group represented by Z is a group represented by the above general formula (3)). More specifically, Z is an organic group having an aldo group such as an ester group containing an aldo group represented by the following general formula (4), an amide group containing an aldo group represented by the following general formula (5);

・・・・・（４）
（式中、Ｒ^１０〜Ｒ^１３は独立して、水素原子、メチル基、またはエチル基である。）

(4)
(In the formula, R ^{10 to} R ¹³ are each independently a hydrogen atom, a methyl group, or an ethyl group.)

・・・・・（５）
（式中、Ｒ^１４〜Ｒ^１７は独立して、水素原子、メチル基、またはエチル基である。）

(5)
(Wherein R ^{14 to} R ¹⁷ are independently a hydrogen atom, a methyl group, or an ethyl group.)

  An organic group having a keto group such as an ester group containing a keto group represented by the following general formula (6) and an amide group containing a keto group represented by the following general formula (7).

(6)
(Wherein R ^{18 to} R ²¹ are independently a hydrogen atom, a methyl group, or an ethyl group.)

・・・・・（７）
（式中、Ｒ^２２〜Ｒ^２５は独立して、水素原子、メチル基、またはエチル基である。）
Ｚとして最も好ましい有機基は、下記式（８）で示される有機基である。

(7)
(In the formula, R ^{22 to} R ²⁵ are each independently a hydrogen atom, a methyl group, or an ethyl group.)
The most preferable organic group as Z is an organic group represented by the following formula (8).

・・・・・（８）

(8)

  Moreover, the said copolymer should just contain 1 or more types of repeating unit (A) and repeating unit (B), respectively. In addition, the said copolymer may contain other repeating units (C) other than a repeating unit (A) and a repeating unit (B).

2. Manufacture of nonspecific adsorption inhibitor Next, the monomer composition used in order to manufacture the said copolymer is demonstrated.

2.1. Monomer (a)
The repeating unit (A) has a structure derived from at least one monomer (a). The monomer (a) is preferably at least one monomer selected from acrylamide and N-substituted monomers of acrylamide.

  Examples of N-substituted monomers for acrylamide include N-methylacrylamide, N, N-dimethylacrylamide, N-hydroxymethylacrylamide, N-hydroxyethylacrylamide, N-methyl-N-hydroxyethylacrylamide, and the like. .

  The monomer (a) is more preferably at least one selected from acrylamide, N-hydroxymethylacrylamide, N-hydroxyethylacrylamide, and N, N-dimethylacrylamide, and more preferably N, N-dimethylacrylamide. It is.

2.2. Monomer (b)
The repeating unit (B) has a structure derived from at least one monomer (b).

  The monomer (b) is preferably (meth) acrylates or (meth) acrylamides represented by the above formula (3) (more specifically, groups represented by the above formulas (4) to (8)). (Meth) acrylates or (meth) acrylamides) having at least one selected from dimethacetone acrylamide from the viewpoint of copolymerization.

2.3. Other monomer (c)
The other repeating unit (C) has a structure derived from at least one monomer (c).

As the monomer (c), preferably,
N-substituted monomers of acrylamide other than the monomer (a) such as N, N-diethylacrylamide, N-isopropylacrylamide, acryloylmorpholine, acryloylpyrrolidine, acryloylpiperidine;
(Meth) such as methoxyethyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, hydroxyethyl (meth) acrylate, glycerol (meth) acrylate, polyoxyethylene (meth) acrylate Acrylate;
Anionic monomers such as acrylic acid, methacrylic acid, styrene sulfonic acid, isoprene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid;
Examples include cationic monomers having primary to quaternary amino groups such as allylamine, aminostyrene, N, N-dimethylaminopropylacrylamide, and methyl chloride quaternary salt of N, N-dimethylaminopropylacrylamide.

  The monomer composition for producing the copolymer is preferably 100% by mass of all monomers, preferably 1 to 99% by mass of monomer (a), 1 to 99% by mass of monomer (b), and other monomers (c ) 0 to 98% by mass, more preferably 10 to 90% by mass of monomer (a), 10 to 90% by mass of monomer (b), and 0 to 80% by mass of other monomer (c).

  If the monomers (a) and (b) are out of the above range, the nonspecific adsorption preventing effect may be inferior.

  Further, the mass ratio of the amount of the monomer (a) used and the amount of the monomer (b) used is more preferably 1:99 to 99: 1, and further preferably 10:90 to 90:10. When the amount of the monomer (b) used is less than 99: 1 with respect to the amount of the monomer (a) used, the non-specific adsorption preventing effect may be inferior. It may become impossible to coat.

  The monomer to be used can be used for copolymerization after being purified as an industrial raw material or unpurified.

  The polymerization of the monomer can be performed by a known polymerization method such as radical polymerization, anionic polymerization, or cationic polymerization, and is preferably radical polymerization because of easy production.

  The polymerization of the monomer is carried out by stirring and heating together with a known solvent, initiator, chain transfer agent and the like. The polymerization time is usually 30 minutes to 24 hours, and the polymerization temperature is about 0 to 120 ° C.

  The aqueous copolymer solution after the polymerization is preferably purified by a dialysis membrane, a dialyzer, an acylizer or the like.

3. Examples and Comparative Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

  In this example, the mass average molecular weight (Mw) and the number average molecular weight (Mn) are TSKgel α-M columns manufactured by Tosoh Corporation, the flow rate is 1 ml / min, and the elution solvent is 0.1 mM sodium chloride aqueous solution / acrylonitrile. It was measured by gel permeation chromatography (GPC) using monodisperse polyethylene glycol as a standard under analysis conditions of a mixed solvent and a column temperature of 40 ° C. Absorbance was measured with a model 680 microplate reader manufactured by Nippon Bio-Rad Laboratories.

3.1. Example 1
3.1.1. Synthesis of non-specific adsorption inhibitor (N-1) 50 g of dimethylacrylamide as monomer (a), 50 g of diacetone acrylamide as monomer (b), and 0.1 g of mercaptopropionic acid as chain transfer agent are mixed with 900 g of water and equipped with a stirrer. Placed in a separable flask. While nitrogen was blown into this, the temperature was raised to 70 ° C., and 4 g of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride was added as an initiator, and then polymerization was continued for 2 hours. The mixture was further heated to 80 ° C. and aged for 3 hours, and then cooled to room temperature. The obtained copolymer solution was purified by a dialyzer and further freeze-dried to obtain 95 g of a non-specific adsorption inhibitor (N-1) of this example.

  The number average molecular weight by GPC of the nonspecific adsorption inhibitor (N-1) was 12,000, and the mass average molecular weight was 44,000.

3.1.2. Measurement of Nonspecific Adsorption Prevention Effect A 0.5% aqueous solution of a nonspecific adsorption inhibitor (N-1) was filled in a 96-well polystyrene plate (hereinafter referred to as “96-well plate”) and incubated at 37 ° C. for 30 minutes. Then, it was washed 5 times with exchange water. Next, a 96-well plate was filled with an aqueous solution of horseradish peroxidase-labeled mouse IgG antibody ("AP124P" manufactured by Millipore), incubated at room temperature for 30 minutes, then washed 3 times with PBS buffer, and TMB (3, 3 ', The color was developed with 5,5′-tetramethylbenzylidene) / hydrogen peroxide solution / sulfuric acid, and the absorbance at 450 nm was measured.

3.2. Examples 2 and 3
The same operation as in Example 1 was performed except that the monomer ratio shown in Table 1 was used as the monomer.

3.3. Comparative Example 1
In Example 1, instead of using 50 g of dimethylacrylamide as the monomer (a) and 50 g of diacetone acrylamide as the monomer (b), only “100 g of dimethylacrylamide” was used as the monomer. Copolymer (X-1) was obtained in the same manner as in “Synthesis of adsorption inhibitor (N-1)”.

  The number average molecular weight by GPC of the copolymerized polymer (X-1) was 14,000, and the mass average molecular weight was 84,000.

  Moreover, the light absorbency at the time of using copolymerization polymer (X-1) was measured by the method similar to "3.1.2. Measurement of nonspecific adsorption | suction prevention effect."

3.4. Comparative Example 2
In Example 1, instead of using 50 g of dimethylacrylamide as the monomer (a) and 50 g of diacetone acrylamide as the monomer (b), only 3 g of diacetone acrylamide was used as the monomer, and “3.1.1. Polymerization was carried out in the same manner as in “Synthesis of specific adsorption inhibitor (N-1)”, but since a white substance insoluble in water was generated and stirring became difficult, the polymerization was stopped.

3.5. Comparative Example 3
In Example 1, using bovine serum albumin (BSA) instead of the non-specific adsorption inhibitor (N-1), in the same manner as “3.1.2. Measurement of non-specific adsorption inhibitory effect”, Absorbance was measured when BSA was used.

3.6. Comparative Example 4
In Example 1, using a commercially available polyoxyethylene alkyl ether instead of the non-specific adsorption inhibitor (N-1), the same method as in “3.1.2. Measurement of non-specific adsorption prevention effect” was used. The absorbance was measured.

3.7. Measurement Results Table 1 shows the measurement results of the nonspecific adsorption preventing effect in the above Examples and Comparative Examples.

Claims (4)

A non-specific adsorption inhibitor comprising a copolymer having a repeating unit (A) represented by the following general formula (1) and a repeating unit (B) represented by the following general formula (2).

(1)
(In the formula, R ¹ represents a hydrogen atom or a methyl group, and R ² represents at least one selected from a hydrogen atom, a methyl group, a hydroxymethyl group, and a hydroxyethyl group.)

(2)
(In the formula, R ³ represents a hydrogen atom or a methyl group, and Z represents an organic group having 4 to 12 carbon atoms having at least one aldo group or keto group.)   The nonspecific adsorption inhibitor according to claim 1, wherein the organic group represented by Z in the general formula (2) has 5 to 9 carbon atoms. The nonspecific adsorption inhibitor according to claim 1 or 2, wherein the organic group represented by Z in the general formula (2) is represented by the following general formula (3).

(3)
(In the formula, X represents an oxygen atom or NH, and Y represents an organic group having 3 to 10 carbon atoms having at least one aldo group or keto group.)   A method for coating an article, comprising the step of bringing the article into contact with a solution containing the nonspecific adsorption inhibitor according to any one of claims 1 to 3.