JP5207012B2 - Non-specific adsorption inhibitor for biological substances and method for coating articles - Google Patents

Description

  The present invention relates to a nonspecific adsorption inhibitor for biologically related substances that prevents nonspecific adsorption of biologically related substances such as various proteins used in clinical diagnostic agents, clinical diagnostic devices, biochips, etc., and the nonspecific adsorption prevention The present invention relates to a method for coating an article using an agent.

  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, the method of using enzyme coloring as a detection method is being switched to a method using fluorescence and chemiluminescence that can obtain 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 specific substances in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. are adsorbed non-specifically to solid phases, instruments, containers, etc. As a result, 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 nonspecific adsorption inhibitor for biologically related substances capable of preventing nonspecific adsorption of biologically related substances such as proteins to solid phase surfaces, instruments and containers used in chemiluminescent immunodiagnostic measurements, and the like. An article coating method using the non-specific adsorption inhibitor is 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 on a biological substance and completed the present invention.

The non-specific adsorption inhibitor for biologically related substances 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)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and Z represents a group represented by the following formula (1a) or (1b)).

(1a)
Wherein R ¹ and R ² are independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or at least one group selected from a hydroxyl group, a carboxyl group, an alkoxy group, an acyloxy group, and an alkoxycarbonyl group. And an alkyl group having 1 to 8 carbon atoms substituted with

(1b)
(Wherein R ³ and R ⁴ are each independently a single bond, methylene, methylene substituted with a hydroxyl group or a carboxyl group, an alkylene group of 2 to 7 carbon atoms, or a carbon number of 2 substituted with a hydroxyl group or a carboxyl group. To 7 alkylene groups (wherein the total number of carbon atoms of R ³ and R ⁴ is 4 to 10), at least one of R ³ and R ⁴ may have an ether bond, and Y is Indicates a single bond, O, or S.)

(2)
(Wherein R ⁵ represents a hydrogen atom or a methyl group, R ⁶ represents a phenyl group or —CO ₂ R ⁷ (wherein R ⁷ represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alicyclic group). Represents a hydrocarbon group or an aromatic hydrocarbon group.)

  In the present invention, the biological substance refers to lipid, protein, saccharide, or nucleic acid.

In the non-specific adsorption inhibitor for a biological substance, in the general formula (1), R ¹ represents a hydrogen atom or a methyl group, and R ² is at least one selected from a hydrogen atom, a methyl group, and a hydroxyethyl group. Can be shown.

  In the non-specific adsorption inhibitor for a biological substance, the repeating unit (B) may have a structure derived from at least one monomer having a solubility in water of less than 20%.

In the non-specific adsorption inhibitor for a biological substance, in the general formula (2), R ⁵ represents a hydrogen atom or a methyl group, R ⁶ represents a —CO ₂ R ⁷ group, and R ⁷ represents a methyl group. It may be at least one selected from a group, an ethyl group, and a methoxyethyl group.

  In the non-specific adsorption inhibitor for a biological substance, the copolymer may further include a repeating unit (C) represented by the following general formula (3).

(3)
(In the formula, R ⁸ represents a hydrogen atom or a methyl group, and R ⁹ represents an organic group having at least one aldo group or keto group.)

  The non-specific adsorption inhibitor for the biological substance may further contain a hydrazide compound (H) having at least two hydrazino groups per molecule.

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

  According to the nonspecific adsorption inhibitor for biologically related substances, a co-polymer having the repeating unit (A) represented by the general formula (1) and the repeating unit (B) represented by the general formula (2). By containing a polymer, it has a high non-specific adsorption preventing effect.

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

1. Nonspecific adsorption inhibitor for biological substances 1.1. Non-specific adsorption inhibitor for bio-related substances The non-specific adsorption inhibitor for bio-related substances according to this embodiment includes a repeating unit (A) represented by the following general formula (1) and the following general formula (2). The copolymer which has a repeating unit (B) represented by these is contained.

(1)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and Z represents a group represented by the following formula (1a) or (1b)).

(1a)
(In the formula, R ¹ and R ² are each independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or a carbon number of 1 substituted with at least one group selected from a hydroxyl group, a carboxyl group, and an alkoxy group. ˜8 alkyl groups.)

(1b)
(Wherein R ³ and R ⁴ are each independently a single bond, methylene, methylene substituted with a hydroxyl group or a carboxyl group, an alkylene group of 2 to 7 carbon atoms, or a carbon number of 2 substituted with a hydroxyl group or a carboxyl group. To 7 alkylene groups (wherein the total number of carbon atoms of R ³ and R ⁴ is 4 to 10), at least one of R ³ and R ⁴ may have an ether bond, and Y is Indicates a single bond, O, or S.)

(2)
(Wherein R ⁵ represents a hydrogen atom or a methyl group, R ⁶ represents a phenyl group or —CO ₂ R ⁷ (wherein R ⁷ represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alicyclic group). Represents a hydrocarbon group or an aromatic hydrocarbon group.)

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

1.2. Physical properties and uses of non-specific adsorption inhibitor for biological substances In the non-specific adsorption inhibitor for biological substances according to this embodiment, the number average molecular weight of the copolymer is usually 1,000 to 1,000,000. Yes, preferably from 2,000 to 100,000, more preferably from 3,000 to 50,000. In addition, the molecular weight distribution of the copolymer typically has a weight average molecular weight / number average molecular weight of 1.5 to 3. When the number average molecular weight of the copolymer is less than the above range, the non-specific adsorption preventing effect may be insufficient. On the other hand, when the number average molecular weight of the copolymer is larger than the above range, the viscosity of the solution This is because there is a case where coating and handling become difficult due to an increase in.

  The copolymer contained in the non-specific adsorption inhibitor of the biological substance 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 biospecific substance non-specific adsorption inhibitor according to the present embodiment has a high non-specific adsorption preventive effect. Specifically, the non-specific adsorption inhibitor for biologically related substances according to the present embodiment can act as follows.

  In the nonspecific adsorption inhibitor for biologically related substances according to the present embodiment, the nonspecific adsorption inhibitor can be adsorbed on the wall surface of a container / equipment or the like by hydrophobic bonding of the repeating unit (B) of the copolymer. Further, non-specific adsorption of proteins, lipids and the like can be prevented by making the wall surface hydrophilic by the repeating unit (A).

  In the non-specific adsorption inhibitor for biological materials according to the present embodiment, the adsorption rate is high by including a copolymer in which the repeating unit (A) and the repeating unit (B) are balanced. It is possible to effectively prevent non-specific adsorption-causing substances such as those from adsorbing to the walls of containers and instruments.

  Moreover, in the nonspecific adsorption inhibitor for biologically related substances according to this embodiment, the repeating unit (B) of the copolymer interacts with a protein, and the repeating unit (A) has a dispersing action on water. Therefore, the protein can be prevented from changing its conformation to become hydrophobic, and the protein can be solubilized in an aqueous solvent.

  The non-specific adsorption inhibitor for biologically related substances according to the present embodiment is, for example, a non-specific adsorption of protein or the like by a method of coating on a container / equipment or a method of adding to a diluent, reaction solvent, or preservative of a diagnostic agent. It has the effect of preventing strongly. That is, the method for coating an article according to an embodiment of the present invention includes a step of bringing the article into contact with a solution containing a nonspecific adsorption inhibitor for a biological substance according to this embodiment.

  Moreover, the nonspecific adsorption inhibitor of the biological substance according to the present embodiment can suppress the signal of the nonspecific specimen by using it as a diluted solution of an immunodiagnostic drug.

  Furthermore, the non-specific adsorption inhibitor for biologically related substances according to the present embodiment includes, for example, labeled antibodies, labeled antigens, enzymes, primary antibodies, primary antigen stabilizers, and proteins contained in plasma preparations used as clinical diagnostic agents. When added to a protein solution as a stabilizing agent such as, a stabilizing agent 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 (1a), examples of the substituted or unsubstituted alkyl group having 1 to 8 carbon atoms represented by R ¹ and R ² include a linear or branched substituted or unsubstituted alkyl group. , A methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, and these groups are, for example, a hydroxyl group, Examples include groups substituted with a functional group such as an alkoxy group.

More specifically, in the above following general formula (1), R ¹ represents a hydrogen atom or a methyl group, R ² is indicative of at least one hydrogen atom, selected from methyl, and hydroxyethyl groups It is preferable.

  Examples of the ring structure corresponding to the general formula (1b) include pyrrolidino group, piperidino group, morpholino group, thiomorpholino group and the like.

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), examples of the substituted or unsubstituted alkyl group having 1 to 12 carbon atoms represented by R ⁷ include a linear or branched substituted or unsubstituted alkyl group.
Methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, and these groups are, for example, hydroxyl, alkoxy And a group substituted with a functional group such as a group.

In the general formula (2), examples of the alicyclic hydrocarbon group represented by R ⁷ include an isobonyl group and a cyclohexyl group, and examples of the aromatic hydrocarbon group represented by R ⁷ include: An example is a benzyl group.

In the general formula (2), ^{R 5} represents a hydrogen atom or a methyl group, a ^{group R 6} is represented by _-CO 2 ^{R 7, R 7} is a methyl group, an ethyl group, and methoxyethyl It is preferable to show at least one selected from the group.

  Further, the copolymer may contain at least one or more repeating units (A) and repeating units (B). In addition, the said copolymer may contain other repeating units (C) other than a repeating unit (A) and a repeating unit (B).

1.5. Repeating unit (C)
In the non-specific adsorption inhibitor for biologically related substances according to this embodiment, the copolymer may further contain a repeating (C) unit represented by the following general formula (3). In the copolymer, the repeating unit (C) can contribute to the improvement of the durability of the coating film (nonspecific adsorption inhibitor layer) to be formed.

(3)
(In the formula, R ⁸ represents a hydrogen atom or a methyl group, and R ⁹ represents an organic group having at least one aldo group or keto group.)

  In the present invention, the aldo group means an aldehyde bonded to a carbon atom, the keto group means a carbonyl group bonded to two carbon atoms, and does not include a carboxyl group or an amide group.

In the above general formula (3), R ⁸ is preferably a hydrogen atom. R ⁹ is preferably an aldo group such as a formyl group, a formylphenyl group, 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) An organic group having

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

(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 acetyl group, an acetylphenyl group, an ester group containing a keto group represented by the following general formula (6), 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)
(In the formula, R ^{22 to} R ²⁵ are each independently a hydrogen atom, a methyl group, or an ethyl group.)

More preferable organic groups as R ⁹ are a formyl group, an acetyl group, and an organic group represented by the following formula (8), and the most preferred organic group is an organic group represented by the following formula (8).

(8)

1.6. Hydrazide compound (H)
The hydrazide compound (H) has at least two hydrazino groups in one molecule. Examples of such hydrazide compounds (H) include oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, sebacic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, Dicarboxylic acid dihydrazides having 2 to 10 carbon atoms, particularly 4 to 6 carbon atoms, such as acid dihydrazide, fumaric acid dihydrazide, itaconic acid dihydrazide; citric acid trihydrazide, nitriloacetic acid trihydrazide, cyclohexanetricarboxylic acid trihydrazide, ethylenediaminetetraacetic acid tetraacetate Trifunctional or higher hydrazides such as hydrazide; ethylene-1,2-dihydrazine, propylene-1,2-dihydrazine, propylene-1,3-dihydrazine, butylene -1,2-dihydrazine, butylene-1,3-dihydrazine, butylene-1,4-dihydrazine, butylene-2,3-dihydrazine, etc. aliphatic dihydrazines having 2 to 4 carbon atoms in total, etc. Water-soluble dihydrazine and at least some hydrazino groups of these polyfunctional hydrazine derivatives are substituted with acetaldehyde, propionaldehyde, butyraldehyde, acetone, methyl ethyl ketone, diethyl ketone, methyl-
Examples thereof include compounds blocked by reacting with carbonyl compounds such as n-propyl ketone, methyl-n-butyl ketone and diacetone alcohol, such as adipic acid dihydrazide monoacetone hydrazone and adipic acid dihydrazide diacetone hydrazone. Among these hydrazide compounds (H), at least one selected from adipic acid dihydrazide, sebacic acid dihydrazide, isophthalic acid dihydrazide, and adipic acid dihydrazide diacetone hydrazone is preferable. A hydrazide compound (H) can be used individually or in mixture of 2 or more types.

  The amount of the hydrazide compound (H) used is such that the molar equivalent ratio of the total amount of aldo groups and keto groups in the copolymer to the hydrazino group of the hydrazide compound (H) is 1: 0.1 to 5, preferably The amount is preferably in the range of 1: 0.5 to 1.5, more preferably 1: 0.7 to 1.2. In this case, when the hydrazino group is less than 0.1 equivalent to the total of 1 equivalent of the aldo group and the keto group, the durability of the formed film (non-specific adsorption inhibitor layer) may be inferior, If it exceeds 5 equivalents, the nonspecific adsorption preventing effect may be reduced.

  The hydrazide compound (H) can be blended in an appropriate process for preparing the non-specific adsorption inhibitor for biologically related substances according to the present embodiment, but in order to maintain the polymerization stability during the production of the copolymer. The total amount of the hydrazide compound (H) is preferably blended after production of the copolymer.

  The hydrazide compound (H) reacts with the keto group and / or aldo group of the copolymer in the drying process after application of the non-specific adsorption inhibitor of the biological substance according to this embodiment, It has a function of forming a hydrophilic network structure and crosslinking the non-specific adsorption inhibitor layer. This crosslinking reaction usually proceeds at room temperature without using a catalyst, but if necessary, a catalyst such as a water-soluble metal salt such as zinc sulfate, manganese sulfate, cobalt sulfate or the like is added, or it is heated and dried. Can be promoted.

2. Manufacture of non-specific adsorption inhibitor for biological substances Next, the monomer composition used to manufacture the copolymer will be described.

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, N-dimethylacrylamide, N, N-diethylacrylamide, N-isopropylacrylamide, N-hydroxyethylacrylamide, acryloylmorpholine, acryloylpyrrolidine, and acryloylpiperidine. .

  The monomer (a) is more preferably at least one selected from acrylamide, N-hydroxyethylacrylamide, and N, N-dimethylacrylamide, and more preferably N, N-dimethylacrylamide or N, N-dimethyl. Combination of acrylamide and N, N-diethylacrylamide.

2.2. Monomer (b)
The repeating unit (B) has a structure derived from at least one monomer (b). Monomer (b) is at least one monomer having a solubility in water of less than 20%.

In the present invention, “a monomer having a solubility in water of less than 20%” means that the monomer is separated from the aqueous phase after the monomer is added and stirred so that the monomer concentration in water at 25 ° C. is 20%. A monomer that can be confirmed visually.

  When the solubility of the monomer (b) in water is less than 20%, a higher nonspecific adsorption preventing effect can be exhibited.

  Examples of the monomer (b) include methoxyethyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, and (meth) acrylic acid. Examples include 2-ethylhexyl, lauryl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, benzyl (meth) acrylate, and styrene. The monomer (b) is more preferably at least one selected from methyl methacrylate, ethyl acrylate, and methoxyethyl acrylate.

2.3. Monomer composition and polymerization In the nonspecific adsorption inhibitor for biologically related substances according to the present embodiment, the copolymer may contain a repeating unit (C) in addition to the repeating units (A) and (B), Furthermore, a repeating unit (D) may be included.

  The monomer (c) is a component for forming the repeating unit (C), and the monomer (d) is a component for forming the repeating unit (D). That is, the repeating unit (C) has a structure derived from at least one monomer (c), and the repeating unit (D) has a structure derived from at least one monomer (d).

  The monomer (c) is preferably selected from acrolein, formylstyrene, vinyl methyl ketone, vinyl phenyl ketone, (meth) acrylate having a group represented by the above formulas (4) to (7), and (meth) acrylamides. And at least one selected from the viewpoint of copolymerization, more preferably at least one selected from acrolein, vinyl methyl ketone, and diacetone acrylamide, and most preferable from the viewpoint of monomer safety. Acetone acrylamide.

  As other monomer (d), 1 to 10% by weight of an anionic monomer, in particular, styrene sulfonic acid, isoprene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, etc. are used as monomer (a) and monomer (b). Further, by producing a copolymer by copolymerizing with the monomer (c) if necessary, the signal suppression effect of a non-specific specimen may be obtained when used as a diluent for an immunodiagnostic drug.

  The monomer composition for producing the copolymer is preferably based on 100% by weight of all monomers, preferably 30% to 99% by weight of monomer (a), 1% to 70% by weight of monomer (b), 0% to monomer (c) 49% by weight, and other monomer (d) 0-49% by weight, more preferably monomer (a) 40-95% by weight, monomer (b) 5-60% by weight, monomer (c) 0-30 % By weight, and other monomer (d) 0-20% by weight. In particular, the monomer composition when durability is required is 100% by weight of the total monomers, preferably 30% to 97% by weight of monomer (a), 1% to 68% by weight of monomer (b), 2% to 49% of monomer (c). % By weight, and other monomer (d) 0-49% by weight.

  If the monomers (a) and (b) are out of the above range, the nonspecific adsorption preventing effect may be inferior. Further, when the monomer (c) is less than 2% by weight, the durability may be inferior, and when it exceeds 49% by weight, the nonspecific adsorption preventing effect may be inferior.

  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 weight 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) 60 g of dimethylacrylamide as monomer (a), 30 g of diethylacrylamide, 10 g of methyl methacrylate as monomer (b), and 1 g of cysteamine hydrochloride as chain transfer agent were mixed in 900 g of water. And put into a separable flask equipped with a stirrer. While nitrogen was blown into this, the temperature was raised to 70 ° C., 2 g of 2,2′-azobis (2-methylpropionamidine) dihydrochloride was added as an initiator, polymerization was continued for 2 hours, and the temperature was further raised to 80 ° C. After warming and aging for 3 hours, it was 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 non-specific adsorption inhibitor (N-1) by GPC was 8,000, and the weight average molecular weight was 16,000.

3.1.2. Measurement of non-specific adsorption preventing effect 0.5% aqueous solution of non-specific adsorption inhibitor (N-1) was filled in a polystyrene 96-well 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, washed 3 times with PBS buffer, and TMB (3, 3 ', The color was developed with 5,5′-tetramethylbenzidene) / 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 60 g of dimethylacrylamide as the monomer (a) and 30 g of diethylacrylamide and 10 g of methyl methacrylate as the monomer (b), only “100 g of diethylacrylamide” was used as the monomer. The copolymer polymer (X-1) was obtained in the same manner as in “1.1. Synthesis of nonspecific adsorption inhibitor (N-1)”.

  The number average molecular weight by GPC of the copolymer (X-1) was 5,200, and the weight average molecular weight was 13,000.

  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 60 g of dimethylacrylamide as the monomer (a) and 30 g of diethylacrylamide and 10 g of methyl methacrylate as the monomer (b), only 100 g of dimethylacrylamide was used as the monomer. The copolymer (X-2) was obtained in the same manner as in “1.1. Synthesis of nonspecific adsorption inhibitor (N-1)”.

  The number average molecular weight by GPC of the copolymerized polymer (X-2) was 9,800, and the weight average molecular weight was 20,000.

  Moreover, the light absorbency at the time of using copolymer copolymer (X-2) was measured by the method similar to "3.1.2. Measurement of nonspecific adsorption | suction prevention effect."

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, instead of using 60 g of dimethylacrylamide as the monomer (a) and 30 g of diethylacrylamide and 10 g of methyl methacrylate as the monomer (b), only 3 g of methyl methacrylate was used as the monomer. 1.1.1 Synthesis of non-specific adsorption inhibitor (N-1) ”was performed, but a large amount of white coagulum was generated several minutes after the addition of the initiator, and thus the polymerization was stopped.

3.7. Comparative Example 5
In Example 1, using commercially available polyvinylpyrrolidone instead of the non-specific adsorption inhibitor (N-1), BSA was prepared in the same manner as in “3.1.2. Measurement of non-specific adsorption prevention effect”. Absorbance was measured when used 3.8. Measurement Results Table 1 shows the measurement results of the nonspecific adsorption preventing effect in the above Examples and Comparative Examples.

  According to Table 1, by using the nonspecific adsorption inhibitor for biologically relevant substances according to Examples 1 to 3, a polymer derived only from diethylacrylamide or dimethylacrylamide corresponding to monomer (a), bovine serum albumin It was confirmed that the amount of non-specific adsorption of the mouse IgG antibody to the 96-well plate can be significantly reduced as compared with the case of using.

3.9. Reference example 1
3.9.1. Synthesis of non-specific adsorption inhibitor (N-4) In Example 1, instead of using 60 g of dimethylacrylamide and 30 g of diethylacrylamide as the monomer (a) and 10 g of methyl methacrylate as the monomer (b), the monomer (a) Except for using 56 g of dimethylacrylamide and 16 g of diethylacrylamide, 8 g of methyl methacrylate as the monomer (b), and 20 g of diacetone acrylamide as the monomer (c), “3.1.1. Non-specific adsorption inhibitor (N-1)” The nonspecific adsorption inhibitor (N-4), which is a copolymer, was obtained in the same manner as in “Synthesis of”.

  The number average molecular weight by non-specific adsorption inhibitor (N-4) by GPC was 9,000, and the weight average molecular weight was 25,000.

  Moreover, the light absorbency at the time of using a nonspecific adsorption inhibitor (N-4) was measured by the method similar to "3.1.2. Measurement of nonspecific adsorption prevention effect".

3.9.2. Measurement of non-specific adsorption preventing effect after washing with surfactant Filled 96-well plate with 0.5% aqueous solution of non-specific adsorption inhibitor (N-4), incubated at 37 ° C. for 30 minutes, and then washed with replacement water The remaining water was blown away with an air gun. This was dried at 40 ° C. for 3 hours. Furthermore, it washed 3 times with polyoxyethylene sorbitan monolaurate which is surfactant. 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, washed 3 times with PBS buffer, and TMB (3, 3 ', The color was developed with 5,5′-tetramethylbenzidene) / hydrogen peroxide solution / sulfuric acid, and the absorbance at 450 nm was measured.

3.10. Reference example 2
1 g of adipic acid dihydrazide is added to 10 g of the nonspecific adsorption inhibitor (N-4), and a 0.55% aqueous solution containing the nonspecific adsorption inhibitor (N-4) and adipic acid dihydrazide is used. Measurement of non-specific adsorption prevention effect and measurement of non-specific adsorption prevention effect after washing with surfactant were carried out.

3.11. Comparative Example 6
Using BSA, the non-specific adsorption preventing effect and the non-specific adsorption preventing effect after washing with a surfactant were measured.

3.12. Measurement Results Table 2 shows the measurement results in Reference Examples 1 and 2 and Comparative Example 6 described above.

According to Table 2, by using the non-specific adsorption inhibitor for biologically relevant substances according to Reference Example 1 and Reference Example 2 , compared with the case of using BSA in Comparative Example 6, after washing with a surfactant. It was confirmed that non-specific adsorption can be reduced. In particular, according to Reference Example 2 , it was confirmed that nonspecific adsorption can be significantly reduced after washing with a surfactant by using a nonspecific adsorption inhibitor containing a hydrazide compound (H).

Claims (10)

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) ;
The said copolymer is a nonspecific adsorption | suction prevention agent of a biological relevant substance which does not contain the repeating unit (C) represented by following General formula (3) .
(1)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and Z represents a group represented by the following formula (1a) or (1b)).
(1a)
Wherein R ¹ and R ² are independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or at least one group selected from a hydroxyl group, a carboxyl group, an alkoxy group, an acyloxy group, and an alkoxycarbonyl group. And an alkyl group having 1 to 8 carbon atoms substituted with
(1b)
(Wherein R ³ and R ⁴ are each independently a single bond, methylene, methylene substituted with a hydroxyl group or a carboxyl group, an alkylene group of 2 to 7 carbon atoms, or a carbon number of 2 substituted with a hydroxyl group or a carboxyl group. To 7 alkylene groups (wherein the total number of carbon atoms of R ³ and R ⁴ is 4 to 10), at least one of R ³ and R ⁴ may have an ether bond, and Y is Indicates a single bond, O, or S.)
(2)
(Wherein R ⁵ represents a hydrogen atom or a methyl group, R ⁶ represents a phenyl group or —CO ₂ R ⁷ (wherein R ⁷ represents a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, an alicyclic group). Represents a hydrocarbon group or an aromatic hydrocarbon group.)
(3)
(In the formula, R ⁸ represents a hydrogen atom or a methyl group, and R ⁹ represents an organic group having at least one aldo group or keto group.) In the general formula (1),
R ¹ represents a hydrogen atom or a methyl group,
The nonspecific adsorption inhibitor for biologically relevant substances according to claim 1, wherein R ² represents at least one selected from a hydrogen atom, a methyl group, and a hydroxyethyl group.   The nonspecific adsorption inhibitor for biologically relevant substances according to claim 1 or 2, wherein the repeating unit (B) has a structure derived from at least one monomer having a solubility in water of less than 20%. In the above general formula (2),
R ⁵ represents a hydrogen atom or a methyl group,
R ⁶ represents a —CO ₂ R ⁷ group,
The nonspecific adsorption inhibitor for biologically relevant substances according to any one of claims 1 to 3, wherein R ⁷ represents at least one selected from a methyl group, an ethyl group, and a methoxyethyl group. The nonspecific adsorption inhibitor for biologically relevant substances according to any one of claims 1 to 4, wherein the copolymer has a number average molecular weight of 1,000 to 1,000,000. The nonspecific adsorption inhibitor for biologically relevant substances according to any one of claims 1 to 5, wherein the copolymer has a (weight average molecular weight / number average molecular weight) value of 1.5 to 3. The repeating unit (A) is formed by polymerizing at least one monomer selected from the group consisting of acrylamide and an N-substituted monomer of acrylamide,
  The N-substituted monomer of the acrylamide is selected from the group consisting of N, N-dimethylacrylamide, N, N-diethylacrylamide, N-isopropylacrylamide, N-hydroxyethylacrylamide, acryloylmorpholine, acryloylpyrrolidine, and acryloylpiperidine. The nonspecific adsorption inhibitor for biologically relevant substances according to any one of claims 1 to 6, which is at least one kind. The repeating unit (B) is methoxyethyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, (meth) acrylic acid 2 -By polymerizing at least one monomer selected from the group consisting of ethylhexyl, lauryl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, benzyl (meth) acrylate, and styrene. The nonspecific adsorption inhibitor for biologically related substances according to any one of claims 1 to 7, which is formed. In the copolymer, the co-polymerization of the monomer (a) forming the repeating unit (A), the monomer (b) forming the repeating unit (B), and the monomer (d) forming another structure The monomer composition in the coalescence is 30% to 99% by weight of monomer (a), 1 to 70% by weight of monomer (b), and 0 to 49% by weight of monomer (d) based on 100% by weight of all monomers. The nonspecific adsorption inhibitor for biologically relevant substances according to any one of 8 to 8. A method for coating an article, comprising the step of bringing the article into contact with a solution containing the nonspecific adsorption inhibitor for a biological substance according to any one of claims 1 to 9 .