JP2018155601A - Blocking agent for biological analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a blocking agent for biological analysis capable of sufficiently covering a function that does not prevent a color from being developed by specific adsorption of a dyeing agent for the target protein, and preventing a non-specific adsorption of a color agent for the protein other than the target by sufficiently covering the protein other than the target, and capable of stably expressing the function for preventing the color development.SOLUTION: A blocking agent for biological analysis includes: a block polymer (a) having an acrylic polymer block formed from an acryl monomer in which an average value of a water/1-octanol partition coefficient (LogP) is equal to or more than 0 and less than 2; and a polyethylene oxide. A mass of the polyethylene oxide/[a total mass of the acryl polymer block and the polyethylene oxide block] contained in the block polymer is 0.01 to 0.3.SELECTED DRAWING: None

Description

  The present invention relates to a blocking agent for biochemical analysis.

In the field of biochemical analysis, methods for detecting and visualizing DNA and proteins using specific adsorption by antigen-antibody reaction are widely known as Southern blotting, Western blotting, ELISA, immunostaining, and the like.
Antibodies cause non-specific adsorption reactions with non-target proteins. In order to prevent such non-specific adsorption, pretreatment is performed to cover the surface of the detection / measurement target with a blocking agent that prevents non-specific adsorption but does not prevent specific adsorption.

  As a blocking agent, biologically derived proteins such as normal serum, bovine serum albumin, gelatin and skim milk are known (Patent Document 1, Non-Patent Document 1).

  Further, as a blocking agent, a surfactant called TWEEN20, a hydroxyalkyl cellulose, polyvinyl alcohol, a copolymer of (meth) acryloylmorpholine and other monomers are also known (Patent Documents 2, 3, 4).

International Publication Number WO2016 / 052690 JP 2004-219111 A Japanese Patent Laid-Open No. 04-019561 JP 2008-209114 A

“Watanabe / Nakane Enzyme Antibody Method” published by Interdisciplinary Planning 2002

However, proteins derived from living organisms as disclosed in Patent Literature 1 and Non-Patent Literature 1 have a potential problem that performance tends to vary.
By the way, the blocking agent specifically adsorbs to the protein that is the target of staining, and does not interfere with the color development when coloring, covers the protein other than the target, and adsorbs the staining agent to the protein other than the target. It is required that no color develops. However, the surfactants disclosed in Patent Documents 2 to 4 do not prevent color development due to specific adsorption of the staining agent to the target protein, or do not sufficiently cover the protein other than the target, and also stain proteins other than the target. There are problems such as the agent adsorbs and develops color.
Furthermore, the copolymer disclosed in Patent Document 5 has a problem in productivity, such as requiring a plurality of reactions and purification accompanying the synthesis of the raw material monomer.
The present invention has the ability to prevent color development due to specific adsorption of the stain to the target protein, or to sufficiently cover the protein other than the target, prevent non-specific adsorption of the stain to the protein other than the target, and prevent color development. It aims at providing the blocking agent for biochemical analysis which can express stably.

That is, the present invention relates to the following [1] to [5].
[1] A block polymer (a) having an acrylic polymer block formed of an acrylic monomer having an average value of water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less and a polyethylene oxide block ,
Biochemical analysis characterized in that the mass of polyethylene oxide block / [total mass of acrylic polymer block and polyethylene oxide block] contained in the block polymer (a) is 0.01 to 0.3 Blocking agent.
[2] The acrylic polymer block and the polyethylene oxide block contained in the block polymer (a) are bonded through any one of the following formulas (IA) to (IC). The blocking agent for biochemical analysis according to [1].

[3] The above-mentioned [1], wherein an acrylic polymer block and a polyethylene oxide block contained in the block polymer (a) are bonded via a structure of the following formula (IA) Blocking agent for biochemical analysis.

[4] A polymer azo polymerization initiator, wherein the block polymer (a) has a polyethylene oxide block having a site represented by the following general formula (II) and has a mass average molecular weight of 5,000 to 100,000. The blocking agent for biochemical analysis according to the above [3], which is a block polymer obtained by polymerizing the acrylic monomer with the use of.

(In the formula, m and n each represent an integer of 1 or more.)

[5] After contacting the immunostaining blocking agent according to any one of [1] to [4] above with the pathological tissue fixed on the surface of the substrate, the staining antibody is applied to the antigen in the pathological tissue. The immunostaining method which adsorb | sucks and detects the said antigen.

  The blocking agent of the present invention does not interfere with the color development due to the specific adsorption of the stain to the target protein, sufficiently covers the protein other than the target, prevents the non-specific adsorption of the stain to the protein other than the target, The performance to prevent can be expressed stably unlike the blocking agent derived from a living body. By using this blocking agent, it becomes possible to perform biochemical analysis using antigen-antibody reactions such as Western blotting and immunohistochemical staining with high sensitivity.

  The blocking agent of the present invention is characterized by containing a block polymer (a).

<Block polymer (a)>
In the present invention, the block polymer (a) means an acrylic polymer block (A1) formed from an acrylic monomer having an average value of water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less, and polyethylene oxide. This refers to a polymer having a structure in which the block (A2) is covalently bonded. By having both the acrylic polymer block (A1) and the polyethylene oxide block (A2) in the molecule, the adsorptivity to protein can be controlled and excellent blocking performance can be obtained.

  The block polymer (a) of the acrylic polymer block (A1) and the polyethylene oxide block (A2) is bonded via the structure (bonding structure) of any of the following formulas (IA), (IB), or (IC). It is preferable. Although the synthesis method will be described later, it is not particularly limited. In addition, although it is preferable that block polymer (a) contains any one or more coupling | bonding structure of following formula (IA)-(IC), you may contain some coupling | bonding structures other than these. A polymer formed by bonding via the structure of the following formula (IA) is more preferable.

An example of a method for synthesizing the block polymer (a) having the bond structure represented by the above formula (IA) will be given.
For example, when an acrylic polymer is synthesized using 4,4′-azobis (4-cyanovaleric acid) (for example, a commercial product “V-501” manufactured by Wako Pure Chemical Industries, Ltd.) as a radical polymerization initiator, An acrylic resin having a carboxyl group is obtained. The acrylic polymer block (A1) and the polyethylene oxide block (A2) are represented by the above formula (IA) by adding polyethylene glycol as a raw material constituting the polyethylene oxide block (A2) to the esterification reaction. A block polymer formed by bonding can be obtained.

  Alternatively, the block polymer (a) having a bond structure represented by the above formula (IA) is a polyethylene oxide block (shown by the following formula (II)) as a radical polymerization initiator when synthesizing an acrylic polymer. A2) and a polymer azo polymerization initiator having a structural unit containing an azo group can be preferably used for the synthesis. In the formula, m and n are each independently an integer of 1 or more. The polymer azo polymerization initiator has a structure in which a polymer segment and an azo group (—N═N—) are repeatedly bonded. In this embodiment, the polymer azo polymerization initiator includes a polyethylene oxide block (A2) as a polymer segment. By using the molecular azo initiator, the block polymer (a) can be easily synthesized.

  When using a polymer azo polymerization initiator, the mass average molecular weight of the block polymer can be adjusted by appropriately changing the ratio of the total number of moles of the acrylic monomer to the number of moles of the azo group contained in the initiator. it can.

The mass average molecular weight of the polymer azo polymerization initiator is preferably about 5,000 to 100,000, and more preferably about 10,000 to 50,000. The molecular weight of the polyethylene oxide block (A2) as the initiator is preferably about 800 to 10,000, and more preferably about 1,000 to 8,000.
Moreover, preferably m is 15-200, More preferably, m is 20-100, Preferably n is 3-50, More preferably, n is 4-30.

Since the polymer azo polymerization initiator has a polyethylene oxide block (A2), it is soluble in water, alcohol, and organic solvent, and a block polymer is synthesized by solution polymerization, emulsion polymerization, or dispersion polymerization. Is possible. In addition, since it has a polymerization initiation part (radical generation part: -N = N-) in the molecular chain skeleton, it is not necessary to use a separate polymerization initiator, and more radically compared to the terminal reactive macromonomer. It has the feature of having high reactivity and stability.
The polymer azo polymerization initiator is: C (CH ₃ ) CN— (CH ₂ ) ₂ —COO— (CH ₂ CH ₂ O) _m —CO— (CH ₂ ) ₂ —C (CH ₃ ) CN A radical as shown below is generated, and an acrylic monomer described later is polymerized. And the main chain which the acrylic polymer block (A1) formed from an acrylic monomer and the part derived from the said radical couple | bonded is formed, and a block polymer is formed. The polyethylene oxide block (A2) is derived from a part of the radical.
Specific examples of the polymer azo polymerization initiator include a polymer azo initiator VPE0201 manufactured by Wako Pure Chemical Industries, Ltd. (the molecular weight of the (CH ₂ CH ₂ O) _m portion of the above formula (II) is about 2000, and n is about 6) And the like.

  In addition to the polymer azo polymerization initiator, an azo initiator such as 2,2'-azobisisobutyronitrile or an organic peroxide initiator such as benzoyl peroxide can be used in combination. By using these initiators in combination, the starting efficiency can be increased, the acrylic polymer block (A1) can be efficiently incorporated into PEG, and the residual monomer can be reduced.

  When synthesizing the block polymer (a), the molecular weight and terminal structure are controlled by using a chain transfer agent such as mercaptans such as lauryl mercaptan and n-dodecyl mercaptan, α-methylstyrene dimer, limonene, etc. May be.

Next, a method for synthesizing the block polymer (a) in which the acrylic polymer block (A1) and the polyethylene oxide block (A2) are bonded via the structure of the above formula (IB) will be described.
For example, the block polymer (a) in which an acrylic polymer block (A1) and a polyethylene oxide block (A2) are bonded via the structure of the above formula (IB) has an acrylic monomer having a hydroxy group. A step of polymerizing using an azo polymerization initiator to obtain an acrylic polymer block having a hydroxy group at the terminal, and a step of urethanizing the acrylic polymer block (A1) and polyethylene glycol with a bifunctional isocyanate compound It can manufacture by the method containing.

For example, 2,2′-azobis [N- (2-hydroxyethyl) -2-methylpropanamide] (for example, a commercial product “VA-086” manufactured by Wako Pure Chemical Industries, Ltd.) is used as a radical polymerization initiator. When an acrylic polymer is synthesized, an acrylic resin having a hydroxyl group at the terminal is obtained. To this, polyethylene glycol is added as a raw material constituting the polyethylene oxide block (A2), and a urethanization reaction is performed using a bifunctional isocyanate compound, whereby an acrylic polymer block (A1) and a polyethylene oxide block (A2) are obtained. A block polymer (a) formed by bonding can be obtained.
Examples of the bifunctional isocyanate compound used in the urethanization reaction include 2,2′-diphenylmethane diisocyanate, 2,4′-diphenylmethane diisocyanate, 4,4′-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, 2, Examples include 6-tolylene diisocyanate, hexamethylene diisocyanate, and isophorone diisocyanate.

Furthermore, a method for synthesizing the block polymer (a) in which the acrylic polymer block (A1) and the polyethylene oxide block (A2) are bonded via the structure of the above formula (IC) will be described.
For example, a block polymer in which an acrylic polymer block (A1) and a polyethylene oxide block (A2) are bonded by the structure of the above formula (IC), an acrylic monomer is used as an azo polymerization initiator having a carboxyl group. It can be produced by a method including a step of polymerizing using and obtaining an acrylic polymer block (A1) having a carboxyl group, and a step of esterifying polyethylene glycol to the acrylic polymer block (A1).

  For example, 2,2′-azobis [N- (2-carboxyethyl) -2-methylpropionamidine] tetrahydrate (for example, a commercial product “VA-057” manufactured by Wako Pure Chemical Industries, Ltd.) as a radical polymerization initiator. Etc.) to synthesize an acrylic polymer, an acrylic resin having a carboxyl group at the end can be obtained. To this, polyethylene glycol is added as a raw material constituting the polyethylene oxide block (A2), and an esterification reaction is performed, whereby the acrylic polymer block (A1) and the polyethylene oxide block (A2) are combined to form a block weight. Combined (a) can be obtained.

  In addition, when obtaining the block polymer (a) formed by bonding via any structure of the formulas (IB) to (IC), when using an azo polymerization initiator that is not a polymer as described above, "Other initiators" described as those that can be used together when using a polymer azo polymerization initiator can also be used in combination as appropriate. Moreover, a chain transfer agent can also be used suitably.

<Acrylic polymer block (A1)>
In the present invention, the acrylic polymer block (A1) refers to a block structure portion containing 10% or more of a (meth) acrylic monomer having at least one acryloyl group and / or methacryloyl group among all monomers. The acrylic polymer block (A1) is composed of monomers having an average value of water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less, so that the obtained polymer has excessive mutual interaction with proteins and tissue fragments. The action can be suppressed and excellent blocking performance can be obtained.

  LogP is one of the numerical values representing the properties of the chemical substance, and is a constant value that does not depend on the addition amount. The target substance is a mixture of water and 1-octanol, and the concentration of each phase is shown in the common logarithm for the case where the aqueous phase and the octanol phase are in an equilibrium state in contact with each other. . When LogP increases, the hydrophobicity tends to increase relatively, and when LogP decreases, the hydrophilicity tends to increase relatively.

  The measurement of LogP can be generally performed by a flask immersion method described in JIS Japanese Industrial Standard Z7260-107 (2000). In addition, LogP can be estimated by a computational chemical method or an empirical method instead of actual measurement. As a method and software used for calculating LogP, publicly known methods can be used, but in the present invention, LogP is obtained by using a program incorporated in the system: ChemdrawPro 11.0 of CambridgeSoft.

The monomer having a water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less is not particularly limited, and examples thereof include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, and n-butyl. Alkyl ester (meth) acrylate such as acrylate; (meth) acryl methoxymethyl (meth) acrylate, 2-methoxyethyl (meth) acrylate, ethoxymethyl (meth) acrylate, ethoxyethyl (meth) acrylate, propoxymethyl (meth) acrylate , Propoxyethyl (meth) acrylate, 2- (2-methoxyethoxy) ethyl (meth) acrylate, 2- [2- (2-methoxyethoxy) ethoxy] ethyl (meth) acrylate, 2- (2-ethoxyethoxy) ethyl (Meth) acrylate, - [2- (2-ethoxyethoxy) ethoxy] ethyl (meth) acrylate, tetrahydrofurfuryl (meth) alkoxy group-containing monomers such as acrylate;
Vinyl group-containing monomers such as (meth) acrylonitrile, vinyl acetate, butadiene, isoprene; amides such as N-vinylpyrrolidone, N-vinyl-ε-caprolactam, 1-vinylimidazole, N-isopropylacrylamide, N, N-dimethylacrylamide Group-containing monomer: (meth) acrylic acid, 2-carboxyethyl (meth) acrylate, or alkylene oxide addition succinic acid (meth) acrylate having a carboxyl group at the terminal where alkylene oxide such as ethylene oxide or propylene oxide is repeatedly added And carboxyl group-containing monomers such as
These may be used alone or in combination of two or more. Of these compounds, n-butyl acrylate and 2-methoxyethyl acrylate are particularly preferable from the viewpoints of economy and operability.

  In this invention, if LogP of the acryl-type monomer to be used is the range of 0-2, it can be used as a raw material of a homopolymer part. Moreover, even if LogP of the (meth) acrylic monomer used is outside the range of 0 to 2, if the mass average value of LogP including other (meth) acrylic monomers is in the range of 0 to 2, It can be used as a raw material for the copolymer part.

  Among monomers outside the range of Log P of 0 to 2, for example, acrylates having an alkyl group having 5 to 20 carbon atoms, methacrylates having an alkyl group having 4 to 20 carbon atoms, vinyl group-containing monomers such as styrene, maleic acid Carboxyl group-containing monomers such as 4-hydroxystyrene, N-hydroxyethyl (meth) acrylamide and other hydroxyl group-containing monomers, polyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, propoxy Polyethylene glycol (meth) acrylate, n-butoxypolyethylene glycol (meth) acrylate, n-pentoxypolyethylene glycol (meth) acrylate, phenoxypolyethylene glycol (meth) acrylate It relates such monomers having polyethylene oxide moieties in the side chain, such as.

  The adhesion between the tissue piece and the antibody can be effectively controlled by the balance between the hydrophilic polyethylene oxide block (A2) constituting the block polymer (a) and the hydrophobic acrylic polymer block (A1). . In the polymer (a), the mass of the polyethylene oxide block (A2) / [total mass of the acrylic polymer block (A1) and the polyethylene oxide block (A2)] is 0.01 to 0.3, 0 .1 to 0.25 is preferable. That is, it is preferable that it is 1-30 mass%, and it is more preferable that it is 10-25 mass%.

<Glass transition temperature>
The glass transition temperature (hereinafter also referred to as Tg) of the block copolymer (a) is preferably -50 ° C to 50 ° C, more preferably -50 to 0 ° C. When Tg is 50 ° C. or higher, there is a possibility that adsorption hardly occurs in the protein.

  The measurement of the glass transition temperature by DSC (differential scanning calorimeter) can be performed as follows. About 2 mg of resin obtained by drying the block copolymer (a) is weighed on an aluminum pan, the test container is set on a DSC measurement holder, and the endothermic peak of the chart obtained under a temperature rising condition of 10 ° C./min is obtained. read. The peak temperature at this time is defined as the glass transition temperature of the present invention.

<Molecular weight>
The mass average molecular weight Mw of the block copolymer (a) is preferably 10,000 to 300,000. If the molecular weight is less than 10,000 or greater than 300,000, there is a possibility that analysis cannot be performed with high sensitivity. On the other hand, when the molecular weight is larger than 300,000, the viscosity is high, which may cause problems in use.
<Measurement method of mass average molecular weight (Mw)>
Mw was measured by Showa Denko GPC (Gel Permeation Chromatography) “GPC-101”. GPC is liquid chromatography that separates and quantifies substances dissolved in a solvent (THF; tetrahydrofuran) based on the difference in molecular size. For the measurement in the present invention, “KF-805L” (manufactured by Showa Denko KK: GPC column: 8 mm ID × 300 mm size) is connected in series to the column, the sample concentration is 1 wt%, the flow rate is 1.0 ml / min, the pressure The measurement was performed under the conditions of 3.8 MPa and a column temperature of 40 ° C., and the mass average molecular weight (Mw) was determined in terms of polystyrene. In the data analysis, a calibration curve, molecular weight, and peak area were calculated using software built in the manufacturer, and a mass average molecular weight was obtained with a retention time of 15 to 30 minutes as an analysis target.

  EXAMPLES The present invention will be described more specifically with reference to the following examples. However, the following examples do not limit the scope of rights of the present invention. In the examples and comparative examples, “part” represents “part by mass”, “mol” represents the amount of substance, and “mol%” represents the ratio of the amount of substance in all monomers.

[Production Example 1]
<Preparation of block polymer (a)>
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen inlet tube, a reflux condenser, and a dropping tube, 150 parts by mass of methyl ethyl ketone (hereinafter abbreviated as MEK) as an organic solvent is charged in a nitrogen stream and stirred at 80 ° C. for 30 minutes. Heated for minutes. 80 parts by mass of methoxyethyl acrylate as a monomer in the dropping tube, 20 parts by mass of 2-ethylhexyl acrylate, 25 parts by mass of VPE0201 (manufactured by Wako Pure Chemical Industries, Ltd .: Macroazo Initiator) as a polymerization initiator, and 10 masses of MEK as a solvent The preparation was added dropwise over 2 hours. After completion of dropping, the mixture was aged for 6 hours. Then, it cooled to room temperature and stopped reaction. Thereafter, MEK was removed with a diaphragm pump to obtain a block polymer (a). Mw of the obtained polymer was 57400.
1 g of the obtained vinyl polymer (a) was put in 99 g of ion-exchanged water at 25 ° C., stirred and dissolved, and then allowed to stand at 25 ° C. for 24 hours. As a result, these resins were not separated or precipitated, indicating that they were completely soluble and water-soluble.

<Preparation of blocking agent>
The block polymer (a) obtained above was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain the blocking agent of Production Example 1 having a concentration of 5% by mass.

[Production Examples 2 to 10]
With the formulation shown in Table 1, the block polymer (a) was synthesized by the same method as in Production Example 1 and dissolved in a PBS solution to obtain blocking agents in Production Examples 2 to 10.

The symbols in the table are as follows.
MEA: Methoxyethyl acrylate, Log P = 0.48
BA: Butyl acrylate, Log P = 1.88
MMA: methyl methacrylate, Log P = 1.11
AA: Acrylic acid, Log P = 0.38
BMA: Butyl methacrylate, Log P = 2.23
2EHA: 2-ethylhexyl acrylate, LogP = 3.53
ISTA: Isostearyl acrylate, Log P = 7.47

<Treatment with blocking agent and performance evaluation>
[Actin adhesion]
An actin solution for evaluation was prepared by diluting human platelet-derived actin (manufactured by Cytoskeleton) with a PBS solution so as to be 1% by mass.
2 μL each of the actin solution for evaluation was dropped into two nitrocellulose membranes (membrane L-08002-010_Asone) at one location using a micropipettor, and allowed to stand and dried.

[Treatment with blocking agent]
Next, the blocking agent prepared above was charged with a nitrocellulose membrane having actin attached thereto, and shaken at room temperature for 1 hour to perform a blocking treatment.
Thereafter, the nitrocellulose membrane was removed from the blocking agent, and the removed nitrocellulose membrane was placed in a PBS solution and shaken at room temperature for 15 minutes. The PBS solution was renewed and the same washing operation was repeated once more to remove excess blocking agent.

[Attachment of two types of antibodies to actin]
Anti-β-actin, monoclonal antibody, and peroxidase bond (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in a PBS solution to obtain a diluted actin antibody solution having a concentration of 0.01% by mass. Similarly, anti-GAPDH, monoclonal antibody, and peroxidase bond (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in a PBS solution to obtain a GAPDH antibody diluted solution having a concentration of 0.01% by mass.
The above-mentioned nitrocellulose membrane from which excess blocking agent was removed was placed in each antibody dilution and shaken at room temperature for 1 hour. Subsequently, the nitrocellulose membrane was taken out from the antibody diluted solution, and the taken out nitrocellulose membrane was placed in a PBS solution and shaken at room temperature for 1 hour to remove excess antibody.
GAPDH is an abbreviation for glyceraldehyde-3-phosphate dehydrogenase.

[Dyeing and evaluation]
DAB tablets (manufactured by Wako Pure Chemical Industries, Ltd.) 10 mg were dissolved in 50 mL of 0.05 mol / L Tris-HCl buffer, and 10 μL of 30% hydrogen peroxide solution was further added to prepare a staining solution.
The two nitrocellulose membranes from which the excess antibody was removed were each covered with a staining solution, and the excess staining solution on the surface was removed with a towel.
According to the following criteria, the staining state of the actin adhesion site in the case of using two types of antibody dilutions was evaluated, and the evaluation results are shown in Table 3.

"When using actin antibody dilution"
○: Clear staining △: Almost no staining ×: No staining “When GAPDH antibody dilution was used”
○: No staining △: With staining ×: With clear staining "Comprehensive evaluation"
○: Stained only when the actin antibody diluted solution was used, and there was no staining when the GAPDH antibody diluted solution was used.
Δ: When using the actin antibody diluted solution, the staining is thin and the contrast with the case using the GAPDH antibody diluted solution is small, or when the actin antibody diluted solution is used, the staining is performed, but the GAPDH antibody diluted solution is used. In some cases, clear staining is also seen.
×: The degree of staining does not change between when the actin antibody dilution is used and when the GAPDH antibody dilution is used (no blocking performance)

  As shown in Table 2, biochemical analysis of actin and an actin antibody could be performed by using the biochemical analysis blocking agent of the present invention. This is because the block polymer (a) contained in the biochemical analysis blocking agent of the present invention appropriately adsorbs to the protein, thereby preventing the antigen-antibody reaction and suppressing the nonspecific adsorption reaction. It is thought that.

On the other hand, in Comparative Example 1 using a polymer having no polyethylene oxide block (A2), clear staining was not obtained when an actin antibody dilution solution was used, and when a GAPDH antibody dilution solution was used. There was no clear difference. Therefore, sufficient sensitivity for analysis could not be obtained. This is presumably because the blocking agent strongly inhibited non-specific adsorption, thereby inhibiting the antigen-antibody reaction itself.
Further, Comparative Example 2 using a polymer having a mass of polyethylene oxide block / [total mass of acrylic polymer block and polyethylene oxide block] larger than 0.3 is also the case when a GAPDH antibody dilution solution is used. Staining was obtained, and no clear difference was observed when the actin antibody dilution was used. Therefore, sufficient sensitivity for analysis could not be obtained. This is considered to be because the performance as a blocking agent was not obtained because the blocking agent was highly water-soluble and could not be sufficiently adsorbed to protein.

Comparative Examples 3 and 4 using a polymer having an average value of water / 1-octanol partition coefficient (LogP) greater than 2 for the acrylic monomer forming the acrylic polymer block (A1) is when an actin antibody diluent is used No clear staining was obtained, and no clear difference was observed when the GAPDH antibody dilution was used. Therefore, sufficient sensitivity for analysis could not be obtained. This is presumably because the blocking agent strongly inhibited non-specific adsorption, thereby inhibiting the antigen-antibody reaction itself.

Claims (5)

A block polymer (a) having an acrylic polymer block formed from an acrylic monomer having an average water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less, and a polyethylene oxide block;
Biochemical analysis characterized in that the mass of polyethylene oxide block / [total mass of acrylic polymer block and polyethylene oxide block] contained in the block polymer (a) is 0.01 to 0.3 Blocking agent. The acrylic polymer block and the polyethylene oxide block contained in the block polymer (a) are bonded through any one of the following formulas (IA) to (IC). The blocking agent for biochemical analysis according to 1.
2. The biochemical analysis according to claim 1, wherein an acrylic polymer block and a polyethylene oxide block contained in the block polymer (a) are bonded via a structure of the following formula (IA). Blocking agent.
The block polymer (a) has a site represented by the following general formula (II), has a polyethylene oxide block, and uses a polymer azo polymerization initiator having a mass average molecular weight of 5,000 to 100,000 The blocking agent for biochemical analysis according to claim 3, which is a block polymer obtained by polymerizing the acrylic monomer.

(In the formula, m and n each represent an integer of 1 or more.) The immunostaining blocking agent according to any one of claims 1 to 4 is brought into contact with the pathological tissue fixed on the surface of the base material, and then the staining antibody is adsorbed to the antigen in the pathological tissue. Immunostaining method to detect.