JP2008215894A - Detection method of protein and detecting method of peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method of a protein or a peptide having high sensitivity and high throughput, having few noises originated in nonspecific adsorption, capable of immobilizing an antibody on an optional position on a substrate surface, and capturing simultaneously a plurality of kinds of target proteins or peptides in a specimen. <P>SOLUTION: This detection method of a protein or a peptide by immobilizing the antibody on the substrate surface, and by capturing specifically the protein or the peptide in the specimen by an interaction with the antibody includes (a) a process for applying a polymer having a phosphorylcholine group on the substrate surface or a polymer acquired by polymerizing ethylene-based unsaturated polymeric monomers having respectively an alkylene glycol residue, (b) a process for immobilizing the antibody on the polymer, (c) a process for labeling the protein or the peptide in the specimen, (d) a process for allowing the specimen acquired in the process (c) and a water-soluble polymer to coexist in the same system, and (e) a process for bringing the specimen acquired in the process (d) into contact with the substrate acquired in the process (b). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a detection method for capturing a labeled protein or peptide using a substance that specifically captures the protein or peptide.

  Attempts to assess gene activity and decipher the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more in-depth about the biological functions of cells. Provide information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level rather than at the gene level. It also includes studies of events that are not encoded by genes such as post-translational modifications of proteins and interactions between proteins.

  Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research is becoming increasingly popular, and as a result, rapid detection of physiologically active substances is increasing. There is a need for higher efficiency (high throughput). A DNA chip has been developed and put into practical use as a molecular array for this purpose. On the other hand, protein chips have been proposed for the detection of the most complex and highly diverse proteins in biological functions, and research has been promoted in recent years. A protein chip is a generic term for a protein or a molecule that captures it immobilized on a chip (micro substrate) surface.

  However, since the current protein chip is generally developed on the extension line of the DNA chip, studies have been made to immobilize a protein or a molecule for capturing the protein on the glass substrate on the surface of the chip (for example, Patent Document 1).

  There is a sandwich method as a method generally used in detection and quantification of specimens such as proteins. In this method, an antibody (primary antibody) is bound to a solid phase, a target protein is trapped by an insolubilized antibody, and then a labeled antibody (secondary antibody) that recognizes and binds to another epitope of the target protein is reacted. Thus, the protein is quantitatively measured by measuring the label (see, for example, Non-Patent Document 1). However, this method requires multiple types of antibodies that do not compete with each other for each target protein when trying to detect a large amount of proteins at once, such as a protein chip. It contains many problems to be solved.

  In addition, after immobilizing a protein-capturing molecule (hereinafter abbreviated as a capturing molecule) on a substrate, it reacts with another protein (corresponding to an antigen in the case of an antigen-antibody reaction) on the surface as in the sandwich method, for example. In addition, when a labeled protein is reacted and finally detected by a detector or the like, a protein other than the molecule, that is, an antigen or a labeled protein is immobilized on a portion where the capture molecule is not immobilized. , It becomes noise at the time of detection, causing a reduction in the signal-to-noise ratio (S / N ratio), and lowering the detection accuracy (see Non-Patent Document 2, for example). In particular, in experiments conducted by the present inventors, serum and plasma used in clinical diagnosis and the like tend to have high non-specific adsorption of contaminating proteins, resulting in high noise and a low S / N ratio. .

  For this reason, in the usual sandwich method, after the primary antibody is immobilized, in order to prevent nonspecific adsorption of the antigen and the secondary antibody, coating with an adsorption inhibitor is performed, but these nonspecific adsorption preventing ability is not sufficient. In addition, since the adsorption inhibitor is coated after immobilizing the primary antibody, it may be coated on the immobilized protein, and there is a problem that it cannot react with the secondary antibody. Therefore, there is a need for a biochip that does not have an adsorption inhibitor coating step after immobilization of the primary antibody and has a small amount of non-specific adsorption of a physiologically active substance.

On the other hand, in terms of the technical aspects of profiling fluctuations in all proteins (proteomes), microfluidics technology that enables the manipulation of ultra-trace amounts of proteins and ultra-small quantities of solutions such as several nanoliters, and on-chip The concept of “lab-on-a-chip” that targets processing, separation, and detection becomes important. In this technology, a physiologically active substance such as a protein as a sample reacts specifically with the capture immobilized in the flow path, and suppresses non-specific adsorption to the inner wall of the flow path other than the capture section. Is required.
JP 2001-116750 A "Enzyme immunoassay", Yuji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, Medical School, 1987, p.44-45 “DNA Microarray Practice Manual”, Yoshihide Hayashizaki, Koji Okazaki, Yodosha, 2000, p.57

  An object of the present invention is to immobilize an antibody at an arbitrary position on the surface of a substrate without coating an adsorption inhibitor, and simultaneously capture a plurality of types of target proteins or peptides in a specimen, and nonspecific adsorption of contaminating proteins. Is to provide a high-sensitivity, high-throughput protein or peptide detection method.

（式中Ｒ₁は水素原子またはメチル基を示し、Ｒ₂は水素原子または炭素数１〜２０のアルキル基を示す。Ｘは炭素数１〜１０のアルキレングリコール残基を示し、ｐは１〜１００の整数を示す。繰り返されるＸは、同一であっても、または異なるアルキレングリコール基の連鎖であってもよい。）
（９）前記アルキレングリコール残基を有するエチレン系不飽和重合性モノマーがメトキシポリエチレングリコール（メタ）アクリレートである（８）記載の蛋白質又はペプチドの検出方法、
（１０）前記のメトキシポリエチレングリコール（メタ）アクリレートのエチレングリコールの平均連鎖が３〜１００である（９）記載の蛋白質又はペプチドの検出方法、
（１１）前記の水溶性ポリマーが、アクリル酸エステル重合体、メタクリル酸エステル重合体、またはアクリル酸エステルとメタクリル酸エステルとの共重合体の少なくともいずれか１種の重合体であり、重合体の分子量が２００〜２００，０００である（１）〜（１０）いずれか記載の蛋白質又はペプチドの検出方法、
（１２）前記水溶性ポリマーがポリビニルアルコールである（１）〜（１０）いずれか記載の蛋白質又はペプチドの検出方法、
（１３）前記水溶性ポリマーがアルキレングリコール、またはアルキレングリコール基を有するポリマーである（１）〜（１０）いずれか記載の蛋白質又はペプチドの検出方法、
（１４）前記アルキレングリコールがエチレングリコールである（１３）記載の蛋白質又はペプチドの検出方法、
（１５）前記アルキレングリコール基を有するポリマーの分子量が２００〜２０，０００であることを特徴とする（１３）記載の蛋白質又はペプチドの検出方法、
（１６）前記アルキレングリコール基を有するポリマーがポリエチレングリコールである（１５）記載の蛋白質又はペプチドの検出方法、
（１７）前記抗体が基体表面にスポット状に固定化されている（１）〜（１６）いずれか記載の蛋白質又はペプチドの検出方法、
（１８）複数種の抗体のスポットが基体表面の同一区画中に存在している（１７）記載の蛋白質又はペプチドの検出方法、
（１９）基体の形状がスライドグラス状である（１）〜（１８）いずれか記載の蛋白質又はペプチドの検出方法、
（２０）基体が微細流路を有しており、微細流路内に抗体が固定化されていることを特徴とする（１）〜（１９）いずれか記載の蛋白質又はペプチドの検出方法、
（２１）基体の材質がプラスチックである（１）〜（２０）いずれか記載の蛋白質又はペプチドの検出方法、
（２２）プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、及びそれらの共重合体よりなる群より選択された少なくとも１種である（２１）記載の蛋白質又はペプチドの検出方法、
である。 That is, the present invention
(1) A method of detecting an antibody by immobilizing an antibody on the surface of the substrate, specifically capturing the protein or peptide in the specimen by interaction with the antibody,
(A) applying a polymer having a phosphorylcholine group on the substrate surface or a polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue;
(B) immobilizing an antibody on the polymer;
(C) a step of labeling the protein or peptide in the specimen,
(D) a step of allowing the sample obtained in step (c) and the water-soluble polymer to coexist in the same system;
(E) contacting the specimen obtained in step (d) with the substrate obtained in step (b);
A method for detecting a protein or peptide, comprising:
(2) The method for detecting a protein or peptide according to (1), wherein the specimen is serum or plasma,
(3) The method for labeling a protein or peptide in a sample is at least one method selected from a fluorescent labeling method, an enzyme labeling method, and a biotin labeling method. Detection method,
(4) Substances used in the fluorescent labeling method are Cy3, Cy5, FITC
(Fluorescein isothiocyanate), and the method for detecting a protein or peptide according to (3), which is at least one selected from rhodamine,
(5) The method for detecting a protein or peptide according to (3), wherein the substance used in the enzyme labeling method is at least one selected from peroxidase, alkaline phosphatase, acid phosphatase, and glucose oxidase,
(6) The method for detecting a protein or peptide according to any one of (1) to (5), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group,
(7) The method for detecting a protein or peptide according to any one of (1) to (6), wherein the polymer having a phosphorylcholine group is a copolymer containing a butyl methacrylate group,
(8) The method for detecting a protein or peptide according to any one of (1) to (5), wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is a monomer represented by the following general formula [1]:

(Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, p represents 1 to 1) Represents an integer of 100. The repeated Xs may be the same or a chain of different alkylene glycol groups.
(9) The method for detecting a protein or peptide according to (8), wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate,
(10) The method for detecting a protein or peptide according to (9), wherein the average chain of ethylene glycol in the methoxypolyethylene glycol (meth) acrylate is 3 to 100,
(11) The water-soluble polymer is at least one polymer of an acrylic ester polymer, a methacrylic ester polymer, or a copolymer of an acrylic ester and a methacrylic ester, The method for detecting a protein or peptide according to any one of (1) to (10), wherein the molecular weight is 200 to 200,000,
(12) The method for detecting a protein or peptide according to any one of (1) to (10), wherein the water-soluble polymer is polyvinyl alcohol,
(13) The method for detecting a protein or peptide according to any one of (1) to (10), wherein the water-soluble polymer is alkylene glycol or a polymer having an alkylene glycol group.
(14) The method for detecting a protein or peptide according to (13), wherein the alkylene glycol is ethylene glycol,
(15) The method for detecting a protein or peptide according to (13), wherein the polymer having an alkylene glycol group has a molecular weight of 200 to 20,000,
(16) The method for detecting a protein or peptide according to (15), wherein the polymer having an alkylene glycol group is polyethylene glycol,
(17) The method for detecting a protein or peptide according to any one of (1) to (16), wherein the antibody is immobilized in the form of a spot on the substrate surface,
(18) The method for detecting a protein or peptide according to (17), wherein a plurality of types of antibody spots are present in the same section of the substrate surface,
(19) The method for detecting a protein or peptide according to any one of (1) to (18), wherein the substrate has a slide glass shape.
(20) The method for detecting a protein or peptide according to any one of (1) to (19), wherein the substrate has a fine channel, and the antibody is immobilized in the fine channel,
(21) The method for detecting a protein or peptide according to any one of (1) to (20), wherein the base material is plastic.
(22) The method for detecting a protein or peptide according to (21), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof. ,
It is.

  According to the method for detecting a protein or peptide of the present invention, without coating an adsorption inhibitor, the antibody is immobilized at an arbitrary position on the surface of the substrate, and a plurality of types of target proteins or peptides in the specimen are simultaneously captured. In addition, nonspecific adsorption of contaminating proteins can be reduced, and proteins or peptides can be detected with high sensitivity and high throughput. This is particularly effective in the case of a specimen having a large amount of nonspecific adsorption of contaminating proteins such as serum and plasma.

  The substrate used in the present invention is characterized in that the substrate surface is coated with a polymer having a phosphorylcholine group or a polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol group. A polymer having a phosphorylcholine group is a polymer having a structure similar to a biological membrane (phospholipid bilayer membrane) and has a property of suppressing nonspecific adsorption of a physiologically active substance (for example, Ishihara K, Tsuji T Kurosaki T, Nakabayashi N, Journal of Biomedical Materials Research, 28 (2), pp.225-232, (1994) 4). A polymer having an ethylenically unsaturated polymerizable monomer having an alkylene glycol group has both the property of suppressing nonspecific adsorption of a physiologically active substance, the property of immobilizing a physiologically active substance, and the property of cross-linking polymer main chains. In the polymer, the alkylene glycol residue plays a role of suppressing nonspecific adsorption of the physiologically active substance.

  Examples of the phosphorylcholine group used in the present invention include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine. Octenyl phosphorylcholine, decenyl phosphorylcholine and the like, and 2-methacryloyloxyethyl phosphorylcholine is preferable.

  In addition, the polymer having a phosphorylcholine group used in the present invention may contain other groups in addition to the phosphorylcholine group, and a binary copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group may be used. preferable.

    The ethylenically unsaturated polymerizable monomer having an alkylene glycol residue used for the polymer used for coating the substrate surface of the present invention is not particularly limited in structure, but is a (meth) acryl group represented by the general formula [1] And a compound consisting of a chain of an alkylene glycol residue X having 1 to 10 carbon atoms.

The alkylene glycol residue X in the formula has 1 to 10 carbon atoms, more preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, and most preferably 2. It is. The repeating number p of the alkylene glycol residue X is an integer of 1 to 100, more preferably an integer of 2 to 100, still more preferably an integer of 2 to 95, and most preferably an integer of 20 to 90. is there. When the number of repetitions is 2 or more and 100 or less, the carbon number of the alkylene glycol residue X to be repeated may be the same or different.
Examples of the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue include methoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and 2-hydroxybutyl (meth). (Meth) acrylates of monosubstituted esters of hydroxyl groups such as acrylate, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-methoxyethyl (meth) acrylate, 2-ethoxyethyl (meth) Acrylate, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, etc. Alkoxy polyethylene glycol methacrylate is preferred.

    The polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue used in the present invention contains an ethylenically unsaturated polymerizable monomer having another group in addition to the alkylene glycol residue as a copolymer component. But you can. For example, an ethylenically unsaturated polymerizable monomer having an alkyl group may be copolymerized, and the ethylenically unsaturated polymerizable monomer having an alkyl group is preferably n-butyl methacrylate, n-dodecyl methacrylate or n-octyl methacrylate. .

    The bonding between the substrate surface and the polymer may be any bonding mode such as covalent bonding, electrostatic interaction, hydrogen bonding, or hydrophobic bonding. In the present invention, the polymer having a phosphorylcholine group on the substrate surface. Or a solution containing a polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol group.

(Base material)
Glass, plastic, metal, etc. can be used as the material of the substrate, but the material of the substrate used in the present invention is preferably a plastic from the viewpoint of ease of surface treatment and mass productivity, and more preferably a thermoplastic resin. preferable. As a thermoplastic resin, a thing with little fluorescence generation amount is preferable. For example, it is preferable to use linear polyolefin such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, saturated cyclic polyolefin, fluorine-containing resin, etc., and saturation that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. It is more preferable to use a cyclic polyolefin. Here, the saturated cyclic polyolefin refers to a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.

(Base shape)
The shape of the substrate used in the present invention is not particularly limited, and examples thereof include a flat plate such as a slide glass and a microbead. Furthermore, it is also preferable that the substrate has a fine channel on the surface and the physiologically active substance is immobilized in the fine channel.

(Immobilization of antibodies)
In the present invention, when immobilizing an antibody on a substrate, a method of spotting a liquid in which the antibody is dissolved or dispersed is preferable.
The pH of the liquid in which the antibody is dissolved or dispersed is preferably 8 to 10, and more preferably pH 9.0 to 9.9. After immobilization, it is preferable to wash with pure water or a buffer solution in order to remove the physiologically active substance that has not been immobilized.
The antibody is preferably immobilized on the substrate surface in the form of spots, and more preferably, a plurality of types of antibody spots are present in the same compartment on the substrate surface.

(Labeling of specimen)
In the present invention, examples of a method for labeling a protein or peptide serving as a specimen include a fluorescent label, an enzyme label, a biotin label, and the like, and any method can be used.
In the case of the fluorescent labeling method, substances such as Cy3, Cy5, FITC (fluorescein isothiocyanate), rhodamine and the like are used.
In the case of the enzyme labeling method, substances such as peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase and the like are used.

(Coexistence of specimen and water-soluble polymer in the same system)
In the present invention, the form of the water-soluble polymer that coexists with the specimen may be any of powder, suspension, and solution, and preferably a solution such as water or a buffer.
In the present invention, the sample coexisting with the water-soluble polymer in the same system is not particularly limited, but those containing many contaminating proteins such as serum can obtain the effect of the present invention.
The coexistence of the sample and the water-soluble polymer in the same system can be carried out, for example, by adding the liquid water-soluble polymer to the sample solution in a solution state so as to coexist in the same solution.

In the present invention, the water-soluble polymer used in the coexistence step in the same system includes acrylic ester polymer, methacrylic ester polymer, copolymer of acrylic ester and methacrylic ester, polyvinyl alcohol, alkylene Polymers having glycol or alkylene glycol groups can be utilized. As the acrylate polymer, for example, a polymer such as methyl acrylate, ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, etc., as the methacrylate polymer, for example, methacrylic acid, butyl methacrylate, isobutyl methacrylate, Polymers such as tertiary butyl methacrylate, cyclohexyl methacrylate, and trimethylolpropane trimethacrylate may be mentioned. Examples of the alkylene glycol include ethylene glycol and propylene glycol. Examples of the polymer having an alkylene glycol group include polyethylene glycol and polypropylene glycol. Among these, ethylene glycol, polyethylene glycol, or polyvinyl alcohol is preferable from the viewpoints of availability and versatility.
The water-soluble polymer used in the above step may be the same as or different from the polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol glycol residue used in the step of applying to the substrate surface. Also good. A mixture of these may also be used.

(Detection of proteins and peptides)
A polymer having a phosphorylcholine group coexists on a substrate on which an antibody is immobilized, and a liquid containing a labeled sample is brought into contact with each other, thereby suppressing nonspecific adsorption of contaminating proteins to the substrate, or The peptide is captured.
In the present invention, the pH of the liquid in which the protein or peptide to be detected is dissolved or dispersed is preferably 6.0 to 7.6. If the pH is less than 6.0, the protein and peptide detected by the acid may be denatured, which is not preferable. Moreover, when pH exceeds 7.6, there exists a possibility that the protein and peptide to detect may denature | alterate, and it is unpreferable.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example)
A saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm) to prepare a solid phase substrate. The solid phase substrate was immersed in a 0.5 wt% ethanol solution of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer to introduce a polymer having a phosphorylcholine group on the substrate surface.
Next, using an automatic spotter, an anti-human interleukin-1β antibody solution, which is a primary antibody adjusted to pH 9.5 at a concentration of 3.3 μmol / L, was spotted on the substrate for 24 hours. It was allowed to stand and immobilized. After immobilization, washing with PBS containing 0.05% Tween 20 was performed.
Subsequently, the whole mixture of antigen human serum proteins was labeled with Cy3 by NHS-Cy3.
Thereafter, a 5% by weight aqueous solution of polyvinyl alcohol (molecular weight 500) was added to the Cy3 labeling solution in an amount of 1/5 of the labeling solution and mixed well. This is brought into contact with the substrate to cause an antigen-antibody reaction on the substrate, and the amount of fluorescence is measured for each spot and the portion other than the spot portion (background), and the S / N ratio (Signal / noise ratio) at that time is measured. Calculated. The results are shown in Table 1.

(Comparative example)
A saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm) to prepare a solid phase substrate. The solid phase substrate was immersed in a 0.5 wt% ethanol solution of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer to introduce a polymer having a phosphorylcholine group on the substrate surface.
Next, using an automatic spotter, an anti-human interleukin-1β antibody solution, which is a primary antibody adjusted to pH 9.5 at a concentration of 3.3 μmol / L, was spotted on the substrate for 24 hours. It was allowed to stand and immobilized. After immobilization, washing with PBS containing 0.05% Tween 20 was performed.
Subsequently, the whole mixture of antigen human serum proteins was labeled with Cy3 by NHS-Cy3.
Thereafter, in order to match the Examples and Cy3-labeled protein concentrations, a PBS aqueous solution was added to the Cy3-labeled solution in an amount of 1/5 of the labeled solution and mixed well. This is brought into contact with the substrate to cause an antigen-antibody reaction on the substrate, and the amount of fluorescence is measured for each spot and the portion other than the spot portion (background), and the S / N ratio (Signal / noise ratio) at that time is measured. Calculated. The results are shown in Table 1.

A biochip scanner “ScanArray” manufactured by Packard BioChip Technologies was used to measure the amount of fluorescence in Examples and Comparative Examples. The measurement conditions were laser output 90%, PMT sensitivity 55%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 10 μm.
In the example, the spot signal intensity is equal to or higher than that of the comparative example, and the fluorescence intensity (background) other than the spot portion is lower than that of the comparative example. However, the result was nearly twice as high as that of the comparative example. That is, it can be said that highly sensitive protein and peptide were detected.

  According to the method for detecting a protein or peptide of the present invention, without coating an adsorption inhibitor, the antibody is immobilized at an arbitrary position on the surface of the substrate, and a plurality of types of target proteins or peptides in the specimen are simultaneously captured. , A polymer having phosphorylcholine groups on the surface of the substrate, or an alkylene glycol residue by coexisting a specimen (especially non-specific adsorption of contaminant proteins such as serum and plasma) and a water-soluble polymer in the same system Because non-specific adsorption of contaminating proteins can be reduced by synergistic effects with polymers obtained by polymerizing ethylenically unsaturated polymerizable monomers with high sensitivity and high-throughput detection of proteins or peptides, various biologics including microfluidics It can be applied to a chip detection method.

Claims (22)

A method of detecting an antibody by immobilizing an antibody on a substrate surface, specifically capturing the protein or peptide in a specimen by interaction with the antibody,
(A) applying a polymer having a phosphorylcholine group on the substrate surface or a polymer obtained by polymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue;
(B) immobilizing an antibody on the polymer;
(C) a step of labeling the protein or peptide in the specimen,
(D) a step of allowing the sample obtained in step (c) and the water-soluble polymer to coexist in the same system;
(E) contacting the specimen obtained in step (d) with the substrate obtained in step (b);
A method for detecting a protein or peptide, comprising: The method for detecting a protein or peptide according to claim 1, wherein the specimen is serum or plasma. The method for detecting a protein or peptide according to claim 1 or 2, wherein the labeling method of the protein or peptide in the sample is at least one method selected from a fluorescent labeling method, an enzyme labeling method, and a biotin labeling method. Substances used in the fluorescent labeling method are Cy3, Cy5, FITC
The method for detecting a protein or peptide according to claim 3, which is at least one selected from (fluorescein isothiocyanate) and rhodamine. The method for detecting a protein or peptide according to claim 3, wherein the substance used in the enzyme labeling method is at least one selected from peroxidase, alkaline phosphatase, acid phosphatase, and glucose oxidase. The method for detecting a protein or peptide according to any one of claims 1 to 5, wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group. The method for detecting a protein or peptide according to any one of claims 1 to 6, wherein the polymer having a phosphorylcholine group is a copolymer containing a butyl methacrylate group. The method for detecting a protein or peptide according to any one of claims 1 to 5, wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is a monomer represented by the following general formula [1].

(Wherein R ₁ represents a hydrogen atom or a methyl group, R ₂ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, p represents 1 to 1) Represents an integer of 100. The repeated Xs may be the same or a chain of different alkylene glycol groups. The method for detecting a protein or peptide according to claim 8, wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate. The method for detecting a protein or peptide according to claim 9, wherein the average chain of ethylene glycol in the methoxypolyethylene glycol (meth) acrylate is 3 to 100. The water-soluble polymer is at least one polymer selected from an acrylic ester polymer, a methacrylic ester polymer, or a copolymer of an acrylic ester and a methacrylic ester, and the molecular weight of the polymer is 200 to 200. It is 200,000, The detection method of the protein or peptide in any one of Claims 1-10. The method for detecting a protein or peptide according to any one of claims 1 to 10, wherein the water-soluble polymer is polyvinyl alcohol. The method for detecting a protein or peptide according to any one of claims 1 to 10, wherein the water-soluble polymer is an alkylene glycol or a polymer having an alkylene glycol group. The method for detecting a protein or peptide according to claim 13, wherein the alkylene glycol is ethylene glycol. The method for detecting a protein or peptide according to claim 13, wherein the polymer having an alkylene glycol group has a molecular weight of 200 to 20,000. The method for detecting a protein or peptide according to claim 15, wherein the polymer having an alkylene glycol group is polyethylene glycol. The method for detecting a protein or peptide according to any one of claims 1 to 16, wherein the antibody is immobilized in a spot shape on the surface of a substrate. The method for detecting a protein or peptide according to claim 17, wherein the spots of a plurality of types of antibodies are present in the same compartment on the substrate surface. The method for detecting a protein or peptide according to any one of claims 1 to 18, wherein the substrate has a slide glass shape. The method for detecting a protein or peptide according to any one of claims 1 to 19, wherein the substrate has a fine channel, and the antibody is immobilized in the fine channel. The method for detecting a protein or peptide according to any one of claims 1 to 20, wherein the substrate is made of plastic. The method for detecting a protein or peptide according to claim 21, wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof.