JP4534817B2 - Protein detection method and peptide detection method - Google Patents

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 bioactive substances 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 by being positioned on the extension line of the DNA chip, studies have been made to immobilize a protein or a molecule for capturing the protein on a 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 detecting 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 of 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.

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 to the substrate surface;
(B) immobilizing an antibody on the polymer;
(C) labeling the protein or peptide in the specimen,
(D) a step of allowing the sample obtained in step (c) and a polymer having a phosphorylcholine group to coexist in the same liquid ;
(E) contacting the sample obtained in step (d) with the substrate obtained in step (b);
A method for detecting a protein or peptide, comprising:
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. A polymer having a phosphorylcholine group is a polymer having a structure similar to a biological membrane (phospholipid bilayer membrane), and has an effect of suppressing 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).

  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.

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

The bonding between the substrate surface and the polymer may be any bonding mode such as covalent bonding, electrostatic interaction, hydrogen bonding, or hydrophobic effect bonding, but from the viewpoint of simplicity of surface treatment, the substrate surface It is preferable that the polymer and the polymer are bonded by a hydrophobic effect.
In the present invention, it is preferable to apply a solution containing a polymer having a phosphorylcholine group on the surface of the substrate.

(Base material)
The base material can be usually glass, metal or the like, but the base material used in the present invention is a plastic from the viewpoint of ease of surface treatment and mass productivity, and in particular, 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 polymer having phosphorylcholine group in the same system)
In the present invention, the form of the polymer having a phosphorylcholine group that coexists with a 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 in the same system with the polymer having a phosphorylcholine group is not particularly limited, but those containing many contaminating proteins such as serum can obtain the effect of the present invention. .
Coexistence of a sample and a polymer having a phosphorylcholine group in the same system can be carried out, for example, by adding the liquid polymer having a phosphorylcholine group to a sample solution in a solution state so as to coexist in the same solution.
In the present invention, the phosphorylcholine group used in the coexistence step in the same system is, for example, 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allyl Examples include phosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine, octenylphosphorylcholine, decenylphosphorylcholine, and the like, and 2-methacryloyloxyethylphosphorylcholine is preferred.
Moreover, the polymer used at the said process may contain other groups other than a phosphorylcholine group, and the binary copolymer of the monomer which has a phosphorylcholine group, and the monomer which has a butylmethacrylate group is preferable.
The phosphorylcholine group and polymer used in the above step may be the same as or different from those used in the step of applying the polymer having a phosphorylcholine group to the substrate surface described above.

(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 the substrate, 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-mouse IgG2a antibody solution, which is a primary antibody adjusted to pH 9.5 at a concentration of 3.3 μmol / L, is spotted on the substrate and allowed to stand for 24 hours. Immobilized. After immobilization, washing with PBS containing 0.05% Tween 20 was performed.
Subsequently, the whole mixture of serum proteins as antigens was Cy3 labeled with NHS-Cy3.
Thereafter, a 5% by weight aqueous solution of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer 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). 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-mouse IgG2a antibody solution, which is a primary antibody adjusted to pH 9.5 at a concentration of 3.3 μmol / L, is spotted on the substrate and allowed to stand for 24 hours. Immobilized. After immobilization, washing with PBS containing 0.05% Tween 20 was performed.
Subsequently, the whole mixture of serum proteins as antigens was Cy3 labeled with NHS-Cy3.
Thereafter, the Cy3-labeled product 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 a portion (background) other than the spot portion. / noise ratio) was 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 65%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 10 μm.
In the examples, the spot signal intensity was almost the same value as the comparative example, but the fluorescence intensity (background) other than the spot portion was lower than that of the comparative example, and the S / N ratio was higher in the example. became. 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. , Synergistic effect between the sample (especially those with many non-specific adsorption of contaminating proteins such as serum and plasma) and the polymer having phosphorylcholine group in the same system, and the polymer having phosphorylcholine group on the gas surface Therefore, non-specific adsorption of contaminating proteins can be reduced, and high-sensitivity and high-throughput protein or peptide can be detected. Therefore, it can be applied to various biochip detection methods including microfluidics.

Claims (13)

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 to the substrate surface;
(B) immobilizing an antibody on the polymer;
(C) labeling the protein or peptide in the specimen,
(D) a step of allowing the sample obtained in step (c) and a polymer having a phosphorylcholine group to coexist in the same liquid ;
(E) contacting the sample 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 is a copolymer containing a butyl methacrylate group. The method for detecting a protein or peptide according to any one of claims 1 to 7, wherein the antibody is immobilized in a spot shape on the surface of the substrate. The method for detecting a protein or peptide according to claim 8, wherein spots of a plurality of types of antibodies are present in the same section of the substrate surface. The method for detecting a protein or peptide according to any one of claims 1 to 9, wherein the substrate has a slide glass shape. The method for detecting a protein or peptide according to any one of claims 1 to 10, wherein the substrate has a fine channel, and an antibody is immobilized in the fine channel. The method for detecting a protein or peptide according to any one of claims 1 to 11, wherein the substrate is made of plastic. The method for detecting a protein or peptide according to claim 12, 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.