JP4353091B2 - Protein 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 decipher the genetic activity and the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more detailed 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 has become increasingly popular, and as a result, rapid detection of bioactive substances has been accelerated. 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 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).

  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 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 part. 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 a physiologically active substance 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 with high sensitivity and high throughput. It is to provide a method for detecting a protein or peptide.

That is, the present invention is as follows.
(1) the antibody is a physiologically active substance fixed to the substrate surface, a method of detecting the protein or the peptide to specifically capture proteins or peptides in the sample by interaction with the antibody,
(A) A step of applying a polymer which is a binary copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group on the surface of the substrate,
(B) immobilizing an antibody on the polymer;
(C) a step of labeling the protein or peptide in the specimen,
(D) a step of bringing the specimen obtained in step (c) into contact with the substrate obtained in step (b);
A method for detecting a protein or peptide, comprising:
(2) The protein or peptide detection method according to (1), wherein the protein or peptide labeling method in the specimen is at least one method selected from a fluorescent labeling method, an enzyme labeling method, and a biotin labeling method.
(3) The method for detecting a protein or peptide according to (2), wherein the substance used in the fluorescent labeling method is at least one selected from Cy3, Cy5, FITC (fluorescein isothiocyanate), and rhodamine.
(4) The method for detecting a protein or peptide according to (2), wherein the substance used in the enzyme labeling method is at least one selected from peroxidase, alkaline phosphatase, acid phosphatase, and glucose oxidase.
(5) The method for detecting a protein or peptide according to any one of (1) to (4), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
(6) The method for detecting a protein or peptide according to any one of (1) to (4), wherein the physiologically active substance is immobilized in a spot shape on the surface of the substrate.
(7) The method for detecting a protein or peptide according to claim 6 , wherein spots of a plurality of types of physiologically active substances are present in the same section of the substrate surface.
(8) The method for detecting a protein or peptide according to any one of (1) to (7), wherein the substrate has a slide glass shape.
(9) The method for detecting a protein or peptide according to any one of (1) to (8), wherein the substrate has a fine channel, and a physiologically active substance is immobilized in the fine channel. .
(10) The method for detecting a protein or peptide according to any one of (1) to (9), wherein the base material is plastic.
(11) The method for detecting a protein or peptide according to (10), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, or a copolymer thereof. .

  According to the protein or peptide detection method of the present invention, the physiologically active substance is immobilized at an arbitrary position on the surface of the substrate without coating with an adsorption inhibitor, and the unnecessary physiologically active substance is adsorbed to other portions. In addition, by suppressing the binding and by labeling all the proteins and peptides in the sample, a plurality of types of target proteins or peptides in the sample can be simultaneously captured, and the protein or peptide can be detected with high sensitivity and high throughput.

  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 include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine, octenylphosphorylcholine, Although senyl phosphorylcholine etc. are mentioned, 2-methacryloyloxyethyl phosphorylcholine is preferable.

  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.

  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.

(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 physiologically active substances)
In the present invention, when the physiologically active substance is immobilized on the substrate, a method of spotting a liquid in which the physiologically active substance is dissolved or dispersed is preferable.
The pH of the liquid in which the physiologically active substance 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.
An antibody is preferably used as the physiologically active substance.
Further, it is preferable that the physiologically active substance is immobilized on the surface of the substrate in the form of spots, and it is further preferable that spots of a plurality of types of physiologically active substances are present in the same section of the substrate surface.

(Labeling of specimen)
Examples of a method for labeling a protein or peptide serving as a specimen include a fluorescent label, an enzyme label, and a biotin label, 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.

(Detection of proteins and peptides)
The protein or peptide in the specimen is supplemented by bringing a liquid containing the labeled specimen into contact with the substrate on which the physiologically active substance is immobilized.
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 9.5. If it is in this range, there is no fear that the protein and peptide to be detected are denatured, which is preferable.

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, an anti-mouse IgG2a antibody, which is a primary antibody having a pH adjusted to 9.5, was spotted on the substrate at the dilution rate (concentration) shown in Table 1 using an automatic spotter and allowed to stand for 24 hours. And fixed. After immobilization, washing with PBS containing 0.05% Tween 20 was performed. Thereafter, the whole mixture of mouse IgG2a antibody and serum protein as antigens was Cy3 labeled with NHS-Cy3 (mouse IgG2a antibody concentration: 19 nmol / L), or only the whole mixture of serum proteins was Cy3 labeled with NHS-Cy3 To cause an antigen-antibody reaction on the substrate, and measure the amount of fluorescence for each spot and the portion (background) other than the spot portion. At that time, the signal ratio with and without the antigen, and the S / N ratio ( Signal / noise ratio) was calculated. The results are shown in Table 1.

(Comparative example)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm). After subjecting the substrate surface to hydrophilization, the substrate was immersed in a 2% aqueous solution of an amino group-containing alkylsilane, and then subjected to heat treatment to introduce amino groups on the surface. This was immersed in a 1% glutaraldehyde aqueous solution to react the surface amino groups with glutaraldehyde to introduce aldehyde groups.
Next, an anti-mouse IgG2a antibody, which is a primary antibody having a pH adjusted to 9.5, was spotted on the substrate at the dilution rate (concentration) shown in Table 1 using an automatic spotter and allowed to stand for 24 hours. And fixed. After immobilization, the substrate is dipped in a solution obtained by diluting Blocking Ace, a blocking agent for immunological experiments manufactured by Dainippon Pharmaceutical Co., Ltd., 4 times with pure water to prevent nonspecific adsorption, and gently shaken at room temperature for 1 hour. did. Thereafter, washing was performed with PBS containing 0.05% Tween20. Subsequently, the whole mixture of antigen mouse IgG2a antibody and serum protein was labeled with CyS with NHS-Cy3 (mouse IgG2a antibody concentration: 19 nmol / L), or only the whole mixture of serum proteins was labeled with CyS with NHS-Cy3. An antigen-antibody reaction is caused on the substrate by contacting the sample, and the amount of fluorescence is measured for each spot and the portion other than the spot (background). At that time, the signal ratio with and without the antigen, and the S / N ratio (Signal / 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 55%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 30 μm.
The example is inferior to the comparative example in the spot signal intensity with the antigen, but the spot signal intensity value without the antigen is lower than the comparative example, and the signal ratio with / without the antigen is about the same or higher than the comparative example. As a result. Furthermore, the fluorescence intensity (background) other than the spot portion was much lower than that of the comparative example, and the S / N ratio was much higher in the example. That is, it can be said that highly sensitive protein and peptide were detected.

＊ 固定化抗体濃度は、
希釈倍率２倍：３．３μｍｏｌ／Ｌ
希釈倍率８倍：０．８３μｍｏｌ／Ｌ

* Immobilized antibody concentration is
Dilution factor 2 times: 3.3 μmol / L
Dilution factor 8 times: 0.83 μmol / L

According to the protein or peptide detection method of the present invention, the physiologically active substance is immobilized at an arbitrary position on the surface of the substrate without coating with an adsorption inhibitor, and the unnecessary physiologically active substance is adsorbed to other portions. And binding, and by labeling all proteins and peptides in the sample, multiple types of target proteins and peptides in the sample can be captured simultaneously, allowing high-sensitivity and high-throughput detection of proteins and peptides. It can be applied to various biochip detection methods including microfluidics.

Claims (11)

The antibody is a physiologically active substance on the substrate surface and fixed, a method of detecting the protein or the peptide to specifically capture proteins or peptides in the sample by interaction with the antibody,
(A) A step of applying a polymer which is a binary copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group on the surface of the substrate,
(B) immobilizing an antibody on the polymer;
(C) a step of labeling the protein or peptide in the specimen,
(D) a step of bringing the specimen obtained in step (c) into contact with the substrate obtained in step (b);
A method for detecting a protein or peptide, comprising: Protein or labeling method is fluorescence labeling of the peptides in the sample, enzymatic labeling, detection method of a protein or peptide of at least one of claims 1, wherein a method selected from biotin-labeling method. The method for detecting a protein or peptide according to claim 2 , wherein the substance used in the fluorescent labeling method is at least one selected from Cy3, Cy5, FITC (fluorescein isothiocyanate), and rhodamine. The method for detecting a protein or peptide according to claim 2 , 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 4 , 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 4, wherein the physiologically active substance is immobilized in a spot shape on the surface of the substrate. The method for detecting a protein or peptide according to claim 6 , wherein spots of a plurality of types of physiologically active substances 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 7 , wherein the substrate has a slide glass shape. The method for detecting a protein or peptide according to any one of claims 1 to 8 , wherein the substrate has a fine channel, and a physiologically active substance is immobilized in the fine channel. The method for detecting a protein or peptide according to any one of claims 1 to 9 , wherein the substrate is made of plastic. The method for detecting a protein or peptide according to claim 10 , wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, or a copolymer thereof.