JP4530895B2 - Solid phase carrier for peptide immobilization and method of using the same - Google Patents

Description

  The present invention relates to a solid phase carrier for arranging and fixing a peptide on the solid phase surface and a method for using the same.

  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).

  On the other hand, immobilizing proteins, which are highly functional molecules, efficiently on the substrate surface while maintaining their functions, and quantitatively detecting their specific interactions also has problems to be overcome. Many are extremely difficult. For this reason, there are an increasing number of situations where peptides are used as relatively stable small molecules that can mimic protein functions. Techniques for synthesizing peptides in a combinatorial manner have been established, and it can be said that a peptide array is a technique suitable for the purpose of high-throughput bioactive substance detection.

Regarding the detection and quantification of analytes such as proteins and peptides, considering the case where peptides are immobilized on a solid support, a method of detecting and quantifying by an ELISA-like antigen-antibody reaction can be considered. That is, the peptide is bound to the solid surface, and then the antibody that recognizes and binds to the immobilized peptide (primary antibody) is reacted, and further, the labeled antibody that recognizes and binds to the primary antibody (secondary antibody) is reacted. This is a method for quantitatively measuring proteins and peptides by measuring the label.
By using this technique, it is possible to search for an epitope recognized by an antibody or screen an antibody that binds to a specific protein, etc., with high throughput. (For example, Non-Patent Document 1)

  Moreover, since a peptide is a fragment of a protein, it is possible to mimic the reaction between an enzyme and a protein by using a peptide at a site where an enzyme acting on a protein recognizes and reacts. That is, it is possible to screen for a substrate or an inhibitor of a specific enzyme using a peptide array prepared from various peptides. (For example, Non-Patent Document 1)

  Although it is a very useful peptide array, since peptides are small molecules, it is difficult to physicochemically adsorb them on a carrier surface like proteins. Therefore, in order to immobilize the peptide, it is necessary to introduce a reactive functional group on the surface of the carrier and immobilize the peptide by a covalent bond. Until now, functional groups have been introduced into the glass surface through several stages of processing. For this reason, it takes time and labor to produce a peptide array, and there are drawbacks such as lack of convenience and versatility.

  In addition, after immobilizing the peptide on the substrate, it reacts with other proteins (corresponding to the primary antibody in the case of ELISA) on the surface, and further reacts with the labeled protein (also corresponding to the secondary antibody). Finally, when detecting with a detector or the like, if a protein other than a peptide, that is, an antibody or a labeled protein is fixed to a portion where the peptide is not fixed, noise is detected at the time of detection, and the signal to noise ratio (S / N ratio) is reduced, and detection accuracy is reduced (see, for example, Non-Patent Document 2).

  For this reason, in order to prevent nonspecific adsorption | suction of an antibody or a labeled protein, the coating of a blocking agent is needed, but these nonspecific adsorption | suction prevention ability is not enough. In addition, since the blocking agent is coated after immobilizing the peptide, it may be coated on the immobilized peptide, resulting in a problem that it is difficult to react with the primary antibody. For this reason, there is a need for a biochip that does not have a blocking step after peptide immobilization and has a small amount of non-specific adsorption of physiologically active substances.

On the other hand, in terms of profiling the fluctuations of all proteins (proteomes), microfluidics technology that enables the manipulation of ultra-trace amounts of proteins and nano-volume solutions such as several nanoliters, 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 that is a sample reacts specifically with the capture immobilized in the flow path and suppresses nonspecific adsorption to the inner wall of the flow path other than the capture section. Is required.
JP 2001-116750 A “DOJIN NEWS No. 109, post-genome 9 from a chemist”, Dojin Chemical Laboratory, 2004 “DNA Microarray Practice Manual”, Yoshihide Hayashizaki, Koji Okazaki, Yodosha, 2000, p.57

  An object of the present invention is to provide a solid phase carrier for immobilizing peptides and a method for using the solid phase carrier, which are simple and can be evaluated in a short time.

The present invention is as follows.
(1) Immobilizing a peptide on a solid surface, allowing a physiologically active substance that reacts specifically with the immobilized peptide to act, and reacting specifically with the peptide, or specifically reacting with the peptide Is a solid phase carrier used when detecting a substance using fluorescence, luminescence or color development, wherein the solid phase material is made of plastic, and the monomer having a phosphorylcholine group on the solid phase surface and the monomer having an active ester group A solid phase carrier for immobilizing a peptide, comprising a polymer substance obtained by copolymerization of
(2) The solid phase carrier for immobilizing a peptide according to (1), wherein the solid phase is a flat substrate, a microwell plate, a substrate having a fine channel, or microbeads.
(3) The solid phase carrier for immobilizing a peptide according to (1) or (2), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
(4) The solid phase carrier for peptide immobilization according to any one of (1) to (3), wherein the active ester group is a p-nitrophenyl ester group or an N-hydroxysuccinimide ester group.
( 5 ) The solid phase carrier for immobilizing a peptide according to ( 1 ), wherein the polymer substance is a copolymer with a monomer further containing a butyl methacrylate group.
( 6 ) Any one of (1) to ( 5 ), 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. Solid phase carrier for immobilizing peptides.
( 7 ) A solid phase carrier in which a peptide is immobilized on the solid phase carrier for peptide immobilization according to any one of (1) to ( 6 ).
( 8 ) A method for using the solid phase carrier for immobilizing a peptide according to any one of (1) to ( 6 ),
(A) a step of immobilizing the peptide on the solid surface,
(B) a step of allowing a physiologically active substance that specifically reacts with the peptide to act, and (c) a physiologically active substance that specifically reacts with the peptide or the specifically reacted peptide that uses fluorescence, luminescence, or color development. Using the solid phase carrier for immobilizing a peptide, comprising the step of detecting by the method.
( 9 ) The method for using a solid phase carrier for immobilizing a peptide according to ( 8 ), wherein the physiologically active substance that specifically reacts with the peptide is a protein.

  According to the solid phase carrier of the present invention, peptides can be efficiently and easily bound to the solid phase carrier, and non-specific adsorption of proteins and fluorescent dyes can be effectively suppressed. Evaluation of peptide arrays and peptide plates, screening of peptide antibodies, and the like are possible.

  The carrier of the present invention is characterized by having a polymer substance having a phosphorylcholine group and an active ester group on the solid surface. A polymer substance having a phosphorylcholine group and an active ester group is a polymer having both the property of suppressing nonspecific adsorption of proteins and fluorescent materials and the property of immobilizing peptides. 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). The active ester plays a role of immobilizing the peptide. Due to the characteristics of the above polymer, it is possible to effectively suppress non-specific adsorption of physiologically active substances and fluorescent substances on the surface of the solid phase carrier. As a result, the peptide immobilized on the surface of the solid phase carrier is specifically inhibited. Only the reacted physiologically active substance can be captured.

  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 more preferable.

Examples of the active ester group used in the present invention include p-nitrophenyl ester group, N-hydroxysuccinimide ester group, succinimide ester group, phthalimide ester group, 5-norbornene-2,3-dicarboximide ester Group, etc. are preferable, and p-nitrophenyl ester group or N-hydroxysuccinimide ester group is more preferable.
The polymer substance used in the present invention is preferably a polymer substance obtained by copolymerizing a monomer having a phosphorylcholine group and a monomer having an active ester group. Furthermore, in addition to the phosphorylcholine group and the active ester group, other groups may be contained, and a copolymer with a monomer containing a butyl methacrylate group is preferred.

(Material of solid support)
The solid phase carrier of the present invention is characterized in that the solid phase material is plastic.
As the plastic, a thermoplastic resin or a thermosetting resin can be used, but the thermoplastic resin is preferable from the viewpoint of production efficiency. As the thermoplastic resin, those that generate a small amount of fluorescence are preferable, and examples thereof include linear polyolefins such as polyethylene and polypropylene, polycarbonates, polystyrenes, polyamides, cyclic polyolefins, and fluorine-containing resins. It is more preferable to use a cyclic polyolefin that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. Here, the cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin.

  Examples of the former are produced by hydrogenating norbornene monomers represented by, for example, norbornene, dicyclopentadiene, and tetracyclododecene, and polymers obtained by ring-opening polymerization of these alkyl-substituted products. It is a saturated polymer.

The latter copolymer is cyclic with α-olefins such as ethylene, propylene, isopropyl, 1-butene, 3-methyl-1-butene, 1-pentene, 3-methyl-1-pentene, 1-hexene and 1-octene. It is a saturated polymer produced by hydrogenating a random copolymer of olefinic monomers. As the copolymer, a copolymer with ethylene is most preferable.
These resins may be used alone, or two or more copolymers or a mixture may be used. Further, in addition to the resin component, an inorganic or organic filler having a fibrous shape, a spherical shape, or other shapes, or various additive components may be included.

(Shape of solid support)
Examples of the shape of the solid phase carrier include a flat substrate, a microwell plate, microbeads, and a substrate having a fine channel shape.
In the case of a carrier with a peptide immobilized on the microbead surface, the surface area on which the peptide can be immobilized is greatly increased compared to the case of immobilizing the peptide on a substrate or plate, resulting in the capture of many proteins for detection purposes. It is possible to increase the S / N ratio.
Further, when the peptide is immobilized in the fine channel, the frequency at which the peptide can capture the protein to be detected increases relatively, so that the reaction time can be shortened.
Furthermore, when the microbeads on which the peptide is immobilized are encapsulated in a fine channel, both of the above-mentioned advantages can be achieved.

(Immobilization of peptides)
In the present invention, when a peptide is immobilized on a solid phase, a method of attaching a liquid for dissolving or dispersing the peptide is preferable. The pH of the liquid in which the peptide is dissolved or dispersed is preferably 8 to 10, more preferably pH 9.0 to 9.5. If it is less than the lower limit, the efficiency of the immobilization reaction is remarkably reduced, and if it exceeds the upper limit, the possibility that the peptide is denatured increases.
After the peptide is attached, due to the properties of the phosphorylcholine group on the surface of the solid phase, washing with water or a buffer containing a surfactant suppresses nonspecific adsorption of proteins and fluorescent substances that can react with the peptide to the solid surface. It becomes possible to do. However, if the protein or fluorescent substance contains many functional groups that can react with the active ester on the solid phase surface, the inactive treatment of the active ester remaining on the solid phase surface other than the peptide immobilized is treated with an alkali compound or It is preferable to carry out with a compound having a primary amino group.
As the alkali compound, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate, calcium hydroxide, magnesium hydroxide, sodium borate, lithium hydroxide, potassium phosphate, etc. are preferably used. Can do.
Examples of the compound having a primary amino group include glycine, 9-amino aquadine, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino-2,3-dihydro-1,4-pentanol, amino Ethanethiol hydrochloride, aminoethanethiolsulfuric acid, 2- (2-aminoethylamino) ethanol, 2-aminoethyl dihydrogen phosphate, aminoethyl hydrogensulfate, 4- (2-aminoethyl) morpholine, 5-aminofluorescein, 6-aminohexanoic acid, aminohexyl cellulose, p-aminohippuric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 5-aminoisophthalic acid, aminomethane, aminophenol, 2-aminooctane, 2 -Aminooctanoic acid, 1-amino-2-propanol, 3-amino- -Propanol, 3-aminopropene, 3-aminopropionitrile, aminopyridine, 11-aminoundecanoic acid, aminosalicylic acid, aminoquinoline, 4-aminophthalonitrile, 3-aminophthalimide, p-aminopropiophenone, aminophenyl Acetic acid, aminonaphthalene and the like can be preferably used, and sodium hydroxide, aminoethanol and glycine are more preferable.

(Sequence of peptide to be immobilized)
In order to increase the reactivity with the active ester group, it is preferable to introduce a cysteine residue or a lysine residue into the peptide to be immobilized. The introduction position of cysteine or lysine residue is preferably at the end of the peptide chain.

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)
Each well of a polystyrene resin 96-well microplate (Sumitomo Bakelite ELISA plate S MS-8496F) was treated with 0.3 weight of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenylcarbonyloxyethyl methacrylate copolymer. A polymer having phosphorylcholine groups was introduced onto the surface of the substrate by immersing in 150 ml of a% ethanol solution.
Next, a well added with 100 ml of somatostatin peptide diluted to 20 mg / ml using a phosphate buffer and adjusted to pH 9.0 and a well added with only 100 ml of phosphate buffer were prepared. After allowing to stand at 2 ° C. for 2 hours to immobilize the peptide, it was washed with PBS containing 0.05% Tween20. Thereafter, an antigen-antibody reaction was caused by using a commercially available rabbit anti-somatostatin antibody (416M manufactured by biomeda) and a commercially available biotin-labeled sheep anti-rabbit IgG antibody (2AB02B manufactured by Serotec), which are primary antibodies, and further commercially available peroxidase-labeled avidin. (An ICN 55898) was used for enzyme labeling. Then, using a color kit for peroxidase (ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.), a color reaction using hydrogen peroxide as a substrate was performed, and TMBZ (3,3 ′, 5,5 ′ tetramethyl bench used as a color substrate was used. The absorbance of gin) was measured. The signal value, blank value, and S / N ratio (Signal / noise ratio) at that time were calculated. The results are shown in Table 1.

(Comparative example)
A 96-well microplate made of polystyrene resin having a carboxyl group directly bonded to its surface (Sumitomo Bakelite ELISA plate carbo MS-8796F) was used as a substrate for peptide immobilization.
100 ml of WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) prepared to 10 mg / ml with PBS (pH 5.8) was added to each well and allowed to stand at 37 ° C. for 2 hours. After the reaction, the wells were prepared by washing with PBS (pH 5.8) and adding 100 ml of somatostatin peptide diluted to 20 mg / ml with PBS (pH 5.8) and 100 ml of PBS (pH 5.8) at 37 ° C. After reacting for 2 hours, washing was performed with PBS containing 0.05% Tween20.
In order to suppress non-specific adsorption of the protein to be reacted with the peptide to the substrate surface, 300 ml of 5% skim milk solution was added to each well as a blocking agent, allowed to stand at room temperature for 2 hours, and then containing 0.05% Tween 20 Washed with PBS.
Thereafter, in the same manner as in Examples, an antigen-antibody reaction was caused by using a commercially available rabbit anti-somatostatin antibody (biomeda 416M) and a commercially available biotin-labeled sheep anti-rabbit IgG antibody (Serotec 2AB02B), and further peroxidase-labeled avidin. (An ICN 55898) was used for enzyme labeling. Then, using a color kit for peroxidase (ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.), a color reaction using hydrogen peroxide as a substrate was performed, and TMBZ (3,3 ′, 5,5 ′ tetramethyl bench used as a color substrate was used. The absorbance of gin) was measured. The signal value, blank value, and S / N ratio (Signal / noise ratio) at that time were calculated. The results are shown in Table 1.

The dilution ratio (concentration) of the used reagent is as follows.
Rabbit anti-somatostatin antibody (10 mg / ml): used at 250-fold dilution Biotin-labeled sheep anti-rabbit IgG antibody (1 mg / ml): used at 5000-fold dilution Peroxidase-labeled avidin (5 mg): used at 1000-fold dilution

The absorbance in Examples and Comparative Examples was measured using a SPECTRAFLUOR manufactured by TECAN at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.
In the example, the signal value was higher than that of the comparative example, and the blank value was lower than that of the comparative example. Regarding the S / N ratio, the Examples were higher than the Comparative Examples, and the peptides could be detected with high sensitivity. Furthermore, the examples do not require any operations for activating the surface of the base material or preventing nonspecific adsorption of proteins, and the operations are simpler than the comparative examples, and the evaluation time is shortened.

  By using the solid phase carrier for immobilizing a peptide of the present invention, the peptide can be immobilized at an arbitrary position of the solid phase carrier without coating with a blocking agent, and unnecessary physiologically active substances and fluorescence to other portions can be immobilized. Adsorption and binding of substances can be suppressed, and high-sensitivity and high-throughput peptide arrays and peptide plates can be evaluated. Moreover, it has a high degree of freedom in shape, and can be applied to various biochip detection methods including microbeads and microfluidics.

Claims (9)

Immobilizing a peptide on a solid phase surface, allowing a physiologically active substance that reacts specifically with the immobilized peptide to act, and fluorescently reacting the physiologically active substance that specifically reacts with the peptide, or the peptide that specifically reacts, A solid phase carrier used for detection using luminescence or color development, the solid phase material is made of plastic, and a monomer having a phosphorylcholine group and a monomer having an active ester group on the solid phase surface are copolymerized A solid phase carrier for immobilizing a peptide, comprising a polymer substance obtained as described above . The solid phase carrier for immobilizing a peptide according to claim 1, wherein the solid phase is a flat substrate, a microwell plate, a substrate having a fine channel, or a microbead. The solid phase carrier for peptide immobilization according to claim 1 or 2, wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group. The solid support for peptide immobilization according to any one of claims 1 to 3, wherein the active ester group is a p-nitrophenyl ester group or an N-hydroxysuccinimide ester group. Monomers and copolymers in which claim 1 peptide immobilization solid support further comprising the polymeric material further butyl methacrylate group. Wherein the plastic is polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefins, polypentene, polyamide, or claim 1 for 5 peptide immobilization of any one is at least one selected from the group consisting of a copolymer thereof Solid support. Claim 1-6 immobilized solid phase carrier peptide to the peptide immobilization solid support according to any one. A method for using the solid phase carrier for immobilizing a peptide according to any one of claims 1 to 6 ,
(A) a step of immobilizing the peptide on the solid phase surface;
(B) a step of causing a physiologically active substance that specifically reacts with the peptide to act; and (c) a physiologically active substance that specifically reacts with the peptide or the peptide that specifically reacts with fluorescence, luminescence, or color development. Using the solid phase carrier for immobilizing a peptide, comprising the step of detecting by the method. The method for using a solid phase carrier for immobilizing a peptide according to claim 8, wherein the physiologically active substance that specifically reacts with the peptide is a protein.