JP2017121205A - Solid phase in which sugar chain is immobilized and classification method of virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solid phase which can measure the interaction with a virus highly sensitively, simply, and inexpensively, and in which sugar chain is immobilized, and to provide a classification method of a virus.SOLUTION: The present invention provides a solid phase in which an α2,3 binding sialic acid-containing sugar chain and/or an α2,6 binding sialic acid-containing sugar chain are immobilized; and a classification method of a virus comprising: contacting a virus with the solid phase in which an α2,3 binding sialic acid-containing sugar chain and/or an α2,6 binding sialic acid-containing sugar chain are immobilized; detecting the virus bound to the solid phase; and classifying the virus based on the binding activity of the virus and the α2,3 binding sialic acid-containing sugar chain or α2,6 binding sialic acid-containing sugar chain.SELECTED DRAWING: None

Description

  The present invention relates to a solid phase having a sugar chain immobilized thereon and a method for classifying viruses.

  Severe acute respiratory syndrome (SARS) and infectious diseases caused by infectious viruses such as influenza have become major social problems. These viruses are known to infect a host by recognizing and binding to a specific sugar chain covering the surface of the host cell.

  Non-Patent Document 1 discloses that the structure of a sugar chain to which a virus can bind varies depending on the type of virus.

Stencel-Baerenwald J. E., et al., The sweet spot: defining virus-sialic acid interactions, Nat. Rev. Microbiology., 12 (11), 739-749, 2014.

  An object of the present invention is to provide a solid phase on which a sugar chain is immobilized, which can measure the interaction with a virus with high sensitivity, simply and inexpensively.

The present invention is as follows.
(1) A solid phase on which an α2,3-linked sialic acid-containing sugar chain and / or an α2,6-linked sialic acid-containing sugar chain is immobilized.
(2) The solid phase according to (1), which is for virus classification.
(3) The solid phase according to (2), wherein the virus is influenza virus, reovirus, adenovirus or rotavirus.
(4) The α2,3-linked sialic acid-containing sugar chain forms an α2,3-linked sialic acid-containing sugar chain-binding peptide, and the α2,6-linked sialic acid-containing sugar chain is an α2,6-linked type. The solid phase according to any one of (1) to (3), which forms a sialic acid-containing sugar chain-binding peptide.
(5) The α2,6-linked sialic acid-containing sugar chain is fixed, and the absorbance measured by the following measurement method is 0.05 or more, according to any one of (1) to (4) Solid phase.
[Measuring method]
20 μg / mL biotinylated Sambucus nigra (SNA) lectin is brought into contact with the region where α2,6-linked sialic acid-containing sugar chains are immobilized on the solid phase at 4 ° C. for 8 hours and then washed. Subsequently, 0.2 μg / mL streptavidin-horseradish peroxidase (HRP) is contacted at room temperature for 60 minutes and then washed. Subsequently, a peroxidase coloring kit (model number “ML-1120T”, manufactured by Sumitomo Bakelite Co., Ltd.) is allowed to contact at room temperature for 15 minutes under light shielding. Subsequently, the absorbance at a wavelength of 450 nm is measured.
(6) The solid phase according to any one of (1) to (5), wherein an asialo sugar chain is further immobilized.
(7) The solid phase according to (6), wherein the asialo sugar chain forms an asialo sugar chain-binding peptide.
(8) A step of bringing a virus into contact with a solid phase on which an α2,3-linked sialic acid-containing sugar chain and / or an α2,6-linked sialic acid-containing sugar chain is immobilized, and detecting the virus bound to the solid phase And a step of classifying the virus based on the binding activity of the virus and the α2,3-linked sialic acid-containing sugar chain or the α2,6-linked sialic acid-containing sugar chain. Classification method.
(9) The virus classification method according to (8), wherein the virus is influenza virus, reovirus, adenovirus or rotavirus.

  According to the present invention, the interaction between a virus and a sugar chain can be measured with high sensitivity, simply and inexpensively.

(A) It is the schematic diagram which showed the structure of hemagglutinin (HA) protein of influenza virus. (B) It is the schematic diagram showing the difference in the coupling | bonding mode of the sugar chain receptor galactose and sialic acid in a bird and a human. (A) Schematic diagram showing the structure of the reovirus outer capsid protein σ1. (B) A schematic diagram showing the binding mode of galactose and sialic acid, a sugar chain receptor to which the outer capsid protein σ1 of T1 reovirus can bind. (C) A schematic diagram showing the binding mode of galactose and sialic acid, a sugar chain receptor to which the outer capsid protein σ1 of T3 reovirus can bind. (A) Schematic diagram showing the structure of the outer capsid protein of D-type adenovirus 37 (Ad37). (B) Schematic diagram showing the binding mode of galactose and sialic acid, a sugar chain receptor to which the outer capsid protein σ1 of Ad37 can bind. (A) Schematic diagram showing the structure of the outer capsid protein of rotavirus. (B) Schematic diagram showing the binding mode of galactose and sialic acid of the sugar chain receptor to which the VP8 ^* subunit of human rotavirus HAL1166 can bind. (A)-(C) The figure which showed an example of the structure of (alpha) 2, 3-bond type sialic acid containing sugar chain binding peptide, (alpha) 2,6 bond type sialic acid containing sugar chain binding peptide, and an asialog sugar chain binding peptide in this embodiment It is. 2 is a graph showing the results of the periodic acid-Schiff base (PAS) method in Test Example 1. FIG. 6 is a graph showing the results of SNA (Sambucus nigra) lectin reaction in Test Example 2. FIG.

<Solid phase with immobilized sugar chains>
In one embodiment, the present invention provides a solid phase on which an α2,3-linked sialic acid-containing sugar chain and / or an α2,6-linked sialic acid-containing sugar chain is immobilized.

  According to the solid phase of the present embodiment, the interaction between the virus and the sugar chain can be measured with high sensitivity, simply and inexpensively.

[Sialic acid-containing sugar chains]
In the present specification, “sialic acid” means a generic name for substances in which the amino group or hydroxy group of neuraminic acid, which is a 9-carbon sugar, is substituted. N-acetylneuraminic acid (Neu5Ac) that has been converted to N-glycolylneuraminic acid (Neu5Gc) modified with glycolic acid.
Sialic acid is widely distributed in the body as a component of glycoconjugates such as glycoproteins, glycolipids, glycopeptides, etc., and is especially present on the cell membrane surface and plays a major role in the specific recognition mechanism of cells. It is said that. The type of sialic acid residue as a substituent strongly affects the activity of metabolic enzymes, particularly sialidase, and affects the conversion rate of glycoconjugates. Complex carbohydrates are involved in a wide variety of cellular functions, for example, the surface of biological organs is protected from enzymatic and immunological attacks by acidic molecules in complex carbohydrates present on the surface of biological organs. . However, on the other hand, glycoconjugates on the surface of living organs become various physiological receptors, become recognition sites for microorganisms and toxins including viruses, and allow infection. Many viruses, particularly influenza viruses, are known to bind to sialic acid residues when infecting cells.
The present inventors have focused on the fact that the sialic acid residues to be bound differ depending on the type of virus, and have completed the present invention.

[Virus]
The solid phase in this embodiment can be used for virus classification.
In this specification, examples of viruses that can be classified include influenza viruses, reoviruses, adenoviruses, rotaviruses, and the like. Among these, influenza virus is preferable and avian influenza virus and human influenza virus are more preferable.

(1. Influenza virus)
Influenza virus is a single-stranded RNA virus belonging to the Orthomyxoviridae family that infects mammals and birds, and recognizes a sugar chain having a specific structure on the surface of a host cell as a receptor (sugar chain receptor). FIG. 1 (A) is a schematic diagram showing the structure of hemagglutinin (HA) protein of influenza virus. FIG. 1 (B) is a schematic diagram showing the difference in binding mode between galactose and sialic acid of sugar chain receptors in birds and humans. When a trimeric hemagglutinin protein adheres to a host cell, most of the avian influenza viruses usually have a galactose and α2,3-linked sialic acid (SAα2,3Gal) having a sugar chain terminal (bird type). In contrast, most human influenza viruses usually accept galactose and α2,6-linked sialic acid (SAα2,6Gal) at the sugar chain end (human-type receptor). It is known to selectively bind to the body.

(2. Reovirus)
A reovirus is a virus that does not have an envelope belonging to the family Reoviridae, and has 10 to 12 linear double-stranded RNAs in its genome. Almost all mammals function as reovirus hosts, but the disease is limited to a very young period.
FIG. 2 (A) is a schematic diagram showing the structure of the reovirus outer capsid protein σ1. Reovirus adherence to host cells is mediated by a trimeric fibrous outer capsid protein σ1 protruding from the surface of the virion. First, the junctional adhesion molecule A (Jam-A) interacts with a region in the globular head of the reovirus σ1 protein. In addition, the sequence of the σ1 shaft domain interacts with cell surface sialic acid.

  Reovirus consists of three serotypes, and type 1 (T1) and type 3 (T3) bind to sugar chains in different ways. FIGS. 2 (B) and (C) are schematic diagrams showing the difference in the binding mode between galactose and sialic acid, a sugar chain receptor to which the outer capsid protein σ1 of T1 reovirus and T3 reovirus can bind. The T1 reovirus σ1 specifically binds to a GM2 (NeuAcα1,3 (GalNAcβ1,4) Galβ1,4Glc) sugar chain and aggregates human and non-human primate erythrocytes. On the other hand, T3 reovirus σ1 binds to a wide range of sialylated sugar chains (galactose and α2,3, α2,6 or α2,8-linked sialic acid sugar chains) and aggregates erythrocytes of various mammals. . T3 reovirus σ1 binds to glycophilin, a sialylated glycoprotein expressed in erythrocytes, but T1 reovirus σ1 does not bind.

  Furthermore, the sugar chain binding sites of T1 reovirus and T3 reovirus are in different domains of the σ1 protein (see FIG. 2 (A)). In the σ1 protein of T1 reovirus, the head domain binds to the GM2 sugar chain, whereas in the σ1 protein of T3 reovirus, the trunk domain is the sugar chain binding region, and galactose and α2,3, α2,6 Alternatively, it binds to α2,8-linked sialic acid sugar chain. In T1 reovirus and T3 reovirus, the head domain of σ1 protein binds to Jam-A, but is different from the GM2 sugar chain binding site of T1 reovirus σ1 protein. Can interact independently with both receptors.

(3. Adenovirus)
An adenovirus is a double-stranded DNA (dsDNA) virus that does not have an envelope belonging to the family Adenoviridae that infects humans, other mammals, and birds. Some adenoviral strains cause conjunctivitis and upper respiratory tract disease in humans, while others rarely cause syndromes in immunocompetent individuals. Adenovirus serotypes, like reovirus, differ in binding to sialic acid. For example, most adenoviruses use protein receptors, but D-type adenovirus 37 (Ad37) is known to aggregate human erythrocytes in a neuraminidase-sensitive manner and bind to sialic acid.

  FIG. 3 (A) is a schematic diagram showing the structure of the outer layer capsid protein of D-type adenovirus 37 (Ad37). Similar to the reovirus σ1 protein, the adenovirus outer capsid protein binds to host cells using fibrous trimeric fibers extending from the 12 vertices of the icosahedron. FIG. 3 (B) is a schematic diagram showing the binding mode of galactose and sialic acid, a sugar chain receptor to which the outer capsid protein of Ad37 can bind. Ad37 outer capsid protein σ1 specifically binds to GD1a (Neu5Acα2,3Galβ1,3GalNAcβ1,4 (Neu5Acα2,3) Galβ1,4Glc) sugar chain, which is a branched hexasaccharide, in α2,3-linked sialic acid It has been known.

(4. Rotavirus)
Rotavirus is a double-stranded RNA virus that does not have an envelope belonging to the family Reoviridae, and is a virus that causes diarrhea in children worldwide. FIG. 4A is a schematic diagram showing the structure of the outer capsid protein of rotavirus. Rotavirus adhesion is sugar chain dependent and is mediated by the trimer outer capsid protein VP4. Rotavirus infectivity is increased by protease cleavage of the N-terminal VP8 ^* and C-terminal VP5 ^* subunits of the VP4 trimer. The VP8 ^* subunit mediates viral adhesion by binding to cell surface sugar chains, and the VP5 ^* subunit causes membrane penetration.

FIG. 4 (B) is a schematic diagram showing the binding mode of galactose and sialic acid, a sugar chain receptor to which the VP8 ^* subunit of human rotavirus HAL1166 can bind. Many animal rotaviruses, including rhesus rotavirus (RRV), bind to receptors containing sialic acid at the end of ganglioside GM3 (Neu5Acα2,3Galβ1,4Glc). Human rotavirus (eg, Wa gene cluster) is a sialic acid linked to one branch of a two-branched sugar chain such as ganglioside GM1 (Galβ1,3GalNAcβ1,4 (Neu5Acα2,3) Galβ1,4Glc). Binds to oxidative receptors. Other human rotavirus strains (eg, HAL1166, etc.) are known to specifically bind to type A HBGAs (histo-blood group antigens). HBGAs are oligosaccharides that are also expressed in erythrocytes, epithelial cells, or mucosal secretions. Furthermore, human P [11] type rotavirus strains that cause diarrhea in newborns bind to HBGAs precursors.

  As described above, since the mode of binding of sialic acid to which the virus can bind and sugar differs depending on the type of virus, the virus can be classified by examining the type of sialic acid-containing sugar chain to which the virus binds. .

[Asialo sugar chain]
In the solid phase of the present embodiment, it is preferable that an asialog sugar chain is further immobilized. Since no virus binds to the asialo sugar chain, the section to which the asialo sugar chain is immobilized in the solid phase of this embodiment can be used as a negative control for the reaction.

  In the present specification, the “asialo sugar chain” means a sugar chain in which sialic acid is not added to the non-reducing end of the sugar chain structure. For example, in the case of an N-linked sugar chain, an asialo sugar chain in which galactose is present at the non-reducing end can be mentioned.

[Amount of sugar chain bound to solid phase]
The amount of sugar chain bound to the solid phase of this embodiment is preferably managed based on the amount of α2,6-linked sialic acid-containing sugar chain. For the amount of α2,6-linked sialic acid-containing sugar chain, the absorbance measured by the following measurement method may be, for example, 0.05 or more, for example, 0.1 or more, for example, 0.5 or more. It may be. Further, the upper limit of the absorbance may be, for example, 2.0.

(Measuring method)
The region where the α2,6-linked sialic acid-containing sugar chain is immobilized on the solid phase is contacted with 20 μg / mL biotinylated SNA lectin at 4 ° C. for 8 hours and then washed. Subsequently, 0.2 μg / mL streptavidin-HRP is contacted at room temperature for 60 minutes and then washed. Subsequently, a peroxidase coloring kit (model number “ML-1120T”, manufactured by Sumitomo Bakelite Co., Ltd.) is allowed to contact at room temperature for 15 minutes under light shielding. Subsequently, the absorbance at a wavelength of 450 nm is measured. Examples of the biotinylated SNA lectin include biotinylated SNA lectin (manufactured by VECTOR) used in Examples described later. Examples of streptavidin-HRP include those in which 1.5 molecules of HRP are bound per molecule of streptavidin.

  The solid phase of this embodiment can classify | categorize the kind of virus correctly because the quantity of the sugar_chain | carbohydrate couple | bonded with the solid phase is the range mentioned above. In addition, when the amount of the sugar chain bound to the solid phase exceeds the above-described range, it may be disadvantageous in cost because the sugar chain is excessively fixed.

[Solid phase]
In the present specification, examples of the solid phase for immobilizing sugar chains include substrates and particles.

(Material of solid phase)
Examples of the solid phase material include inorganic substances such as silica, alumina, glass, and metal. Moreover, a thermoplastic resin etc. are mentioned as an organic polymer substance. When a solid phase carrier with a sugar chain fixed is used for fluorescence observation, the thermoplastic resin preferably has a small amount of fluorescence generation. Examples of the resin that generates a small amount of fluorescence include linear polyolefin resins such as polyethylene and polypropylene; cyclic polyolefin resins; and fluorine-containing resins. Among them, it is preferable to use a saturated cyclic polyolefin resin that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. In this specification, the saturated cyclic polyolefin resin refers to a homopolymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.

(1. Solid phase substrate)
Specific examples of the solid phase substrate include a multiwell plate, a plate-like substrate, and a film. Among these, a multiwell plate is preferable. Multiwell plates include those in which an arbitrary number of wells are arranged. Examples of the number of wells include 24, 96, 384, 1536, etc., per plate.

(2. Solid phase particles)
Of the solid phase carriers, the particulate form is called solid phase particles. The shape of the solid phase particles is preferably a sphere, and more preferably polymer particles having an average particle size of 0.1 μm or more and 500 μm or less. It is considered that the carrier particles having a particle size in such a range can be easily collected by centrifugation, a filter or the like, and have a sufficient surface area, so that the reaction efficiency with sugar chains is high. When the average particle size is 500 μm or less, the surface area is not too small and the reaction efficiency with the sugar chain is high. When the average particle size is 0.1 μm or more, the particles can be efficiently collected by the filter, and there is no possibility that the pressure loss at the time of liquid passage becomes large when the particles are packed in a column.

  The surface of the solid phase carrier may be coated with a polymer compound having a functional group for immobilizing a sugar chain (hereinafter sometimes referred to as “immobilization group”). The polymer compound preferably has a polymer compound having an ethylenically unsaturated polymerizable monomer unit as a constituent unit as a basic structure. Examples of the ethylenically unsaturated polymerizable monomer units include (meth) acrylic resins, olefin resins (polyethylene, polypropylene, polycycloolefin, etc.), styrene resins (polystyrene, acrylonitrile-styrene copolymer, methyl methacrylate-styrene). Copolymer, styrene-butadiene-styrene block copolymer, acrylonitrile-butadiene-styrene copolymer, methyl methacrylate-butadiene-styrene copolymer), chlorine-containing resin (polyvinyl chloride, polyvinylidene chloride, etc.), Examples include monomer units that can constitute a resin such as a fluorine-containing resin. The polymer compound that can constitute the coating layer may be a polymer compound that contains one type selected from the above units alone, as long as it has an immobilizing group, or a plurality of types may be combined. A high molecular compound may be sufficient.

  The immobilization group is not particularly limited as long as it is a group that can be covalently or non-covalently bonded to a sugar chain. For example, the immobilized group is chemically active (that is, activated so as to have high reactivity with a sugar chain). ) Group, receptor group, ligand group and the like. The sugar chain may be modified so as to allow covalent bonding or non-covalent bonding with the immobilization group.

  Specific examples include activated carboxyl derivative groups, carboxyl groups, aldehyde groups, epoxy groups, vinyl sulfone groups, biotinyl groups, thiol groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, acrylate groups, Examples include, but are not limited to, a maleimide group, a hydrazide group, an aminooxy group, an azide group, an amide group, a sulfonate group, avidin, streptavidin, and a metal chelate. Among these, from the viewpoint of reactivity with an amino group, an aldehyde group, an activated carboxyl derivative group, an epoxy group, and a vinyl sulfone group are preferable, and a biotinyl group having a high binding constant is also preferable. In particular, in the case of bonding via an amino group bonded to a sugar chain, an activated carboxyl derivative group is preferred from the balance between reactivity with the amino group and storage stability. On the other hand, an aminooxy group or a hydrazide group is preferred when the sugar chain is bound via its reducing end because of high reactivity.

  Examples of the monomer from which the unit having such an immobilizing group is derived include an ethylenically unsaturated monomer represented by the following general formula [1A].

In the ethylenically unsaturated monomer of the above formula [1A], an activated carboxyl derivative group is bonded to a (meth) acryl group via an alkylene glycol residue or an alkyl group linking group. More specifically, in the formula [1A], R ₁ represents a hydrogen atom or a methyl group, and X ₁ is an alkylene glycol residue or alkylene group which may have 1 to 10 carbon atoms. W represents an activated carboxyl derivative group. p may be an integer from 1 to 100.

  The alkylene glycol residue itself has the property of suppressing nonspecific adsorption of proteins. For this reason, the ethylenically unsaturated monomer of the above formula [1A] has both the property of immobilizing sugar chains and the property of suppressing nonspecific adsorption of sugar chains when the linking group X is an alkylene glycol residue. .

In the formula [1A], when the linking group X ₁ is an alkylene glycol residue, the carbon number of X ₁ may be 1 or more and 10 or less, preferably 1 or more and 6 or less, more preferably 2 or more and 4 or less. More preferably, it is 2 or more and 3 or less, and most preferably 2.

The alkylene glycol residue refers to an alkyleneoxy group (—R) that remains after the condensation reaction of the hydroxyl group at one or both ends of alkylene glycol (HO—R—OH, where R is an alkylene group) with another compound. -O-, wherein R is an alkylene group. For example, the alkylene glycol residue in the case of methylene glycol (HO—CH ₂ —OH) is a methyleneoxy group (—CH ₂ —O—), and the alkylene in the case of ethylene glycol (HO—CH ₂ CH ₂ —OH). The glycol residue is an ethyleneoxy group (—CH ₂ CH ₂ —O—).

Repeating number p of X ₁ may be an integer between 1 and 100, inclusive, when X ₁ is alkylene glycol residues, more preferably 2 or more and 50 or less integer, more preferably 2 to 30 integer And most preferably an integer of 2 or more and 20 or less. When the polymer compound is composed of a plurality of types of units having different numbers of p, p is specified as an average value in the entire polymer compound. When the repetition number p is 2 or more, the repeated X ₁ may be the same or different.

In the formula [1A], when the linking group X ₁ is an alkylene group, the total number of carbon atoms in the p alkylene groups ((X ₁ ) p) is preferably 1 or more and 100 or less, and preferably 1 or more and 20 or less. It is more preferable that The structure of the alkylene group is not particularly limited, and the alkylene group may be linear, branched or cyclic.

  The activated carboxylic acid derivative group (W) is a group in which a carboxyl group is activated, and a leaving group A (excluding a hydroxyl group) is bonded to a carbonyl group as shown in the following formula [1B]. It is a group.

  The activated carboxylic acid-derived group represented by the above formula [1B] is a group activated for a nucleophilic reaction by having a highly acidic electron-withdrawing group as the leaving group A. Specifically, as the activated carboxylic acid-derived group, a carboxyl group of a carboxylic acid such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, fumaric acid, an active ester, an acid anhydride, an acid halide, Examples include groups converted to activated amides.

More specifically, examples of the active ester group include a group having OCR ₂ or ONR ₃ as a leaving group A as represented by the following formula [1B-1] or the following formula [1B-2]. .

In the above formula [1B-1] and formula [1B-2], R ₂ and R ₃ may each be a monovalent organic group, which may be linear, branched, or cyclic. Good. In the above formula [1B-1], R ₂ may be a divalent group that forms an aliphatic ring together with C, or may be a group that forms an aromatic ring together with C. Further, in the above formula [1B-2], R ₃ may be a divalent group that forms an aliphatic ring together with N, or may be a group that forms an aromatic ring together with N. In the above formulas [1B-1] and [1B-2], except that R ₂ and R ₃ together with C or N form an aromatic ring, C and N include R ₂ and Depending on the valence of R ₃ , H not shown is bonded.

As a more specific example of the active ester group, a substituted or unsubstituted OCR ₂ group represented by the above formula [1B-1] is a p-nitrophenyl group (see the following formula [1B-1-1]) or the like A phenol active ester group; and the ONR ₃ group represented by the above formula [1B-2] is an N-hydroxysuccinimide group (see the following formula [1B-2-1]), an N-hydroxyphthalimide group, an N-hydroxy-5 -N-hydroxyimide active ester groups such as norbornene-2,3-dicarboximide, etc. Among these, a p-nitrophenol active ester group or an N-hydroxysuccinimide active ester group is preferable, and a p-nitrophenol active ester group is particularly preferable from the viewpoint of a balance between storage stability and high reactivity.

  Other examples of active esters also include thioester groups corresponding to the active ester groups. Preferably, a substituted or unsubstituted thiophenol ester group is mentioned.

  The active ester group is preferably used because it has excellent reactivity under mild conditions. The mild conditions include, for example, neutral or alkaline conditions, specifically pH 7.0 or more and 10.0 or less, more specifically pH 7.6 or more and 9.0 or less, and more specifically pH 8.0. can do.

Examples of the acid anhydride group include groups represented by the following formulas [1B-3-1] and [1B-3-2].

  Examples of the acid halogen group include a group having a halogen such as —Cl and —F as the leaving group A in the above formula [1B].

Examples of the activated amide group include groups represented by the following formula [1B-4-1] and the like.

A particularly preferred compound as the ethylenically unsaturated polymerizable monomer from which the unit having an immobilizing group is derived is p-nitrophenyloxycarbonyl-poly (ethylene glycol) methacrylate represented by the following formula. In the following formula, the ethylene glycol repeat number p is preferably 2 or more and 20 or less.

  When the immobilizing group has a primary amino group such as an aminooxy group or a hydrazide group, it may be difficult to obtain a polymer compound by polymerizing monomers having these groups. In such a case, polymerization is performed using a monomer in which a primary amino group is protected with a protective group in advance or a monomer having a functional group that can be converted to a primary amino group, and after obtaining a polymer compound, the protective group is removed. The primary amino group may be introduced by chemical reaction.

The monomer in which the primary amino group is protected with a protecting group is not particularly limited, and examples thereof include an ethylenically unsaturated monomer represented by the following general formula [2A].

In the ethylenically unsaturated monomer of the above formula [2A], a (meth) acryl group and an oxylamino group or a hydrazide group are bonded via a linking group X ₂ containing an alkylene glycol residue. More specifically, in the formula [2A], R ₄ represents a hydrogen atom or a methyl group, and X ₂ is interrupted by —O—, —S—, —NH—, —CO—, —CONH—. A hydrocarbon chain having 1 to 20 carbon atoms, or an alkylene glycol group having 1 to 20 carbon atoms or a repeating structure thereof, Z represents an oxygen atom or —NH group (amino group), and Y represents a protecting group .

The linking group X ₂ in the formula [2A] is not particularly limited, but may be a hydrocarbon chain having 2 to 9 carbon atoms or an alkylene glycol group which may be interrupted by —NH— or —CONH— or a repetition thereof. It may be a structure.

  The protecting group Y is not limited as long as it can protect the amino group, and can be arbitrarily used. For example, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like can be mentioned.

Specific examples of the monomer include those represented by the following formula [2A-1].

The monomer having a functional group that can be converted into a primary amino group is not particularly limited in structure, but may be, for example, a monomer having an alkoxy group. Examples of the alkoxy group include a methoxy group, an ethoxy group, a propoxy group, and a t-butoxy group. Specific examples of the monomer having an alkoxy group include those represented by the following formula [2A-2].

  As a method for returning the functional group, for example, a method of producing a hydrazide group by reacting an alkoxy group with a hydrazine compound is preferable. Examples of the hydrazine compound include hydrazine monohydrate.

  The ratio of the unit having an immobilizing group is not particularly limited. For example, when the polymer compound has a hydrophilic group, an immobilization group, and a hydrophobic group, it is preferably 1 mol% or more and 30 mol% or less, more preferably, relative to the total number of units constituting the polymer compound. 1 mol% or more and 20 mol% or less, more preferably 2 mol% or more and 15 mol% or less. Further, for example, when the polymer compound has a hydrophilic group, an immobilizing group, and a crosslinking group, it is preferably 1 mol% or more and 99.7 mol% or less with respect to the total number of units constituting the polymer compound, Preferably they are 1 mol% or more and 70 mol% or less, Most preferably, they are 1 mol% or more and 50 mol%.

  Moreover, the high molecular compound can have a hydrophilic group. The hydrophilic group has an excellent nonspecific adsorption suppressing effect. Specific examples include a phosphorylcholine group and an alkylene glycol residue. For example, a hydrophilic group can be introduced into the polymer compound by copolymerizing a monomer having these and an ethylenically unsaturated polymerizable monomer from which the unit having an immobilizing group is derived.

  Examples of the monomer having a phosphorylcholine group include (meth) acryloyloxyalkylphosphorylcholines such as 2-methacryloyloxyethylphosphorylcholine and 6-methacryloyloxyhexylphosphorylcholine; 2-methacryloyloxyethoxyethylphosphorylcholine and 10-methacryloyloxyethoxynonylphosphorylcholine. (Meth) acryloyloxyalkoxyalkylphosphorylcholine; alkenylphosphorylcholine such as allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine, octenylphosphorylcholine, and decenylphosphorylcholine.

  Among the above, 2-methacryloyloxyethyl phosphorylcholine is preferable. Thereby, nonspecific adsorption | suction can be suppressed more reliably.

  The proportion of the unit having a phosphorylcholine group is not particularly limited, but is preferably 5 mol% or more and 95 mol% or less, more preferably 10 mol% or more and 90 mol% or less, and still more preferably 15 mol based on the total number of units constituting the polymer compound. % Or more and 90 mol% or less.

  Examples of the monomer from which the unit having an alkylene glycol residue is derived include an ethylenically unsaturated monomer represented by the following general formula [3A].

Ethylenically unsaturated monomer of formula [3A] is (meth) acrylic group comprises an alkylene glycol residue X _3. More specifically, in the formula [3A], R ₅ represents a hydrogen atom or a methyl group, and R ₆ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. X ₃ represents an alkylene glycol residue that may have 1 to 10 carbon atoms, and q represents an integer that may have 1 to 100 carbon atoms.

The number of carbon atoms of the alkylene glycol residue X ₃ in the formula [3A] may be 1 or more and 10 or less, preferably 1 or more and 6 or less, more preferably 2 or more and 4 or less, and further preferably 2 or more and 3 or less. And most preferably 2. The number of repetitions q of the alkylene glycol residue X ₃ is not particularly limited, but may be an integer of 1 to 100, preferably an integer of 2 to 100, more preferably 2 to 95. And most preferably an integer of 5 or more and 90 or less. When the polymer compound constituting the coating layer is composed of a plurality of types of units having different numbers of q, q is specified as an average value in the entire polymer compound. When the number of repetitions q is 2 or more, X ₃ may be the same or different.

  More specific examples of the ethylenically unsaturated polymerizable monomer from which the unit having an alkylene glycol residue is derived include methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) ) Acrylate and mono-substituted ester thereof, 2-hydroxypropyl (meth) acrylate and mono-substituted ester thereof, 2-hydroxybutyl (meth) acrylate and mono-substituted ester thereof, glycerol mono (meth) acrylate, polypropylene (Meth) acrylate having 2-glycol as a side chain, 2-methoxyethyl (meth) acrylate, 2-ethoxyethyl (meth) acrylate, methoxydiethylene glycol (meth) acrylate, etoxy Diethylene glycol (meth) acrylate, ethoxy polyethylene glycol (meth) acrylate.

  Among these, methoxypolyethylene glycol methacrylate or ethoxypolyethylene glycol methacrylate is preferable because of non-specific adsorption of sugar chains and availability. In particular, methoxypolyethylene glycol (meth) acrylate or ethoxypolyethylene glycol (meth) acrylate having an average number of ethylene glycol residue repeats of 5 or more and 90 or less is more preferably used from the viewpoint of good operability (handling) during synthesis. It is done.

  The proportion of units having an alkylene glycol residue is not particularly limited, but is preferably 0 mol% or more and 95 mol% or less, more preferably 30 mol% or more and 95 mol% or less, and still more preferably, with respect to the total number of units constituting the polymer compound. It is 50 mol% or more and 90 mol% or less.

  Moreover, the high molecular compound may have a hydrophobic group. The hydrophobic group can ensure the adsorptivity to the solid phase carrier. Specific examples include linear, branched, and cyclic alkyl groups. Carbon number of an alkyl group is 1-20, for example, Preferably it is 4-20. As for the hydrophobic group, for example, the hydrophobic group is introduced into the polymer compound by copolymerizing a monomer having these and an ethylenically unsaturated polymerizable monomer from which the unit having the immobilizing group is derived. be able to.

  As a monomer from which the unit having such a hydrophobic group is derived, n-butyl (meth) acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, neopentyl (meth) acrylate, cyclohexyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) Acrylate, heptyl (meth) acrylate, n-octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate N-decyl (meth) acrylate, iso-decyl (meth) acrylate, n-dodecyl (meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meth) acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n- Examples include octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, and isobonyl (meth) acrylate. Among these, particularly preferred groups include cyclohexyl (meth) acrylate, n-butyl methacrylate, n-dodecyl methacrylate, and n-octyl methacrylate.

  The proportion of units of the hydrophobic group is not particularly limited, but is preferably 20 mol% or more and 95 mol% or less, more preferably 30 mol% or more and 90 mol% or less, still more preferably 30 mol%, based on the total number of units constituting the polymer compound. More than 80 mol%.

Moreover, the high molecular compound may have a crosslinking group. The crosslinking group can impart insolubility to the polymer compound by, for example, crosslinking the main chain of the polymer compound. Alternatively, the solid phase carrier surface can be cross-linked and firmly bonded.
The cross-linking group is not particularly limited as long as it is a group that cross-links the polymer compounds or a group that cross-links the polymer compound and the solid phase carrier surface.
Such a crosslinkable group can be generated by polymerizing an ethylenically unsaturated polymerizable monomer having a crosslinkable functional group and then reacting the crosslinkable functional group to crosslink the polymer compounds. .

  The crosslinkable functional group is not particularly limited as long as it is a crosslinkable group that does not react during the polymerization reaction of the ethylenically unsaturated polymerizable monomer. Examples thereof include a functional group that generates a silanol group by hydrolysis, an epoxy group, a (meth) acryl group, and a glycidyl group. Among these, a functional group that generates a silanol group by hydrolysis, an epoxy group, and a glycidyl group are preferable because the crosslinking treatment is easy. Furthermore, a functional group that generates a silanol group by hydrolysis is preferable because it can be crosslinked at a lower temperature.

  The functional group that generates a silanol group by hydrolysis is a group that readily undergoes hydrolysis and forms a silanol group when contacted with water. For example, a halogenated silyl group, an alkoxysilyl group, a phenoxysilyl group, an acetoxysilyl group Etc. Among these, an alkoxysilyl group, a phenoxysilyl group, and an acetoxysilyl group are preferable because they do not contain halogen. Furthermore, an alkoxysilyl group is most preferable because it easily generates a silanol group.

  The ethylenically unsaturated polymerizable monomer having a functional group that generates a silanol group by hydrolysis has a general formula in which a (meth) acryl group and an alkoxysilyl group are bonded directly or via an alkyl chain having 1 to 20 carbon atoms. It is preferable that it is an ethylenically unsaturated polymerizable monomer represented by 4A].

The ethylenically polymerizable unsaturated monomer of the above formula [4A] includes an alkyl chain X ₄ having a (meth) acryl group of 1 to 20 carbon atoms. More specifically, in the formula [4A], R ₇ represents a hydrogen atom or a methyl group, and A _1-3 represent a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. X ₄ represents an alkyl group having 1 to 20 carbon atoms.

  Examples of the ethylenically unsaturated polymerizable monomer containing an alkoxysilyl group include 3- (meth) acryloxypropenyltrimethoxysilane, 3- (meth) acryloxypropylbis (trimethylsiloxy) methylsilane, and 3- (meth). Acryloxypropyldimethylmethoxysilane, 3- (meth) acryloxypropyldimethylethoxysilane, 3- (meth) acryloxypropylmethyldimethoxysilane, 3- (meth) acryloxypropylmethyldiethoxysilane, 3- (meth) acryl Roxypropyltrimethoxysilane, 3- (meth) acryloxypropyltriethoxysilane, 3- (meth) acryloxypropyltris (methoxyethoxy) silane, 8- (meth) acryloxyoctanyltrimethoxysilane, 11- (meth) A And the like Lilo carboxymethyl undecene sulfonyl trimethoxysilane the (meth) acryloxy alkyl silane compound. Among them, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, and 3-methacryloxypropyldimethylethoxysilane having an alkylene glycol residue are ethylenically unsaturated polymerizable monomers. It is preferable from the viewpoint of excellent copolymerizability and easy availability. These ethylenically unsaturated polymerizable monomers having an alkoxysilyl group are used alone or in combination of two or more.

  The ratio of the unit of the crosslinking group is not particularly limited, but is preferably 1 mol% or more and 20 mol% or less, more preferably 2 mol% or more and 15 mol% or less, and still more preferably, with respect to the total number of units constituting the polymer compound. It is 2 mol% or more and 10 mol% or less.

  When the polymer compound is obtained by copolymerization of the above-described monomers having two or more components, the bonding method may be any form such as random, block, or graft.

  The coating of the polymer compound is performed, for example, by applying the polymer compound dissolved in an organic solvent at a concentration of 0.05 to 10% by mass on the substrate by dipping, spraying, spin coating, or the like. It can be performed by drying at room temperature of about ℃ or under heating. Examples of the organic solvent include 2-butanone, ethanol, methanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl ethyl ketone, and the like.

  The bonding between the surface of the substrate and the polymer compound may be any bonding mode such as a covalent bond, an electrostatic interaction, a hydrogen bond, and a bond due to a hydrophobic effect.

[Method of immobilizing sugar chains]
The method for immobilizing the sugar chain may be either a covalent bond or a non-covalent bond, and can be determined by a person skilled in the art according to a known method depending on the functional group of the solid phase to be immobilized. Of these, immobilization by a covalent bond is preferable because the sugar chain is hardly detached. For example, a method in which a solution containing a sugar chain or a sugar chain modifying substance is brought into contact with a solid phase having a functional group covalently bonded to the sugar chain, and the like can be mentioned. The functional group covalently bonded to the sugar chain or the sugar chain modifying substance is not particularly limited as long as it has sugar chain immobilization ability. For example, when binding to a solid phase via an amino group of a sugar chain or a sugar chain modifying substance, an activated carboxyl derivative group is preferable because the sugar chain can be efficiently immobilized. In this case, the solvent for dissolving the sugar chain or the sugar chain modifying substance is not particularly limited, but a general buffer solution having a pH of 7.0 to 10.0 may be used. For example, a phosphate buffer solution or a Tris buffer solution may be used. Etc. When binding to a solid phase via an amino group of a sugar chain or a sugar chain modifying substance, dehydration condensation with a carboxyl group of the solid phase may be performed. The solvent in this case may be a solution containing a condensing agent such as carbodiimide, and the pH is preferably pH 3-8, more preferably pH 4-6. On the other hand, when binding to a solid phase via a reducing end of a sugar chain or a sugar chain modifying substance, an aminooxy group or a hydrazide group is preferable because the sugar chain can be efficiently immobilized. In this case, the solvent for dissolving the sugar chain or the sugar chain-modifying substance is not particularly limited, but preferably a buffer solution having a pH of 2 to 7, more preferably a buffer solution having a pH of 4 to 6.

[Blocking operation]
After the sugar chain immobilization, it is preferable to inactivate the immobilizing group that was not involved in the immobilization of the sugar chain after removing the reaction solution. The inactivation treatment can be performed by changing to another group that does not have a binding property with a sugar chain according to the type of the immobilization group. For example, when the immobilization group is an active ester group, an aldehyde group, or the like, it can be inactivated with a compound having an alkali compound, a primary amino group, or the like. When the immobilizing group is an aminooxy group, hydrazide group or the like, it can be inactivated with an acid anhydride such as acetic anhydride or succinic anhydride.

  Examples of the compound having a primary amino group include methylamine, ethylamine, butylamine, glycine, 9-aminoacazine, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino2,3-dihydro-1, 4-pentanol, aminoethanethiol 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-aminooctane 1-amino-2-propanol, 3-amino-1-propanol, 3-aminopropene, 3-aminopropionitrile, aminopyridine, 11-aminoundecanoic acid, aminosalicylic acid, aminoquinoline, 4-aminophthalonitrile, 3 -Aminophthalimide, p-aminopropiophenone, aminophenylacetic acid, aminonaphthalene and the like. Of these, aminoethanol and glycine are preferable.

  In this embodiment, the α2,3-linked sialic acid-containing sugar chain may form an α2,3-linked sialic acid-containing sugar chain-binding peptide, and the α2,6-linked sialic acid-containing sugar The chain may form an α2,6-linked sialic acid-containing sugar chain-binding peptide, and the above asialo sugar chain may form an asialo sugar chain-binding peptide.

  By utilizing the amino group of the sugar chain-binding peptide for binding to the solid phase, the sugar chain can be sufficiently exposed.

  The length of a peptide is not specifically limited, For example, 5-30 amino acid residues may be sufficient, for example, 5-20 amino acid residues may be sufficient, for example, 5-10 amino acid residues may be sufficient.

  5A to 5C show the structures of the α2,3-linked sialic acid-containing sugar chain-binding peptide, the α2,6-linked sialic acid-containing sugar chain-binding peptide, and the asialo sugar chain-binding peptide in this embodiment. It is the figure which showed an example. According to these sugar chain-binding peptides, functional groups on the solid phase (for example, carboxyl group, active ester structure, etc.), α2,3-linked sialic acid-containing sugar chain-binding peptides, α2,6-linked sialic acid-containing Each sugar chain can be immobilized on the substrate by the amino group of the sugar chain-binding peptide and the asialo sugar chain-binding peptide forming an amide bond.

<Virus classification method>
In one embodiment, the present invention comprises a step of bringing a virus into contact with a solid phase to which an α2,3-linked sialic acid-containing sugar chain or an α2,6-linked sialic acid-containing sugar chain is immobilized, and a virus bound to the solid phase. And a step of classifying the virus based on the binding activity between the virus and at least one of the α2,3-linked sialic acid-containing sugar chain and the α2,6-linked sialic acid-containing sugar chain. Provide a virus classification method.

  In the present specification, examples of viruses that can be classified include the aforementioned influenza viruses, reoviruses, adenoviruses, rotaviruses, and the like. Among these, from the viewpoint of high demand for classification, influenza virus is preferable, and avian influenza virus and human influenza virus are more preferable.

  The virus classification method of this embodiment will be described in detail below. First, the virus is brought into contact with a solid phase on which an α2,3-linked sialic acid-containing sugar chain or an α2,6-linked sialic acid-containing sugar chain is immobilized. The sample containing the virus is not particularly limited, and examples thereof include a subject's blood, a body fluid such as saliva, a biological sample such as urine, a suspension of cells or virus itself, drinking water, sewage and the like.

  Next, the virus bound to the solid phase is detected. The detection method is not particularly limited. For example, a method using an antiviral antibody, for example, measuring the change in absorbance by irradiating the solid phase with light of a certain wavelength from before binding to after binding. And the like.

  Subsequently, the viruses are classified based on the binding activity between the virus and the α2,3-linked sialic acid-containing sugar chain or the α2,6-linked sialic acid-containing sugar chain. As described above, since the mode of binding of sialic acid to which the virus can bind and sugar differs depending on the type of virus, the virus can be classified by examining the type of sialic acid-containing sugar chain to which the virus is bound. . For example, influenza viruses bound to α2,3-linked sialic acid-containing sugar chains can be classified as avian influenza viruses. In addition, influenza viruses bound to α2,6-linked sialic acid-containing sugar chains can be classified as human influenza viruses.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Example 1] Production of sugar chain-immobilized plate Synthesis of Polymer Compound Solution (1) Synthesis of Polymer Compound First, a polymer compound used for the coating layer was prepared. 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as “MPC”), n-butyl methacrylate (hereinafter referred to as “BMA”), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (hereinafter referred to as “MEONP”) Was dissolved in ethanol to 1 mol / L. In addition, each molar ratio in a monomer mixed solution is 25: 70: 5 in order of MPC, BMA, and MEONP. Further, 2,2-azobisisobutyronitrile (hereinafter referred to as “AIBN”, manufactured by Wako Pure Chemical Industries, Ltd.) is added to 0.01 mol / L and stirred until uniform. Thus, a monomer mixed solution was prepared.

  Then, it was made to react at 60 degreeC under argon gas atmosphere for 6 hours, the reaction solution was dripped in the diethyl ether / chloroform mixed solvent (volume ratio 80/20), and the high molecular compound was obtained by collecting precipitation. The above-mentioned MEONP was synthesized as shown in (2) below.

(2) Synthesis of p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP) 0.01 mol of polyethylene glycol monomethacrylate (manufactured by NOF Corporation, “Blenmer PE-200”) was dissolved in 20 mL of chloroform, and then −30 Cooled to ° C. While maintaining the temperature at −30 ° C., 0.01 mol of p-nitrophenyl chloroformate (manufactured by Aldrich), 0.01 mol of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) and 20 mL of chloroform were added to this solution. The homogeneous solution was slowly added dropwise. After reacting at −30 ° C. for 1 hour, the solution was further stirred at room temperature for 2 hours. Thereafter, the salt was removed from the reaction solution by filtration, and the solvent was distilled off to obtain MEONP.

2. Preparation of sugar chain plate To a 96-well plate made of cycloolefin, a 0.3 wt% ethanol solution of the above polymer compound was dispensed at 300 μL / well and allowed to stand at room temperature for 30 minutes or more. Next, after recovering the polymer compound solution from the well, 300 μL of a washing solution (75% ethanol) was dispensed and allowed to stand for 3 minutes. The solution was removed and dried.

  Subsequently, a sugar chain solution to be immobilized was prepared. Examples of sugar chains include α2,3-linked sialic acid-containing sugar chain-binding peptides, α2,6-linked sialic acid-containing sugar chain-binding peptides having the structures shown in FIGS. 5 (A), (B), and (C), respectively. Asialo sugar chain-binding peptide was used. Using 5M potassium phosphate buffer, each glycopeptide solution was divided into 8 types, 0, 0.09, 0.19, 0.38, 0.75, 1.50, 3.00, 6.00 μg / mL. The concentration was adjusted so as to obtain a concentration of.

  Subsequently, 100 μL / well of each concentration of glycopeptide solution was dispensed and allowed to stand at room temperature for 2 hours. Thereafter, the solution was removed, dispensed 300 μL / well with ultrapure water, and then removed three times.

  A blocking solution (monoethanolamine) was dispensed at 300 μL / well and allowed to stand at room temperature for 60 minutes. Thereafter, the solution was removed, dispensed 300 μL / well with ultrapure water, and then removed three times.

[Test Example 1] Detection of immobilized glycans by periodic acid-Schiff base (PAS) method 100 μL / well of 10 μg / mL sodium periodate aqueous solution was added to the plate prepared in Example 1. The solution was dispensed, covered with a plate seal, and allowed to stand at 37 ° C. for 30 minutes. Subsequently, the plate was washed 3 times with pure water. Next, biotinamidohexanoic acid hydrazide was diluted with 300 mM sodium acetate buffer to a concentration of 50 μM, and 100 μL was dispensed. The lid was covered with a plate seal and allowed to stand at 37 ° C. overnight. Subsequently, the plate was washed 3 times with pure water.
Next, streptavidin-HRP (model number “18-152”, manufactured by Millipore) was diluted with a 1% BSA / PBS solution so as to be 0.2 μg / mL, and dispensed by 100 μL each. The lid was covered with a plate seal and allowed to stand at room temperature for 60 minutes. Subsequently, the plate was washed 3 times with pure water.
Next, a coloring kit for peroxidase (model number “ML-1120T”, manufactured by Sumitomo Bakelite Co., Ltd.) was dispensed at 100 μL / well and allowed to stand at room temperature for 15 minutes under light shielding. Subsequently, 100 μL / well of 0.5 M sulfuric acid was dispensed, and the absorbance at 450 nm was measured using an absorbance meter (infinite M200, manufactured by Tecan). The results are shown in Table 1 and FIG. It was confirmed that all three types of glycopeptides were immobilized.

[Test Example 2] SNA (Sambucus nigra) lectin reaction test The plate prepared in Example 1 was allowed to react with SNA lectin known to bind to α2,6-linked sialic acid-containing sugar chains. SNA lectin was used as a substitute for virus. Specifically, biotinylated SNA lectin (manufactured by VECTOR) was diluted with the solution to 20 μg / mL on the plate prepared in Example 1, and 100 μL was dispensed. Covered with a plate seal and allowed to stand at 4 ° C. overnight. Subsequently, the plate was washed 3 times with pure water.
Next, streptavidin-HRP (model number “18-152”, manufactured by Millipore) was diluted with a 1% BSA / PBS solution so as to be 0.2 μg / mL, and dispensed by 100 μL each. The lid was covered with a plate seal and allowed to stand at room temperature for 60 minutes. Subsequently, the plate was washed 3 times with pure water.
Next, a color development kit for peroxidase (model number “ML-1120T”, manufactured by Sumitomo Bakelite Co., Ltd.) was dispensed at 100 μL / well and allowed to stand at room temperature for 15 minutes under light shielding. Subsequently, 100 μL / well of 0.5 M sulfuric acid was dispensed, and the absorbance at 450 nm was measured using an absorbance meter (infinite M200, manufactured by Tecan Corporation). The results are shown in Table 2 and FIG.

  From Table 2 and FIG. 7, it was confirmed that SNA lectin reacts only with α2,6 sugar chain sialyl sugar chain.

  ADVANTAGE OF THE INVENTION According to this invention, the solid phase with which the sugar_chain | carbohydrate was fix | immobilized which can measure interaction with a virus with high sensitivity simply and cheaply can be provided.

Claims (9)

  A solid phase on which an α2,3-linked sialic acid-containing sugar chain and / or an α2,6-linked sialic acid-containing sugar chain is immobilized.   The solid phase according to claim 1, which is used for virus classification.   The solid phase according to claim 2, wherein the virus is influenza virus, reovirus, adenovirus or rotavirus.   The α2,3-linked sialic acid-containing sugar chain forms an α2,3-linked sialic acid-containing sugar chain-binding peptide, and the α2,6-linked sialic acid-containing sugar chain contains an α2,6-linked sialic acid The solid phase according to any one of claims 1 to 3, which forms a sugar chain-binding peptide. The solid phase according to any one of claims 1 to 4, wherein the α2,6-linked sialic acid-containing sugar chain is immobilized, and the absorbance measured by the following measurement method is 0.05 or more.
[Measuring method]
20 μg / mL biotinylated Sambucus nigra (SNA) lectin is brought into contact with the region where α2,6-linked sialic acid-containing sugar chains are immobilized on the solid phase at 4 ° C. for 8 hours and then washed. Subsequently, 0.2 μg / mL streptavidin-horseradish peroxidase (HRP) is contacted at room temperature for 60 minutes and then washed. Subsequently, a peroxidase coloring kit (model number “ML-1120T”, manufactured by Sumitomo Bakelite Co., Ltd.) is allowed to contact at room temperature for 15 minutes under light shielding. Subsequently, the absorbance at a wavelength of 450 nm is measured.   Furthermore, the solid phase as described in any one of Claims 1-5 to which the asialo sugar chain was fixed.   The solid phase according to claim 6, wherein the asialo sugar chain forms an asialo sugar chain-binding peptide. contacting the virus with a solid phase on which an α2,3-linked sialic acid-containing sugar chain and / or an α2,6-linked sialic acid-containing sugar chain is immobilized;
Detecting the virus bound to the solid phase;
Classifying the virus based on the binding activity between the virus and the α2,3-linked sialic acid-containing sugar chain or the α2,6-linked sialic acid-containing sugar chain;
A method for classifying viruses.   The virus classification method according to claim 8, wherein the virus is influenza virus, reovirus, adenovirus or rotavirus.

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