WO2013035727A1 - Sugar chain detection method using photocrosslinking agent and molecular probe - Google Patents

Sugar chain detection method using photocrosslinking agent and molecular probe Download PDF

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WO2013035727A1
WO2013035727A1 PCT/JP2012/072574 JP2012072574W WO2013035727A1 WO 2013035727 A1 WO2013035727 A1 WO 2013035727A1 JP 2012072574 W JP2012072574 W JP 2012072574W WO 2013035727 A1 WO2013035727 A1 WO 2013035727A1
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sugar chain
molecular probe
photocrosslinking agent
sugar
binding
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PCT/JP2012/072574
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French (fr)
Japanese (ja)
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富雄 矢部
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国立大学法人岐阜大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

  • the present invention relates to a sugar chain detection method, and more particularly to a sugar chain detection method using a photocrosslinking agent and a molecular probe.
  • Glycosaminoglycan sugar chain (sulfated sugar chain) is a generic term for heparan sulfate and chondroitin sulfate that are universally expressed on the surface of higher animal cells and can be used in blood and urine such as mucopolysaccharidosis. It is an indicator molecule for a diagnostic marker.
  • an antibody reaction that is recognized by the structure of a protein is widely used as the indicator recognition mechanism.
  • sulfated sugar chains are in vivo macromolecules having a negative charge composed of acidic sugars and sulfate groups. Therefore, in the detection method using an antibody, non-specific interaction with a negatively charged molecule occurs, and it is difficult to detect a very small amount of sample.
  • Patent Document 1 The inventor himself has applied for a patent for a molecular probe useful for highly sensitive recognition of the sulfated sugar chain.
  • An object of the present invention is to provide a sugar chain detection method that solves such problems.
  • the molecular probe is labeled with a photoreactive crosslinking agent using a chemical modification method, and the rotational speed of the interaction between the probe and the glycosaminoglycan sugar chain is made permanent by photocrosslinking.
  • the photoreactive crosslinking agent used in the present invention is originally used for the purpose of protein-molecule interaction analysis (Stauffer, DA, and Karlin, A. (1994) Biochemistry 33, 6840-6849. Holmgren, M., et al. (1996) Neuropharmacology 35, 797-804.Liu, Y., et al. (1996) Neuron 16, 859-867.).
  • a method for quantifying sugar chains there has been used in a method for quantifying sugar chains.
  • the gist of the present invention is as follows. (1) A method for detecting a sugar chain using a molecular probe capable of specifically binding to a sugar chain, wherein a molecule probe labeled with a photocrosslinking agent capable of binding to a sugar chain is brought into contact with a sample, and then light And linking the sugar chain specifically bound to the molecular probe. (2) The method according to (1), further comprising visualizing a sugar chain specifically bound to the molecular probe. (3) The method according to (1) or (2), wherein light is irradiated on a solid phase on which a sugar chain to be detected is fixed. (4) The method according to any one of (1) to (3), wherein a sugar chain is quantitatively detected by a competition method.
  • a saccharide comprising contacting a test substance labeled with a photocrosslinking agent capable of binding to a sugar chain with a sugar chain, and then irradiating light to crosslink the sugar chain specifically bound to the test substance.
  • a screening method for substances that specifically bind to chains A molecular probe capable of specifically binding to a sugar chain is contacted with a sample labeled with a photocrosslinking agent capable of binding to the sugar chain, and then irradiated with light, specifically to the molecular probe.
  • a kit for detecting a sugar chain comprising the following (a) or (b).
  • (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) The molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain (9)
  • the kit according to (8), further comprising a solid phase on which a sugar chain to be detected is fixed.
  • a screening kit for a substance that specifically binds to a sugar chain comprising the following (a) or (b): (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) A molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain (12) A test kit for a disease in which a sugar chain serves as a diagnostic marker, comprising the following (a) or (b): (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  • detection method of the present invention 1. Detection error due to nonspecific binding of detection reagent (molecular probe) to sulfated sugar chain 2. Difficult to detect the complex when the binding force between the target molecule and the probe is very small. 3. The target of photocrosslinking is limited.
  • the conventional detection method can solve the problem that a competing method that can be easily kitted cannot be used.
  • GAG glycosaminoglycan
  • HP heparin
  • CS-A chondroitin sulfate A
  • CS-C chondroitin sulfate C.
  • heparin / heparan sulfate has been removed by enzyme treatment
  • those stained with 10E4 (anti-heparan sulfate antibody) those stained with hematoxylin / eosin.
  • a molecular probe HappY-Cys
  • a photoreactive crosslinking reagent Mts-Atf-LC-Biotin
  • top Stained image of skin tissue section (cat-derived) with HappY probe. Center: Stained image of the skin tissue section (derived from cat) with the HappY probe (treated with glycosylation enzyme). Bottom: Stained image of skin tissue section (cat-derived) with commercially available antibody (10E4). Top: Stained image of mastocytoma (cat-derived) skin tissue section with toluidine blue. Center: Stained image of mastocytoma (cat-derived) skin tissue section with HappY probe. Bottom: Stained image of mastocytoma (cat-derived) skin tissue section with commercially available antibody (10E4).
  • the present invention is a method for detecting a sugar chain using a molecular probe capable of specifically binding to a sugar chain, wherein a molecule probe labeled with a photocrosslinking agent capable of binding to a sugar chain is contacted with a sample, The method is provided comprising irradiating light to crosslink a sugar chain specifically bound to the molecular probe.
  • detection includes quantification.
  • the sugar chain to be detected may be any sugar chain, but it should be a molecular probe that can specifically bind, and examples include glycosaminoglycans. Can do.
  • Glycosaminoglycans are sugar chains that have a repeating structure of amino sugars, including those with sulfate groups added, and disaccharides of uronic acid or galactose, and are almost the cell surface and extracellular matrix of higher animals than nematodes. Exists in a free state or bound to a protein. Glycosaminoglycans have a long chain structure of disaccharides consisting of amino sugars (galactosamine, glucosamine, etc.) and uronic acids (glucuronic acid, iduronic acid, etc.) or galactose, and may be acetylated or sulfated. .
  • the sulfation site is a sulfate group transferred to a specific hydroxyl group (O-sulfation) or amino group (N-sulfation) of the constituent sugar.
  • Glycosaminoglycan is negatively charged by the sulfate group or the carboxyl group of uronic acid.
  • glycosaminoglycans hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, chondroitin and the like are known.
  • Heparin is a kind of heparan sulfate, which has a repeating structure of disaccharide units of D-glucuronic acid or L-iduronic acid and N-acetyl-D-glucosamine, and has an average of 2 to 3 sulfate groups per disaccharide. Although it is a polymer, it has a higher degree of sulfation than heparan sulfate.
  • Heparin In vivo, heparin interacts with coagulation proteins involved in anticoagulation. Heparin is used as one of anticoagulants and is used for treatment of thromboembolism, disseminated intravascular coagulation syndrome, artificial dialysis, prevention of coagulation in extracorporeal circulation, and the like. Heparin binds to antithrombin III, thereby activating the anticoagulant action of antithrombin III and suppressing the coagulation system.
  • Antithrombin III is a glycoprotein that inhibits the blood clotting activity of thrombin by forming a complex with thrombin.
  • hyaluronic acid is not bound to the core protein and has no sulfate group.
  • Hyaluronic acid is present in the extracellular matrix in vivo such as joints, vitreous body, skin, and brain.
  • the sugar chain (for example, glycosaminoglycan) to be detected may be present in a free state or in a state in which it is bound to another substance such as a core protein. It may be.
  • the molecular probe used in the detection method of the present invention is not particularly limited as long as it can specifically bind to a sugar chain and can be labeled with a photocrosslinking agent capable of binding to the sugar chain.
  • a photo-crosslinking agent capable of binding to a sugar chain is 2- ⁇ N2- [N6- (4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl) -N6- (6-biotinamidocaproyl) -L-lysinylamido ] ⁇ ethyl methanethiosulfonate (Mts-Atf-LC-Biotin) (Pierce Bioctechnology), it should have a thiol group (SH) that can bind to the methanethiosulfonate group of Mts-Atf-LC-Biotin
  • SH thiol group
  • Such a molecular probe can be obtained by introducing a free thiol
  • glycosaminoglycan examples of the substance that specifically recognizes glycosaminoglycan include the following. Heparin and heparan HappY that specifically binds to a sulfuric acid (H eparin- a ssociated p e p tide Y, 8 May 19, 2008 28th Annual Meeting of the Japanese sugar Society Annual Meeting (Tsukuba) oral presentations and Abstracts p.79 (Amino acid sequence: RTRGSTREFRTG) (SEQ ID NO: 3) -HappY with Cys added (HappY-Cys) (amino acid sequence: RTRGSTREFRTGC) (SEQ ID NO: 1) -CS-56 (anti-chondroitin sulfate antibody; Avnur Z.
  • MO-224 as an antibody that specifically recognizes glycosaminoglycan
  • Anti-chondroitin sulfate antibody Yamagata, M. et al., J. Biol. Chem. 262, 4146-4152, 1987
  • HepSS-1 anti-heparan sulfate antibody
  • Kure, S. and Yoshie O., J. Immunol. 137, 3900-3908, 1986
  • Antithrombin III that specifically recognizes heparin (Bjork, I. and Lindahl, U., Mol. Cell. Biochem.
  • glycosaminoglycans The substance specifically recognizing glycosaminoglycans is the phage display method reported in August 28, 2008 at the 28th Annual Meeting of the Japanese Society of Carbohydrates (Tsukuba) and p. 79, Kuppevelt et al. (Van Kuppevelt, TH et al., J. Biol. Chem. 273, 12960-12966, 1998) and the like.
  • sugars other than glycosaminoglycans a large number of sugar-specific recognition proteins represented by lectins are widely known and can be used.
  • a site that does not interfere with binding so as not to interfere with the structure-specific binding force of the substance that specifically recognizes a sugar chain may be introduced into the C-terminus of the peptide or protein.
  • the introduction of a free thiol group can be performed, for example, by adding a cysteine residue during peptide synthesis or by modifying the amino acid side chain using a thiol group introduction reagent.
  • the addition of a cysteine residue can be performed by a peptide solid phase synthesis method using cysteine protected with a 9-fluorenylmethoxy group (Fmoc group).
  • the molecular probe has a free thiol group, it can be labeled with a photocrosslinker having a methanethiosulfonate group, such as Mts-Atf-LC-Biotin. Is bonded to a sugar chain (“weak bond”), and then irradiated with light to crosslink the sugar chain, thereby forming a “strong bond” (see FIG. 1).
  • this thiol group can be used to generate light with a methanethiosulfonate group such as Mts-Atf-LC-Biotin. Since it can be labeled with a cross-linking agent, it is not necessary to introduce a thiol group into the molecular probe in advance.
  • the detection method of the present invention enables highly reliable detection even after sufficient washing by crosslinking the sugar chain recognized by the molecular probe with a photocrosslinking agent.
  • the photocrosslinking agent may be any one as long as it can crosslink sugar chains by light irradiation and can bind to a molecular probe.
  • Examples of the photocrosslinking agent used in the present invention include those having the following three functions in the molecule. 1. Methanethiosulfonate group (disulfide bond with thiol group of molecular probe) 2. Phenyl azide group (can be chemically bonded to CH or C-NH 2 of sugar chain by UV irradiation) 3.
  • Photocrosslinking agents include 2- ⁇ N2- [N6- (4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl) -N6- (6-biotinamidocaproyl) -L-lysinylamido] ⁇ ethyl methanethiosulfonate (Mts-Atf-LC-Biotin) (Pierce Bioctechnology), 2- [N2- (4-azido-2,3,5,6-tetrafluorobenzoyl) -N6- (6-biotinamidocaproyl) -L-lysinyl] ethyl methanethiosulfonate (Mts-Atf-Biotin) (Pierce Bioctechnology).
  • Mts sulfhydryl-reactive methanethiosulfonate group
  • Atf tetrafluorophenyl azide group.
  • Molecular weight 953.11 Biotin-Mts spacer arm: 29.3mm Biotin-Atf Spacer Arm: 35.2mm
  • the molecular probe should be reacted with a photocrosslinking agent before contacting with the sample (label).
  • the photo-crosslinking agent may be sufficiently saturated, and the ratio of the photo-crosslinking agent to the molecular probe is 1: 1 to 10: 1, preferably 1: 1 to 5: 1, more preferably 1: 1 to 2: 1. It is good to add so that it may become mass ratio.
  • the photocrosslinking agent may be added after being dissolved in a solvent such as DMSO or DMF.
  • the photocrosslinking agent may be used after being dissolved in an amount of 1 to 200 ⁇ mM, preferably 10 to 100 ⁇ mM, more preferably 25 to 50 ⁇ mM with respect to 100 ⁇ L of the solution.
  • the reaction between the photocrosslinking agent and the molecular probe may be performed under appropriate conditions.
  • the reaction between Mts-Atf-LC-Biotin (photocrosslinking agent) and HappY-Cys (HappY with cysteine added) is performed in the dark at room temperature for 1-2 hours or at 4 ° C for 4-8 hours. It is better to react under conditions.
  • the molecular probe is allowed to react with the photocrosslinking agent, excess photocrosslinking agent is removed by dialysis or an ion exchange column, and then the molecular probe is brought into contact with the sample.
  • sample to be contacted with the molecular probe examples include, but are not limited to, various biological samples such as serum, plasma, pleural effusion, ascites, urine, joint fluid, culture fluid, cerebrospinal fluid, and tissue homogenate. I don't mean.
  • the sugar chain and the molecular probe in the sample to be detected should have a mass ratio of 1: 0.1 to 1: 100, preferably 1: 1 to 1:50, more preferably 1: 5 to 1:20. It is good to contact.
  • a solution in which the molecular probe is dissolved may be added to the sample, and the molecular probe is 0.01 to 100 nmol, preferably 0.05 to 50 nmol, more preferably 1 to 100 ⁇ L of the solution. It is recommended to use it after dissolving in an amount of ⁇ 10 nmol.
  • the sample should be used as appropriate by diluting it to an appropriate concentration with an appropriate solution such as phosphate buffered saline. Good.
  • the solution for dissolving the molecular probe may contain bovine serum albumin, and the concentration of bovine serum albumin is suitably 2 to 10% (% by weight).
  • Bovine serum albumin is generally used as a blocking agent and can also be used in that role in the detection method of the present invention.
  • the molecular probe binds to the solid phase (for example, plastic well), which is the reaction field, in a non-specific manner, but when a small amount of molecular probe is used, it has a large effect on the detection amount. Effect of lowering the proportion of molecular probes that bind to a solid phase (for example, plastic wells) (in principle, they are almost unbound). It is thought that there is.
  • the main premise is that bovine serum albumin does not bind to the sugar chain to be detected, but the heparin sugar chain has already been confirmed.
  • a sample is brought into contact with a molecular probe labeled with a photocrosslinking agent, and the temperature is 4 to 37 ° C., preferably 4 to 25 ° C., more preferably 15 to 25 ° C., 0.5 to 24 hours, preferably 0.5 to 16 hours. More preferably, the binding reaction between the sugar chain in the sample and the molecular probe is performed for 0.5 to 3 hours.
  • the irradiation light and the irradiation time may be any wavelength and time at which a bond is generated between the sugar chain to be detected and the photocrosslinking agent.
  • the photocrosslinking agent is Mts-Atf-LC-Biotin,
  • the glycosaminoglycan sugar chain can be cross-linked by irradiating ultraviolet rays of 300 nm to 370 nm for 1 to 15 minutes on ice.
  • the detection method of the present invention it is preferable to visualize the sugar chain specifically bound to the molecular probe.
  • any method may be used to visualize the sugar chain specifically bound to the molecular probe.
  • a dye for example, fluorescein isothiocyanate; FITC ), Texas red, Cy3, Cy5, Cy7, etc.
  • streptavidin labeled with enzymes honeyeradish peroxidase (HRP), alkaline phosphatase (AP), etc.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • DAB color reaction when detecting with streptavidin labeled with HRP, it is detected by DAB color reaction.
  • the detection method of the present invention can be used by either a competitive method or a non-competitive method.
  • a competitive method or a non-competitive method it is good to crosslink the sugar chain bound to the molecular probe by irradiating light on the solid phase on which the sugar chain to be detected is fixed. .
  • the molecular probe is competed by competition between the sugar chain immobilized on the solid phase and the sugar chain in the sample. After allowing the sample and the molecular probe to act on the immobilized sugar chain, the free molecular probe and the molecular probe bound to the sugar chain in the sample are removed by washing. A calibration curve can be created with a molecular probe, and the amount of sugar chain in the sample can be determined from the amount of fluorescence decrease due to competition (that is, quantitative detection is possible).
  • the sugar chain in the sample When used in a non-competitive method, the sugar chain in the sample is immobilized on a solid phase, brought into contact with a molecular probe, the sugar chain to be detected is bound to the molecular probe, and then the sugar chain is irradiated by light irradiation. And then sugar chains can be detected by fluorescence. In this case, the sugar chain in the sample can be directly detected. That is, a calibration curve is created with a molecular probe, and the amount of sugar chain in the sample can be determined from the amount of fluorescence.
  • solid phase examples include, but are not limited to, microplate wells, plastic tubes, beads, and the like. If the solid surface is coated with streptavidin, the sugar chain labeled with biotin can be immobilized. Biotin labeling of sugar chains can be performed using a commercially available biotinylation reagent.
  • a sugar chain specifically bound to a molecular probe and crosslinked with a photocrosslinking agent can be visualized by staining.
  • HappY with Cys added labeled with Mts-Atf-LC-Biotin (photocrosslinking agent) and paraffin-embedded tissue (small intestine or kidney) section at room temperature Incubate for 30 minutes, and then irradiate with ultraviolet light for 15 minutes on ice. After washing to remove uncrosslinked HappY, label with streptavidin-HRP and stain by DAB color reaction. did.
  • the sugar chain detection method of the present invention can be applied to screening of substances that specifically bind to sugar chains, examination of diseases in which sugar chains are diagnostic markers, and the like.
  • the present invention comprises contacting a test substance labeled with a photocrosslinking agent capable of binding to a sugar chain with a sugar chain, and then irradiating light to crosslink the sugar chain specifically bound to the test substance.
  • a screening method for a substance that specifically binds to a sugar chain is provided.
  • the test substance may be any substance, such as protein, peptide, vitamin, hormone, polysaccharide, oligosaccharide, monosaccharide, low molecular weight compound, nucleic acid (DNA, RNA, oligonucleotide, mononucleotide, etc.), lipid, other than the above Natural compounds, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof and the like. If the test substance does not have a functional group capable of binding to the photocrosslinking agent, such a functional group may be introduced in advance.
  • the present invention also provides a method in which a molecular probe capable of specifically binding to a sugar chain is contacted with a sample labeled with a photocrosslinking agent capable of binding to the sugar chain, and then irradiated with light, Provided is a method for testing a disease in which a sugar chain serves as a diagnostic marker, which comprises cross-linking a specifically bound sugar chain.
  • Hyaluronic acid is a diagnostic marker for diseases such as liver disease, rheumatoid arthritis, osteoarthritis of the knee, and cancer.
  • Chondroitin sulfate is a diagnostic marker for diseases such as thyroid disease, collagen disease, diabetes, and traumatic knee arthropathy. Heparan sulfate is known to be effective as a diagnostic marker for diseases such as diabetic nephropathy, and keratan sulfate is known to be effective as a diagnostic marker for diseases such as traumatic knee joint disease.
  • the disease can be examined. The presence or absence of disease can be determined by comparing the amount of sugar chains in a sample from a subject (eg, serum, plasma, pleural effusion, ascites, urine, joint fluid, etc. collected from the subject) with a healthy person.
  • the present invention also provides a kit for detecting a sugar chain, comprising the following (a) or (b).
  • (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  • the photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
  • the kit of the present invention may further contain a standard sugar chain.
  • solid phases microplates, plastic tubes, beads, etc.
  • reagents for visualizing sugar chains specifically bound to molecular probes streptavidin, biotinylation reagents, etc.
  • buffers washing solutions, frames, seals
  • Instruction manuals, blocking agents and the like may also be included.
  • a standard sugar chain may be immobilized on the solid phase.
  • a calibration curve and the like may be described.
  • the present invention also provides a screening kit for a substance that specifically binds to a sugar chain, including the following (a) or (b).
  • (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  • the photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
  • the screening kit of the present invention may further contain a standard sugar chain.
  • a solid phase microplate, plastic tube, beads, etc.
  • streptavidin a standard sugar chain
  • biotinylation reagent a standard sugar chain
  • buffer solution a washing solution
  • frame, seal a standard sugar chain
  • instruction manual may include screening criteria.
  • the present invention also provides a test kit for a disease comprising a sugar chain as a diagnostic marker, comprising the following (a) or (b).
  • a) Photocrosslinking agent and molecular probe capable of binding to sugar chain (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  • the photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
  • the test kit of the present invention may further include a standard sugar chain.
  • a solid phase microplate, plastic tube, beads, etc.
  • streptavidin a standard sugar chain
  • biotinylation reagent a standard sugar chain
  • buffer solution a washing solution
  • frame, seal a standard sugar chain
  • instruction manual may describe disease evaluation and / or differentiation criteria.
  • the advantages of the present invention are listed below. 1. Since the molecular probe that specifically recognizes the target sugar chain is irreversibly cross-linked in a state of being bound to the sugar chain, sufficient washing after the reaction is possible, and more reliable and specific detection is possible. (Molecular probes that bind to sugar chains have weak binding power, and sulfated sugar chains are more difficult to detect because they are more limited by their negative charges.) 2.
  • the photocrosslinking agent used in this detection method can be easily labeled as long as it is a probe containing a thiol group such as cysteine. Therefore, it is possible to operate with a quantification kit in which a sugar chain to be detected is solid-phased in advance. 3.
  • the photocrosslinking agent recognizes CH groups and NH groups and performs a crosslinking reaction. Therefore, not only sulfated sugar chains but a wide range of crosslinking effects can be expected. 4). It is easy to develop popular types such as quantification kits using a simple competitive method for operation. The measurement of fluorescence intensity with fluorescent dyes is not only extremely sensitive but also excellent in quantification. Therefore, the development of structure-specific molecular probes has led to the development of sugar chains with specific structures (for example, sulfated sugar chains). ) Only can be measured quantitatively.
  • Photocrosslinking agent Thermo, Heparin: Nacalai Tesque, CS-A: Seikagaku, CS-C: Seikagaku, Paraffin section: Geno staff, Anti-heparan sulfate antibody (10E4): Seikagaku, Small intestine tissue section: Geno Staff, kidney tissue section: Geno staff, cat skin tissue section: Gifu University, provided by Associate Professor Hiroki Sakai, Mastocytoma (cat-derived) skin tissue section; Gifu University, Associate Professor Hiroki Sakai (Procedure preparation method) Happy the (H eparin- a ssociated p e p tide Y) and named molecular probe (WO / 2011/048922) to the C-terminus of a state obtained by adding Cys peptide (Happy-Cys) were synthesized by the Fmoc
  • HappY-Cys The amino acid sequence of HappY is shown in SEQ ID NO: 3, and the base sequence of the DNA encoding it is shown in SEQ ID NO: 2.
  • Example 1 The heparin sugar chain was detected by a molecular probe (HappY-Cys) labeled with a photocrosslinking agent (competitive method, detected by FITC).
  • a PBS-tween (0.025%) solution containing heparin introduced with biotin at the reducing end was dispensed into a 96-well plate coated with streptavidin and allowed to react at room temperature for 1 hour or at 4 ° C. overnight. After washing with PBS-tween to remove unbound heparin, excess biotin was blocked with 5% BSA / PBS-tween containing streptavidin. Next, excess streptavidin was blocked with 5% BSA / PBS-tween containing biotin.
  • HappY-Cys Disperse heparin and chondroitin sulfate at known concentrations in a microtube in the dark, add labeled HappY-Cys diluted with 5% BSA / PBS-tween, mix, and then coat heparin 96 Dispense into hole plates. After incubating for 15 minutes at room temperature in the dark, a plate was placed on ice and irradiated with UV 365 nm for 15 minutes from a distance of 2.5 cm. After washing 6 times with 2M NaCl, it was washed 3 times with PBS-tween. Fluorescein-streptavidin diluted with PBS-tween was dispensed and incubated for 30 minutes at room temperature, protected from light.
  • the relative fluorescence intensity of each sample was shown as a value ( ⁇ FI) obtained by subtracting the fluorescence intensity of the well (blank) of only the reaction solution not containing labeled HappY-Cys from the measured value.
  • ⁇ FI fluorescence intensity obtained by subtracting the fluorescence intensity of the well (blank) of only the reaction solution not containing labeled HappY-Cys from the measured value.
  • chondroitin sulfate (CS-A and CS-C), to which HappY-Cys cannot specifically bind, has a relative fluorescence intensity compared to the control even in a solution containing 1000 ng that can be significantly quantified with heparin. HappY-Cys showed that heparin was specifically quantified.
  • Example 2 Paraffin-embedded tissue sections (tissue: small intestine) were stained with hematoxylin for staining intracellular nuclei and eosin for staining cytoplasm. As shown in FIG. 4, epithelial cells forming the villi of the small intestine tissue were detected.
  • This tissue section was stained with an anti-heparan sulfate antibody (10E4), which is known to detect heparan sulfate, according to the usual immunohistochemical staining method. Then, heparan sulfate in the basement membrane existing inside the villi (bottom of epithelial cells) was stained (FIG. 4).
  • an anti-heparan sulfate antibody (10E4) which is known to detect heparan sulfate, according to the usual immunohistochemical staining method. Then, heparan sulfate in the basement membrane existing inside the villi (bottom of epithelial cells) was stained (FIG. 4).
  • HappY-Cys labeled with a photoreactive cross-linking reagent was used to incubate the tissue section and HappY-Cys at room temperature for 30 minutes, and then an ultraviolet lamp of 365 nm from a distance of 2.5 cm on ice. was irradiated for 15 minutes to carry out a photocrosslinking reaction. After washing with PBS to remove uncrosslinked HappY-Cys probe, it was labeled with streptavidin-HRP and detected by DAB color reaction.
  • HappY-Cys does not specifically recognize heparan sulfate / heparin sugar chains and binds to tissues non-specifically
  • heparin / heparan sulfate specific degrading enzyme heparinase, After treating with heparitinase I and heparitinase II
  • staining was similarly performed with HappY-Cys.
  • HappY-Cys As a result, when the sugar chain was removed, no stained images with HappY-Cys were obtained, and it was revealed that HappY-Cys specifically detected heparan sulfate and heparin sugar chains (FIG. 4).
  • Example 3 Paraffin-embedded tissue sections (tissue: kidney) were stained with hematoxylin that stains intracellular nuclei and eosin that stains cytoplasm. As shown in FIG. 5, renal bodies of the kidney tissue were detected.
  • This tissue section was stained with an anti-heparan sulfate antibody (10E4), which is known to detect heparan sulfate, according to the usual immunohistochemical staining method.
  • heparan sulfate in the basement membrane was stained (FIG. 5).
  • HappY-Cys labeled with a photoreactive cross-linking reagent was used to incubate the tissue section and HappY-Cys at room temperature for 30 minutes, and then an ultraviolet lamp of 365 nm from a distance of 2.5 cm on ice. was irradiated for 15 minutes to carry out a photocrosslinking reaction. After washing with PBS to remove uncrosslinked HappY-Cys probe, it was labeled with streptavidin-HRP and detected by DAB color reaction.
  • Example 4 Skin tissue section (cat-derived) and HappY labeled with photoreactive cross-linking reagent were incubated at room temperature for 30 minutes, and then irradiated with a 365-nm UV lamp (40 watts) from ice at a distance of 2.5 cm for 15 minutes on ice. Went. After washing with 1 M NaCl and PBS to remove uncrosslinked HappY probe, it was labeled with streptavidin-HRP, and sugar chains were detected by DAB color reaction.
  • tissue sections were treated with heparin / heparan sulfate specific degrading enzymes (heparinase, heparitinase I, heparitinase II), and sugar chains After removal, staining was similarly performed using a HappY probe.
  • HappY specifically detected heparan sulfate and heparin sugar chains by the removal of sugar chains, and HappY clearly detected heparin sulfate and heparin sugar chains (center of FIG. 6).
  • glycosaminoglycan sugar chains heparan sulfate
  • Example 5 To examine the distribution of mast cells, we first performed toluidine blue staining to examine the distribution of mast cells using mastocytoma (cat-derived) skin tissue sections composed of mast cells. Mastocytoma is a common skin malignancy in old dogs and cats. As a result of toluidine blue staining, it was confirmed that mast cells were present in one plane in the observed tissue (FIG. 7 upper).
  • Skin tissue section (derived from cat mastocytoma) and HappY labeled with photoreactive cross-linking reagent were incubated at room temperature for 30 minutes, and then irradiated with a 365-nm UV lamp (40 watts) from 2.5 cm distance for 15 minutes on ice. A photocrosslinking reaction was performed. After washing with 1M NaCl and PBS to remove the uncrosslinked HappY probe, it was labeled with streptavidin-HRP and the sugar chain was detected by DAB color reaction.
  • mast cells present in the cat mastocytoma were not recognized at all by the HappY probe, and no stained image was seen (center of FIG. 7).
  • Mast cells are cells that originally contain a lot of heparin recognized by HappY, and this result suggests that heparin in mast cells in mastocytoma has a special structure.
  • the heparin biosynthesized by mast cells in mastocytoma is a heparin with a special structure that is not recognized by HappY, and should be used for diagnosis of mastocytoma in the skin. I think you can. For example, by combining staining with a HappY probe with toluidine blue staining, it may be possible to distinguish between normal mast cells and tumorized mast cells.
  • SEQ ID NO: 1 shows the amino acid sequence (RTRGSTREFRTGC) of a peptide obtained by adding Cys to HappY. This peptide can specifically bind to glycosaminoglycans (heparin, heparan sulfate) and has a free thiol group.
  • SEQ ID NO: 2 shows glycosaminoglycan (heparin, heparan sulfate) which specifically recognize the peptide, HappY (H eparin- a ssociated p e p tide Y) nucleotide sequence of DNA encoding a (ShijijieishijishijitijijiGTCGACCCGGGAATTCCGGACCGGT).
  • SEQ ID NO: 3 shows the glycosaminoglycan (heparin, heparan sulfate) specifically recognize peptides, the amino acid sequence of HappY (H eparin- a ssociated p e p tide Y) a (RTRGSTREFRTG).

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Abstract

　Although competitive detection methods are generally considered to be effective as a simplified method of detecting sugar chains, such methods are unsuited to quantitative determination. Provided is a sugar chain detection method that resolves such issues encountered when using conventional techniques.　The method for detecting sugar chains using a molecular probe that can bind specifically to the sugar chains comprises bringing into contact with a specimen a substance that labels the molecular probe using a photocrosslinking agent that can bind to the sugar chains, and then irradiating the specimen with light and crosslinking the sugar chains which have specifically binded to the molecular probes. A kit for detecting sugar chains contains (a) and (b) below. (a) A photocrosslinking agent and molecular probe that can bind to the sugar chains (b) A molecular probe that is labelled using a crosslinking agent that can bind to the sugar chains Provided are a screening method for substances that bind specifically to a sugar chain, and a kit therefor, and a disease testing method in which the sugar chain acts as a diagnostic marker, and a kit therefor.

Description

光架橋剤と分子プローブを用いた糖鎖の検出方法Sugar chain detection method using photocrosslinking agent and molecular probe
　本発明は、糖鎖の検出方法に関し、より詳細には、光架橋剤と分子プローブを用いた糖鎖の検出方法に関する。 The present invention relates to a sugar chain detection method, and more particularly to a sugar chain detection method using a photocrosslinking agent and a molecular probe.
　グリコサミノグリカン糖鎖（硫酸化糖鎖）は、高等動物細胞表面に普遍的に発現しているヘパラン硫酸やコンドロイチン硫酸などの総称であり、ムコ多糖症などの血中、尿中で使用できる診断マーカーの指標分子である。 Glycosaminoglycan sugar chain (sulfated sugar chain) is a generic term for heparan sulfate and chondroitin sulfate that are universally expressed on the surface of higher animal cells and can be used in blood and urine such as mucopolysaccharidosis. It is an indicator molecule for a diagnostic marker.
　指標の認識機構は、タンパク質の構造で識別する抗体反応が広く用いられている。しかし、硫酸化糖鎖は、酸性糖や硫酸基から構成される負電荷を有する生体内高分子である。そのため、抗体を利用した検出方法では、非特異的に負電荷分子と相互作用することが起こり、微量なサンプルを検出するのが困難である。 An antibody reaction that is recognized by the structure of a protein is widely used as the indicator recognition mechanism. However, sulfated sugar chains are in vivo macromolecules having a negative charge composed of acidic sugars and sulfate groups. Therefore, in the detection method using an antibody, non-specific interaction with a negatively charged molecule occurs, and it is difficult to detect a very small amount of sample.
　上記の硫酸化糖鎖の高感度認識に有用な分子プローブは、本発明者自身が特許出願している（特許文献１）。 The inventor himself has applied for a patent for a molecular probe useful for highly sensitive recognition of the sulfated sugar chain (Patent Document 1).
ＷＯ２０１１／０４８９２２パンフレットWO2011 / 048922 pamphlet
　しかしながら、ＷＯ２０１１／０４８９２２の発明は、硫酸化糖鎖の定性的検出の点では優れている一方、使用している分子プローブと糖鎖間の相互作用が非常に速く、結合と解離を繰り返す（回転速度が速い＝“弱い結合”）ために、より簡便に硫酸化糖鎖を検出する方法として一般に有効とされる競合的検出法（抗原―抗体結合において、標識された抗原を混入させることで、未知の試料中の抗原が抗体と結合する量が逆転して測定される方法）による定量には適さないことが問題点であった。 However, while the invention of WO2011 / 048922 is excellent in terms of qualitative detection of sulfated sugar chains, the interaction between the molecular probe used and the sugar chain is very fast and repeats binding and dissociation (rotation) Fast speed = “ weak binding” ), so that it is a more effective method for detecting sulfated glycans in a more convenient manner (by mixing a labeled antigen in antigen-antibody binding, The problem is that it is not suitable for quantification by a method in which the amount of an antigen in an unknown sample bound to an antibody is reversed.
　本発明は、かかる問題点を解決する糖鎖の検出方法を提供することを目的とする。 An object of the present invention is to provide a sugar chain detection method that solves such problems.
　そこで、本発明では、“弱い結合”を“強固”にする改良に着眼した。具体的には、化学的修飾法を利用して光反応性架橋剤で分子プローブを標識し、プローブとグリコサミノグリカン糖鎖との相互作用の回転速度の速さを、光架橋により永続的に固定してしまうことで通常方法では困難な糖鎖の検出を可能とした。本発明で利用する光反応架橋剤は、本来タンパク質分子間相互作用分析の目的で利用されている（Stauffer, D.A., and Karlin, A. (1994) Biochemistry 33, 6840-6849.Holmgren, M., et al. (1996) Neuropharmacology 35, 797-804.Liu, Y., et al. (1996) Neuron 16, 859-867.）。しかしながら、糖鎖の定量法に利用された事例はない。 Therefore, in the present invention, we focused on an improvement to make “weak bond” “strong”. Specifically, the molecular probe is labeled with a photoreactive crosslinking agent using a chemical modification method, and the rotational speed of the interaction between the probe and the glycosaminoglycan sugar chain is made permanent by photocrosslinking. This makes it possible to detect sugar chains that are difficult to obtain by conventional methods. The photoreactive crosslinking agent used in the present invention is originally used for the purpose of protein-molecule interaction analysis (Stauffer, DA, and Karlin, A. (1994) Biochemistry 33, 6840-6849. Holmgren, M., et al. (1996) Neuropharmacology 35, 797-804.Liu, Y., et al. (1996) Neuron 16, 859-867.). However, there has been no case where it has been used in a method for quantifying sugar chains.
　本発明の要旨は以下の通りである。
（１）糖鎖に特異的に結合できる分子プローブを用いて糖鎖を検出する方法であって、糖鎖に結合できる光架橋剤で分子プローブを標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む、前記方法。
（２）さらに、前記分子プローブに特異的に結合した糖鎖を可視化することを含む（１）記載の方法。
（３）検出の対象となる糖鎖が固定されている固相の上で、光を照射する（１）又は（２）記載の方法。
（４）競合法で糖鎖が定量的に検出される（１）～（３）のいずれかに記載の方法。
（５）染色により糖鎖が可視化される（１）～（３）のいずれかに記載の方法。
（６）糖鎖に結合できる光架橋剤で標識した被験物質を糖鎖と接触させ、その後、光を照射して、前記被験物質に特異的に結合した糖鎖を架橋することを含む、糖鎖に特異的に結合する物質のスクリーニング法。
（７）糖鎖に特異的に結合することができる分子プローブを糖鎖に結合できる光架橋剤で標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む、糖鎖が診断マーカーとなる疾患の検査法。
（８）下記の(a)又は(b)を含む、糖鎖を検出するためのキット。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ
（９）さらに、検出の対象となる糖鎖が固定された固相を含む（８）記載のキット。
（１０）さらに、分子プローブに特異的に結合した糖鎖を可視化するための試薬を含む（８）又は（９）記載のキット。
（１１）下記の(a)又は(b)を含む、糖鎖に特異的に結合する物質のスクリーニングキット。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ
（１２）下記の(a)又は(b)を含む、糖鎖が診断マーカーとなる疾患の検査キット。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ The gist of the present invention is as follows.
(1) A method for detecting a sugar chain using a molecular probe capable of specifically binding to a sugar chain, wherein a molecule probe labeled with a photocrosslinking agent capable of binding to a sugar chain is brought into contact with a sample, and then light And linking the sugar chain specifically bound to the molecular probe.
(2) The method according to (1), further comprising visualizing a sugar chain specifically bound to the molecular probe.
(3) The method according to (1) or (2), wherein light is irradiated on a solid phase on which a sugar chain to be detected is fixed.
(4) The method according to any one of (1) to (3), wherein a sugar chain is quantitatively detected by a competition method.
(5) The method according to any one of (1) to (3), wherein the sugar chain is visualized by staining.
(6) A saccharide comprising contacting a test substance labeled with a photocrosslinking agent capable of binding to a sugar chain with a sugar chain, and then irradiating light to crosslink the sugar chain specifically bound to the test substance. A screening method for substances that specifically bind to chains.
(7) A molecular probe capable of specifically binding to a sugar chain is contacted with a sample labeled with a photocrosslinking agent capable of binding to the sugar chain, and then irradiated with light, specifically to the molecular probe. A method for testing a disease in which a sugar chain serves as a diagnostic marker, which comprises cross-linking bound sugar chains.
(8) A kit for detecting a sugar chain, comprising the following (a) or (b).
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) The molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain (9) The kit according to (8), further comprising a solid phase on which a sugar chain to be detected is fixed.
(10) The kit according to (8) or (9), further comprising a reagent for visualizing a sugar chain specifically bound to the molecular probe.
(11) A screening kit for a substance that specifically binds to a sugar chain, comprising the following (a) or (b):
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) A molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain (12) A test kit for a disease in which a sugar chain serves as a diagnostic marker, comprising the following (a) or (b):
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
　本発明の検出方法では、１．硫酸化糖鎖に非特異的に検出試薬（分子プローブ）が結合することによる検出誤差を生じる点、２．標的分子とプローブの結合力が微小な場合の両者複合体の検出が困難な点、３．光架橋の標的が限定される点、４．従来の検出法はキット化が容易な競合法が利用できない点、といった課題を解決することができる。
　本明細書は、本願の優先権の基礎である日本国特許出願、特願2011‐195558の明細書および／または図面に記載される内容を包含する。 In the detection method of the present invention, 1. Detection error due to nonspecific binding of detection reagent (molecular probe) to sulfated sugar chain 2. Difficult to detect the complex when the binding force between the target molecule and the probe is very small. 3. The target of photocrosslinking is limited. The conventional detection method can solve the problem that a competing method that can be easily kitted cannot be used.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2011-195558, which is the basis of the priority of the present application.
光架橋剤で標識した分子プローブを糖鎖と結合させた（“弱い結合”）後、光を照射して、糖鎖を架橋することにより、“強固な結合”とすることができることを模式的に示した図である。Schematic that a molecular probe labeled with a photocrosslinking agent can be linked to a sugar chain ("weak bond"), and then irradiated with light to crosslink the sugar chain, resulting in a "strong bond". It is the figure shown in. 本発明の検出方法を競合法で用いる態様を模式的に示した図である。It is the figure which showed typically the aspect which uses the detection method of this invention by a competition method. 光架橋剤により標識したHappY-Cysペプチドとヘパリン表示量を混合した後、UV照射により固定化ヘパリンを光架橋し、検出された結合HappY-Cysペプチド量（競合法、FITCにより検出）を示すグラフである。コンドロイチン硫酸とは競合しなかった。GAG: グリコサミノグリカン、HP:ヘパリン、CS-A:コンドロイチン硫酸Ａ、CS-C:コンドロイチン硫酸Ｃ。Graph showing the amount of bound HappY-Cys peptide (competitive method, detected by FITC) after mixing HappY-Cys peptide labeled with photocrosslinker and heparin display amount, then photocrosslinking immobilized heparin by UV irradiation It is. It did not compete with chondroitin sulfate. GAG: glycosaminoglycan, HP: heparin, CS-A: chondroitin sulfate A, CS-C: chondroitin sulfate C. パラフィン包埋組織切片（組織：小腸）を用いて、光反応性架橋試薬（Mts-Atf-LC-Biotin）を標識した分子プローブ（HappY-Cys）によって、ヘパラン硫酸・ヘパリン糖鎖の局在部位を染色によって検出した結果を示す。酵素処理によりヘパリン／ヘパラン硫酸を除去したもの、10E4（抗ヘパラン硫酸抗体）を用いて組織染色したもの、ヘマトキリシン／エオジン染色したものも示す。Using a paraffin-embedded tissue section (tissue: small intestine) and a molecular probe (HappY-Cys) labeled with a photoreactive cross-linking reagent (Mts-Atf-LC-Biotin), the localization sites of heparan sulfate and heparin sugar chains The result of having been detected by staining is shown. Also shown are those from which heparin / heparan sulfate has been removed by enzyme treatment, those stained with 10E4 (anti-heparan sulfate antibody), and those stained with hematoxylin / eosin. パラフィン包埋組織切片（組織：腎臓）を用いて、光反応性架橋試薬（Mts-Atf-LC-Biotin）を標識した分子プローブ（HappY-Cys）によって、ヘパラン硫酸・ヘパリン糖鎖の局在部位を染色によって検出した結果を示す。10E4（抗ヘパラン硫酸抗体）を用いて組織染色したもの、ヘマトキリシン／エオジン染色したものも示す。Using a paraffin-embedded tissue section (tissue: kidney), a molecular probe (HappY-Cys) labeled with a photoreactive crosslinking reagent (Mts-Atf-LC-Biotin) is used to localize heparan sulfate and heparin sugar chains. The result of having been detected by staining is shown. Also shown are those stained with 10E4 (anti-heparan sulfate antibody) and stained with hematoxylin / eosin. 上：HappYプローブによる皮膚組織切片（ネコ由来）の染色像を示す。中央：HappYプローブによる皮膚組織切片（ネコ由来）の染色像（糖鎖分解酵素処理済）を示す。下：市販抗体（10E4）による皮膚組織切片（ネコ由来）の染色像を示す。Top: Stained image of skin tissue section (cat-derived) with HappY probe. Center: Stained image of the skin tissue section (derived from cat) with the HappY probe (treated with glycosylation enzyme). Bottom: Stained image of skin tissue section (cat-derived) with commercially available antibody (10E4). 上：トルイジンブルーによる肥満細胞腫（ネコ由来）皮膚組織切片の染色像を示す。中央：HappYプローブによる肥満細胞腫（ネコ由来）皮膚組織切片の染色像を示す。下：市販抗体（10E4）による肥満細胞腫（ネコ由来）皮膚組織切片の染色像を示す。Top: Stained image of mastocytoma (cat-derived) skin tissue section with toluidine blue. Center: Stained image of mastocytoma (cat-derived) skin tissue section with HappY probe. Bottom: Stained image of mastocytoma (cat-derived) skin tissue section with commercially available antibody (10E4).
　以下、本発明の実施の形態についてより詳細に説明する。 Hereinafter, embodiments of the present invention will be described in more detail.
　本発明は、糖鎖に特異的に結合できる分子プローブを用いて糖鎖を検出する方法であって、糖鎖に結合できる光架橋剤で分子プローブを標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む、前記方法を提供する。本発明において、検出には定量も含まれる。 The present invention is a method for detecting a sugar chain using a molecular probe capable of specifically binding to a sugar chain, wherein a molecule probe labeled with a photocrosslinking agent capable of binding to a sugar chain is contacted with a sample, The method is provided comprising irradiating light to crosslink a sugar chain specifically bound to the molecular probe. In the present invention, detection includes quantification.
　本発明の検出方法において、検出の対象となる糖鎖は、いかなる糖鎖であってもよいが、特異的に結合できる分子プローブが存在するものであるとよく、グリコサミノグリカンを例示することができる。 In the detection method of the present invention, the sugar chain to be detected may be any sugar chain, but it should be a molecular probe that can specifically bind, and examples include glycosaminoglycans. Can do.
　グリコサミノグリカンは、硫酸基が付加したものを含むアミノ糖とウロン酸またはガラクトースの二糖の繰り返し構造を有する糖鎖であり、線虫以上の高等動物のほぼすべての細胞表面および細胞外マトリクスに遊離状態あるいはタンパク質に結合した状態で存在する。グリコサミノグリカンは、アミノ糖（ガラクトサミン、グルコサミンなど）とウロン酸（グルクロン酸、イズロン酸など）又はガラクトースからなる二糖の繰り返し長鎖構造を持ち、アセチル化や硫酸化されている場合もある。硫酸化部位は構成糖の特定の水酸基（O-硫酸化）またはアミノ基（N-硫酸化）に硫酸基が転移されたものである。 Glycosaminoglycans are sugar chains that have a repeating structure of amino sugars, including those with sulfate groups added, and disaccharides of uronic acid or galactose, and are almost the cell surface and extracellular matrix of higher animals than nematodes. Exists in a free state or bound to a protein. Glycosaminoglycans have a long chain structure of disaccharides consisting of amino sugars (galactosamine, glucosamine, etc.) and uronic acids (glucuronic acid, iduronic acid, etc.) or galactose, and may be acetylated or sulfated. . The sulfation site is a sulfate group transferred to a specific hydroxyl group (O-sulfation) or amino group (N-sulfation) of the constituent sugar.
　グリコサミノグリカンは、硫酸基やウロン酸のカルボキシル基により負に荷電している。グリコサミノグリカンとしては、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸、ヘパリン、コンドロイチンなどが知られている。 Glycosaminoglycan is negatively charged by the sulfate group or the carboxyl group of uronic acid. As glycosaminoglycans, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, chondroitin and the like are known.
　コンドロイチン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸、ヘパリンなどは、プロテオグリカンのコアタンパクに共有結合した形で、細胞表面や細胞外マトリックスに存在する。ヘパリンは、ヘパラン硫酸の一種であり、D-グルクロン酸又はL-イズロン酸とN-アセチル-D-グルコサミンの二糖単位の繰り返し構造をとり、二糖あたり平均２～３個の硫酸基を持つ高分子であるが、ヘパラン硫酸より硫酸化の度合いが高い。生体内において、ヘパリンは、抗凝固作用に関与する凝固系のタンパク質と相互作用する。ヘパリンは、抗凝固薬の一つとして利用されており、血栓塞栓症、播種性血管内凝固症候群の治療、人工透析、体外循環での凝固防止などに用いられている。ヘパリンは、アンチトロンビンIIIと結合することによって、アンチトロンビンIIIの抗凝血作用を賦活化し、凝固系を抑制する。アンチトロンビンIIIは、トロンビンと複合体を形成することによって、トロンビンの血液凝固活性を阻害する糖タンパク質である。 Chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin and the like are present on the cell surface and extracellular matrix in a form covalently bound to the core protein of proteoglycan. Heparin is a kind of heparan sulfate, which has a repeating structure of disaccharide units of D-glucuronic acid or L-iduronic acid and N-acetyl-D-glucosamine, and has an average of 2 to 3 sulfate groups per disaccharide. Although it is a polymer, it has a higher degree of sulfation than heparan sulfate. In vivo, heparin interacts with coagulation proteins involved in anticoagulation. Heparin is used as one of anticoagulants and is used for treatment of thromboembolism, disseminated intravascular coagulation syndrome, artificial dialysis, prevention of coagulation in extracorporeal circulation, and the like. Heparin binds to antithrombin III, thereby activating the anticoagulant action of antithrombin III and suppressing the coagulation system. Antithrombin III is a glycoprotein that inhibits the blood clotting activity of thrombin by forming a complex with thrombin.
　ヒアルロン酸は、他のグリコサミノグリカンと異なり、コアタンパクに結合しておらず、また硫酸基も持たない。ヒアルロン酸は、関節、硝子体、皮膚、脳などの生体内の細胞外マトリックスに存在する。 Unlike other glycosaminoglycans, hyaluronic acid is not bound to the core protein and has no sulfate group. Hyaluronic acid is present in the extracellular matrix in vivo such as joints, vitreous body, skin, and brain.
　本発明において、検出の対象とする糖鎖（例えば、グリコサミノグリカン）は、遊離の状態で存在しているものであってもよいし、コアタンパクなどの他の物質と結合している状態のものであってもよい。 In the present invention, the sugar chain (for example, glycosaminoglycan) to be detected may be present in a free state or in a state in which it is bound to another substance such as a core protein. It may be.
　本発明の検出方法に用いる分子プローブは、糖鎖に特異的に結合することができ、かつ糖鎖に結合できる光架橋剤で標識可能なものであればよい。例えば、糖鎖に結合できる光架橋剤が2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl)-N6-(6-biotinamidocaproyl)-L-lysinylamido]}ethyl methanethiosulfonate  (Mts-Atf-LC-Biotin)(Pierce Bioctechnology)である場合、Mts-Atf-LC-Biotin のメタンチオスルフォネート基と結合できるチオール基(SH)を持つものであるとよく、このような分子プローブは、糖鎖を特異的に認識する物質に、必要により、遊離のチオール基を導入することにより得られる。 The molecular probe used in the detection method of the present invention is not particularly limited as long as it can specifically bind to a sugar chain and can be labeled with a photocrosslinking agent capable of binding to the sugar chain. For example, a photo-crosslinking agent capable of binding to a sugar chain is 2- {N2- [N6- (4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl) -N6- (6-biotinamidocaproyl) -L-lysinylamido ]} ethyl methanethiosulfonate (Mts-Atf-LC-Biotin) (Pierce Bioctechnology), it should have a thiol group (SH) that can bind to the methanethiosulfonate group of Mts-Atf-LC-Biotin Such a molecular probe can be obtained by introducing a free thiol group into a substance that specifically recognizes a sugar chain, if necessary.
　糖鎖がグリコサミノグリカンである場合、グリコサミノグリカンを特異的に認識する物質としては、以下のものを挙げることができる。
・ヘパリンやヘパラン硫酸に特異的に結合するHappY（Heparin-associated peptide Y, 2008年8月19日第28回日本糖質学会年会（つくば）口頭発表及び要旨集p.79）（アミノ酸配列：RTRGSTREFRTG）（配列番号３）
・HappYにCysを付加したもの(HappY-Cys)（アミノ酸配列：RTRGSTREFRTGC）（配列番号１）
・グリコサミノグリカンを特異的に認識する抗体として，CS-56（抗-コンドロイチン硫酸抗体；Avnur Z. and Geiger, B.，Exp. Cell Res. 158，321-332，1985），MO-224（抗-コンドロイチン硫酸抗体；Yamagata, M. et al.，J. Biol. Chem. 262，4146-4152，1987），HepSS-1（抗-ヘパラン硫酸抗体；Kure, S. and Yoshie, O.，J. Immunol. 137，3900-3908，1986）などがある。
・ヘパリンを特異的に認識するアンチトロンビンIII（Bjork, I. and Lindahl, U.，Mol. Cell. Biochem. 48，161-182，1982）
　グリコサミノグリカンを特異的に認識する物質は、2008年8月19日第28回日本糖質学会年会（つくば）口頭発表及び要旨集p.79に報告されているファージディスプレイ法、Kuppeveltらの方法（Van Kuppevelt, T. H. et al.，J. Biol. Chem. 273，12960-12966，1998）などに報告されているスクリーニング法などにより、探索することができる。 When the sugar chain is a glycosaminoglycan, examples of the substance that specifically recognizes glycosaminoglycan include the following.
Heparin and heparan HappY that specifically binds to a sulfuric acid (H eparin- a ssociated p e p tide Y, 8 May 19, 2008 28th Annual Meeting of the Japanese sugar Society Annual Meeting (Tsukuba) oral presentations and Abstracts p.79 (Amino acid sequence: RTRGSTREFRTG) (SEQ ID NO: 3)
-HappY with Cys added (HappY-Cys) (amino acid sequence: RTRGSTREFRTGC) (SEQ ID NO: 1)
-CS-56 (anti-chondroitin sulfate antibody; Avnur Z. and Geiger, B., Exp. Cell Res. 158, 321-232, 1985), MO-224 as an antibody that specifically recognizes glycosaminoglycan (Anti-chondroitin sulfate antibody; Yamagata, M. et al., J. Biol. Chem. 262, 4146-4152, 1987), HepSS-1 (anti-heparan sulfate antibody; Kure, S. and Yoshie, O., J. Immunol. 137, 3900-3908, 1986).
Antithrombin III that specifically recognizes heparin (Bjork, I. and Lindahl, U., Mol. Cell. Biochem. 48, 161-182, 1982)
The substance specifically recognizing glycosaminoglycans is the phage display method reported in August 28, 2008 at the 28th Annual Meeting of the Japanese Society of Carbohydrates (Tsukuba) and p. 79, Kuppevelt et al. (Van Kuppevelt, TH et al., J. Biol. Chem. 273, 12960-12966, 1998) and the like.
　グリコサミノグリカン以外の糖については，レクチンに代表されるような糖特異的認識蛋白質が広く多数知られており、それらを利用することができる。 Regarding sugars other than glycosaminoglycans, a large number of sugar-specific recognition proteins represented by lectins are widely known and can be used.
　糖鎖を特異的に認識する物質に遊離のチオール基を導入する場合には、糖鎖を特異的に認識する物質の構造特異的な結合力を妨げることがないよう、結合に干渉しない部位（例えば、糖鎖を特異的に認識する物質がペプチド又はタンパク質である場合には、ペプチド又はタンパク質のC末端など）に導入するとよい。遊離のチオール基の導入は、例えば、ペプチド合成時のシステイン残基の付加、チオール基導入試薬を用いてアミノ酸側鎖を修飾するなどにより行なうことができる。システイン残基の付加は、9-フルオレニルメトキシ基（Fmoc基）で保護したシステインを用いたペプチド固相合成法により、行なうことができる。 When a free thiol group is introduced into a substance that specifically recognizes a sugar chain, a site that does not interfere with binding so as not to interfere with the structure-specific binding force of the substance that specifically recognizes a sugar chain ( For example, when the substance that specifically recognizes the sugar chain is a peptide or protein, it may be introduced into the C-terminus of the peptide or protein. The introduction of a free thiol group can be performed, for example, by adding a cysteine residue during peptide synthesis or by modifying the amino acid side chain using a thiol group introduction reagent. The addition of a cysteine residue can be performed by a peptide solid phase synthesis method using cysteine protected with a 9-fluorenylmethoxy group (Fmoc group).
　分子プローブが遊離のチオール基を有していれば、Mts-Atf-LC-Biotinなどのメタンチオスルフォネート基を有する光架橋剤で標識することができるので、光架橋剤で標識した分子プローブを糖鎖と結合させた（“弱い結合”）後、光を照射して、糖鎖を架橋することにより、“強固な結合”とすることができる（図１参照）。また、分子プローブがタンパク質やペプチドであり、すでに遊離のチオール基を含有している場合は、このチオール基を利用して、Mts-Atf-LC-Biotinなどのメタンチオスルフォネート基を有する光架橋剤で標識できるため、あらかじめ分子プローブにチオール基を導入する必要はない。 If the molecular probe has a free thiol group, it can be labeled with a photocrosslinker having a methanethiosulfonate group, such as Mts-Atf-LC-Biotin. Is bonded to a sugar chain (“weak bond”), and then irradiated with light to crosslink the sugar chain, thereby forming a “strong bond” (see FIG. 1). In addition, when the molecular probe is a protein or peptide and already contains a free thiol group, this thiol group can be used to generate light with a methanethiosulfonate group such as Mts-Atf-LC-Biotin. Since it can be labeled with a cross-linking agent, it is not necessary to introduce a thiol group into the molecular probe in advance.
　本発明の検出方法は、分子プローブが認識する糖鎖を光架橋剤により架橋することにより、十分な洗浄後でも信頼性の高い検出を可能にする。 The detection method of the present invention enables highly reliable detection even after sufficient washing by crosslinking the sugar chain recognized by the molecular probe with a photocrosslinking agent.
　光架橋剤は、光照射により糖鎖を架橋でき、かつ分子プローブに結合できるものであれば、いかなるものであってもよい。 The photocrosslinking agent may be any one as long as it can crosslink sugar chains by light irradiation and can bind to a molecular probe.
　本発明で利用する光架橋剤の一例として、以下の3つの機能を分子内に有するものを挙げることができる。
１．メタンチオスルフォネート基（分子プローブのチオール基とジスルフィド結合する）
２．フェニルアジド基（紫外線照射により糖鎖のC-H又はC-NH2と化学的に結合可能する）
３．ビオチン（色素、酵素標識ストレプトアビジンなどによる定量時の可視化に利用できる）
　光架橋剤の具体例として、2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl)-N6-(6-biotinamidocaproyl)-L-lysinylamido]}ethyl methanethiosulfonate (Mts-Atf-LC-Biotin)(Pierce Bioctechnology)、2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethyl methanethiosulfonate (Mts-Atf-Biotin)(Pierce Bioctechnology)などを挙げることができる。Mts:スルフヒドリル反応性メタンチオスルフォネート基、Atf:テトラフルオロフェニルアジド基。
Mts-Atf-LC-Biotin
2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl)-N6-(6-biotinamidocaproyl)-L-lysinylamido]}ethyl methanethiosulfonate 
分子量：953.11
Biotin -Mtsスペーサーアーム：29.3Å
Biotin -Atfスペーサーア－ム：35.2Å
Mts-Atfスペーサーア－ム：21.8Å Examples of the photocrosslinking agent used in the present invention include those having the following three functions in the molecule.
1. Methanethiosulfonate group (disulfide bond with thiol group of molecular probe)
2. Phenyl azide group (can be chemically bonded to CH or C-NH 2 of sugar chain by UV irradiation)
3. Biotin (can be used for visualization during quantification with dyes, enzyme-labeled streptavidin, etc.)
Specific examples of photocrosslinking agents include 2- {N2- [N6- (4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl) -N6- (6-biotinamidocaproyl) -L-lysinylamido]} ethyl methanethiosulfonate (Mts-Atf-LC-Biotin) (Pierce Bioctechnology), 2- [N2- (4-azido-2,3,5,6-tetrafluorobenzoyl) -N6- (6-biotinamidocaproyl) -L-lysinyl] ethyl methanethiosulfonate (Mts-Atf-Biotin) (Pierce Bioctechnology). Mts: sulfhydryl-reactive methanethiosulfonate group, Atf: tetrafluorophenyl azide group.
Mts-Atf-LC-Biotin
2- {N2- [N6- (4-azido-2,3,5,6-tetrafluorobenzoyl-6-aminocaproyl) -N6- (6-biotinamidocaproyl) -L-lysinylamido]} ethyl methanethiosulfonate
Molecular weight: 953.11
Biotin-Mts spacer arm: 29.3mm
Biotin-Atf Spacer Arm: 35.2mm
Mts-Atf Spacer Arm: 21.8mm
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
Mts-Atf-Biotin
2-[N2-(4-azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethyl methanethiosulfonate
分子量：839.95
Biotin -Mtsスペーサーアーム：29.3Å
Biotin -Atfスペーサーア－ム：30.7Å
Mts-Atfスペーサーア－ム：11.1Å Mts-Atf-Biotin
2- [N2- (4-azido-2,3,5,6-tetrafluorobenzoyl) -N6- (6-biotinamidocaproyl) -L-lysinyl] ethyl methanethiosulfonate
Molecular weight: 839.95
Biotin-Mts spacer arm: 29.3mm
Biotin-Atf Spacer Arm: 30.7mm
Mts-Atf Spacer Arm: 11.1mm
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
　分子プローブは、試料と接触させる前に、光架橋剤と反応させておくとよい（標識）。 The molecular probe should be reacted with a photocrosslinking agent before contacting with the sample (label).
　光架橋剤は十分飽和量を加えるとよく、光架橋剤と分子プローブの比が、１：１～10：１、好ましくは１：１～５：１、より好ましくは１：１～２：１の質量比となるように添加するとよい。 The photo-crosslinking agent may be sufficiently saturated, and the ratio of the photo-crosslinking agent to the molecular probe is 1: 1 to 10: 1, preferably 1: 1 to 5: 1, more preferably 1: 1 to 2: 1. It is good to add so that it may become mass ratio.
　光架橋剤は、DMSO、DMFなどの溶媒に溶解して、添加するとよい。光架橋剤は、溶液100μLに対して、１～200 mM、好ましくは10～100 mM、より好ましくは25～50 mMの量で溶解して、使用するとよい。 The photocrosslinking agent may be added after being dissolved in a solvent such as DMSO or DMF. The photocrosslinking agent may be used after being dissolved in an amount of 1 to 200 μmM, preferably 10 to 100 μmM, more preferably 25 to 50 μmM with respect to 100 μL of the solution.
　光架橋剤と分子プローブとの反応は、適切な条件下で行うとよい。例えば、Mts-Atf-LC-Biotin（光架橋剤）とHappY-Cys（HappYにシステインが付加したもの）との反応は、暗所，室温で１～２時間または４℃で４～８時間の条件下で反応させるとよい。 The reaction between the photocrosslinking agent and the molecular probe may be performed under appropriate conditions. For example, the reaction between Mts-Atf-LC-Biotin (photocrosslinking agent) and HappY-Cys (HappY with cysteine added) is performed in the dark at room temperature for 1-2 hours or at 4 ° C for 4-8 hours. It is better to react under conditions.
　分子プローブを光架橋剤と反応させ、過剰な光架橋剤を透析やイオン交換カラムにより取り除いた後，分子プローブを試料と接触させるとよい。 The molecular probe is allowed to react with the photocrosslinking agent, excess photocrosslinking agent is removed by dialysis or an ion exchange column, and then the molecular probe is brought into contact with the sample.
　分子プローブと接触させる試料としては、血清、血漿、胸水、腹水、尿、関節液、培養液、脳脊髄液，組織のホモジネートなどの種々の生体試料を例示することができるが、これらに限定されるわけではない。 Examples of the sample to be contacted with the molecular probe include, but are not limited to, various biological samples such as serum, plasma, pleural effusion, ascites, urine, joint fluid, culture fluid, cerebrospinal fluid, and tissue homogenate. I don't mean.
　検出の対象となる試料中の糖鎖と分子プローブは、1：0.1～1：100、好ましくは1：1～1：50、より好ましくは1：5～1：20の質量比となるように接触させるとよい。 The sugar chain and the molecular probe in the sample to be detected should have a mass ratio of 1: 0.1 to 1: 100, preferably 1: 1 to 1:50, more preferably 1: 5 to 1:20. It is good to contact.
　分子プローブと試料を接触させるためには、分子プローブを溶解した溶液を試料に添加するとよく、分子プローブは、溶液100μLに対して、0.01～100 nmol、好ましくは0.05～50 nmol、より好ましくは1～10 nmolの量で溶解して、使用するとよい。試料は、その物性や糖鎖の含有量・種類などに応じて、必要により、リン酸緩衝生理食塩水などの適当な溶液で適当な濃度に希釈して用いることにより、使用量を適宜調整するとよい。分子プローブを溶解する溶液は、ウシ血清アルブミンを含むとよく、ウシ血清アルブミンの濃度は、2～10％（重量％）が適当である。ウシ血清アルブミンは一般的にブロッキング剤として使用されており、本発明の検出法でもその役割で使用することができる。すなわち、反応の場となる固相（例えば、プラスチックウェル）には、非特異的に分子プローブが少なからず結合してしまうが、少量の分子プローブを使用したときにはそれが検出量に対して大きな影響を与えてしまうので、ウシ血清アルブミンを相対的に多量に存在させ、固相（例えば、プラスチックウェル）に結合してしまう分子プローブの割合を下げる（原理的にはほとんど結合しない状態である）効果があると考えられる。大前提として検出対象の糖鎖にはウシ血清アルブミンが結合しないことが条件となるが、ヘパリン糖鎖については、すでにそれは確認している。 In order to bring the molecular probe into contact with the sample, a solution in which the molecular probe is dissolved may be added to the sample, and the molecular probe is 0.01 to 100 nmol, preferably 0.05 to 50 nmol, more preferably 1 to 100 μL of the solution. It is recommended to use it after dissolving in an amount of ˜10 nmol. Depending on its physical properties and sugar chain content / type, etc., the sample should be used as appropriate by diluting it to an appropriate concentration with an appropriate solution such as phosphate buffered saline. Good. The solution for dissolving the molecular probe may contain bovine serum albumin, and the concentration of bovine serum albumin is suitably 2 to 10% (% by weight). Bovine serum albumin is generally used as a blocking agent and can also be used in that role in the detection method of the present invention. In other words, the molecular probe binds to the solid phase (for example, plastic well), which is the reaction field, in a non-specific manner, but when a small amount of molecular probe is used, it has a large effect on the detection amount. Effect of lowering the proportion of molecular probes that bind to a solid phase (for example, plastic wells) (in principle, they are almost unbound). It is thought that there is. The main premise is that bovine serum albumin does not bind to the sugar chain to be detected, but the heparin sugar chain has already been confirmed.
　光架橋剤で標識した分子プローブと試料を接触させ、4～37℃、好ましくは、4～25℃、より好ましくは、15～25℃で、0.5～24時間、好ましくは、0.5～16時間、より好ましくは、0.5～3時間、試料中の糖鎖と分子プローブとの結合反応を行わせるとよい。 A sample is brought into contact with a molecular probe labeled with a photocrosslinking agent, and the temperature is 4 to 37 ° C., preferably 4 to 25 ° C., more preferably 15 to 25 ° C., 0.5 to 24 hours, preferably 0.5 to 16 hours. More preferably, the binding reaction between the sugar chain in the sample and the molecular probe is performed for 0.5 to 3 hours.
　分子プローブと試料を接触させ、検出の対象となる試料中の糖鎖と分子プローブを結合させた後、光照射により架橋する。 ¡Molecular probe and sample are brought into contact, sugar chains in the sample to be detected are bound to the molecular probe, and then crosslinked by light irradiation.
　照射する光とその照射時間は、検出の対象となる糖鎖と光架橋剤の間に結合が生じる波長と時間であればよく、例えば、光架橋剤がMts-Atf-LC-Biotinであり、糖鎖がグリコサミノグリカン糖鎖である場合、氷上で、300nm～370nmの紫外線を１～１５分間照射すれば、グリコサミノグリカン糖鎖を架橋することができる。 The irradiation light and the irradiation time may be any wavelength and time at which a bond is generated between the sugar chain to be detected and the photocrosslinking agent. For example, the photocrosslinking agent is Mts-Atf-LC-Biotin, When the sugar chain is a glycosaminoglycan sugar chain, the glycosaminoglycan sugar chain can be cross-linked by irradiating ultraviolet rays of 300 nm to 370 nm for 1 to 15 minutes on ice.
　本発明の検出方法では、さらに、分子プローブに特異的に結合した糖鎖を可視化するとよい。 In the detection method of the present invention, it is preferable to visualize the sugar chain specifically bound to the molecular probe.
　分子プローブに特異的に結合した糖鎖の可視化は、いかなる方法を用いてもよいが、例えば、光架橋剤が分子内にビオチン部分を有する場合、色素（例えば、フルオレセインイソチオシアネート（fluorescein isothiocyanate; FITC）、Texas red，Cy3，Cy5，Cy7など）、酵素（ホースラディッシュペルオキシダーゼ（HRP）、アルカリフォスファターゼ（AP）など）などで標識したストレプトアビジンにより可視的な検出をすることができる。例えば、FITCで標識したストレプトアビジンにより検出する場合は、485 nmの光で励起し、520 nmの蛍光を検出する。また、HRPで標識したストレプトアビジンにより検出する場合は、DAB呈色反応により検出する。 Any method may be used to visualize the sugar chain specifically bound to the molecular probe. For example, when the photocrosslinking agent has a biotin moiety in the molecule, a dye (for example, fluorescein isothiocyanate; FITC ), Texas red, Cy3, Cy5, Cy7, etc.), streptavidin labeled with enzymes (horseradish peroxidase (HRP), alkaline phosphatase (AP), etc.), etc., can be used for visual detection. For example, when detecting with streptavidin labeled with FITC, excitation is performed with 485 nm light and fluorescence at 520 nm is detected. In addition, when detecting with streptavidin labeled with HRP, it is detected by DAB color reaction.
　本発明の検出法は、競合法又は非競合法のいずれの方法でも用いることができる。競合法又は非競合法のいずれの場合であっても、検出の対象となる糖鎖が固定されている固相の上で、光を照射して、分子プローブと結合した糖鎖を架橋するとよい。 The detection method of the present invention can be used by either a competitive method or a non-competitive method. In either the competitive method or the non-competitive method, it is good to crosslink the sugar chain bound to the molecular probe by irradiating light on the solid phase on which the sugar chain to be detected is fixed. .
　競合法で用いる場合には、検出の対象となる糖鎖と同じ糖鎖を固定した固相を準備しておき、分子プローブ（光架橋剤で標識されている）と試料を接触させ、検出の対象となる糖鎖と分子プローブを結合させた後、糖鎖を固定した固相に分子プローブと試料の混合物を添加し、次いで、光照射により、糖鎖を架橋させた後、蛍光により、糖鎖を検出することができる（図２）。固相に固定する糖鎖は、既知量でなくとも、一定量固定されていれば十分である。競合法の場合、固相に固定された糖鎖と試料中の糖鎖との競合により、分子プローブを奪い合うことになる。試料と分子プローブを固定化糖鎖に作用させた後、遊離の分子プローブと、試料中の糖鎖と結合した分子プローブを洗浄により除去する。分子プローブで検量線を作成し、競合した分の蛍光の減少量から試料中の糖鎖量を求めることができる（すなわち、定量的な検出が可能である。）
　非競合法で用いる場合には、試料中の糖鎖を固相に固定した後、分子プローブと接触させ、検出の対象となる糖鎖と分子プローブを結合させた後、光照射により、糖鎖を架橋し、次いで、蛍光により、糖鎖を検出することができる。この場合、試料中の糖鎖を直接検出することができる。すなわち、分子プローブで検量線を作成し、蛍光量から試料中の糖鎖量を求めることができる。 When using in the competitive method, prepare a solid phase with the same sugar chain as the target sugar chain immobilized, bring the molecular probe (labeled with a photocrosslinker) into contact with the sample, After binding the target sugar chain and the molecular probe, the mixture of the molecular probe and the sample is added to the solid phase to which the sugar chain is fixed, and then the sugar chain is cross-linked by light irradiation, and then the sugar is The strand can be detected (Figure 2). Even if the sugar chain to be immobilized on the solid phase is not a known amount, it is sufficient if a certain amount is immobilized. In the case of the competition method, the molecular probe is competed by competition between the sugar chain immobilized on the solid phase and the sugar chain in the sample. After allowing the sample and the molecular probe to act on the immobilized sugar chain, the free molecular probe and the molecular probe bound to the sugar chain in the sample are removed by washing. A calibration curve can be created with a molecular probe, and the amount of sugar chain in the sample can be determined from the amount of fluorescence decrease due to competition (that is, quantitative detection is possible).
When used in a non-competitive method, the sugar chain in the sample is immobilized on a solid phase, brought into contact with a molecular probe, the sugar chain to be detected is bound to the molecular probe, and then the sugar chain is irradiated by light irradiation. And then sugar chains can be detected by fluorescence. In this case, the sugar chain in the sample can be directly detected. That is, a calibration curve is created with a molecular probe, and the amount of sugar chain in the sample can be determined from the amount of fluorescence.
　固相としては、マイクロプレートのウェル、プラスチックチューブ、ビーズなどを例示することができるが、これらに限定されるわけではない。固相表面をストレプトアビジンでコーティングしておけば、ビオチン標識した糖鎖を固定できる。糖鎖のビオチン標識化は、市販のビオチン化試薬を用いて行なうことができる。 Examples of the solid phase include, but are not limited to, microplate wells, plastic tubes, beads, and the like. If the solid surface is coated with streptavidin, the sugar chain labeled with biotin can be immobilized. Biotin labeling of sugar chains can be performed using a commercially available biotinylation reagent.
　また、分子プローブに特異的に結合し、かつ光架橋剤で架橋した糖鎖を染色により、可視化することもできる。 Also, a sugar chain specifically bound to a molecular probe and crosslinked with a photocrosslinking agent can be visualized by staining.
　例えば、後述の実施例では、HappYにCysを付加したもの（分子プローブ）をMts-Atf-LC-Biotin（光架橋剤）で標識したものとパラフィン包埋組織（小腸又は腎臓）切片を室温で30分間インキュベーションし、その後、氷上で紫外線を15分間照射して架橋反応を行った後、洗浄して未架橋のHappYを除去した後、ストレプトアビジン-HRPにて標識し、DAB呈色反応により染色した。 For example, in the examples described below, HappY with Cys added (molecular probe) labeled with Mts-Atf-LC-Biotin (photocrosslinking agent) and paraffin-embedded tissue (small intestine or kidney) section at room temperature Incubate for 30 minutes, and then irradiate with ultraviolet light for 15 minutes on ice. After washing to remove uncrosslinked HappY, label with streptavidin-HRP and stain by DAB color reaction. did.
　本発明の糖鎖検出法は、糖鎖に特異的に結合する物質のスクリーニング、糖鎖が診断マーカーとなる疾患の検査などに応用することができる。 The sugar chain detection method of the present invention can be applied to screening of substances that specifically bind to sugar chains, examination of diseases in which sugar chains are diagnostic markers, and the like.
　従って、本発明は、糖鎖に結合できる光架橋剤で標識した被験物質を糖鎖と接触させ、その後、光を照射して、前記被験物質に特異的に結合した糖鎖を架橋することを含む、糖鎖に特異的に結合する物質のスクリーニング法を提供する。 Therefore, the present invention comprises contacting a test substance labeled with a photocrosslinking agent capable of binding to a sugar chain with a sugar chain, and then irradiating light to crosslink the sugar chain specifically bound to the test substance. A screening method for a substance that specifically binds to a sugar chain is provided.
　被験物質は、いかなる物質であってもよく、タンパク質、ペプチド、ビタミン、ホルモン、多糖、オリゴ糖、単糖、低分子化合物、核酸（DNA、RNA、オリゴヌクレオチド、モノヌクレオチド等）、脂質、上記以外の天然化合物、合成化合物、植物抽出物、植物抽出物の分画物、それらの混合物などを挙げることができる。被験物質に光架橋剤と結合できる官能基がない場合には、予めそのような官能基を導入しておくとよい。 The test substance may be any substance, such as protein, peptide, vitamin, hormone, polysaccharide, oligosaccharide, monosaccharide, low molecular weight compound, nucleic acid (DNA, RNA, oligonucleotide, mononucleotide, etc.), lipid, other than the above Natural compounds, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof and the like. If the test substance does not have a functional group capable of binding to the photocrosslinking agent, such a functional group may be introduced in advance.
　また、本発明は、糖鎖に特異的に結合することができる分子プローブを糖鎖に結合できる光架橋剤で標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む、糖鎖が診断マーカーとなる疾患の検査法を提供する。 The present invention also provides a method in which a molecular probe capable of specifically binding to a sugar chain is contacted with a sample labeled with a photocrosslinking agent capable of binding to the sugar chain, and then irradiated with light, Provided is a method for testing a disease in which a sugar chain serves as a diagnostic marker, which comprises cross-linking a specifically bound sugar chain.
　ヒアルロン酸は、肝疾患、慢性関節リウマチ、変形性膝関節症、癌などの疾患の診断マーカーとして、コンドロイチン硫酸は、甲状腺疾患、膠原病、糖尿病、外傷性膝関節症などの疾患の診断マーカーとして、ヘパラン硫酸は、糖尿病性腎症などの疾患の診断マーカーとして、ケラタン硫酸は、外傷性膝関節症などの疾患の診断マーカーとして有効であることが知られているので、本発明により、これらの疾患の検査が可能となる。被験者からの試料（例えば、被験者から採取した血清、血漿、胸水、腹水、尿、関節液など）中の糖鎖の量を健常者と比較することにより、罹患の有無を判定することができる。 Hyaluronic acid is a diagnostic marker for diseases such as liver disease, rheumatoid arthritis, osteoarthritis of the knee, and cancer. Chondroitin sulfate is a diagnostic marker for diseases such as thyroid disease, collagen disease, diabetes, and traumatic knee arthropathy. Heparan sulfate is known to be effective as a diagnostic marker for diseases such as diabetic nephropathy, and keratan sulfate is known to be effective as a diagnostic marker for diseases such as traumatic knee joint disease. The disease can be examined. The presence or absence of disease can be determined by comparing the amount of sugar chains in a sample from a subject (eg, serum, plasma, pleural effusion, ascites, urine, joint fluid, etc. collected from the subject) with a healthy person.
　本発明は、下記の(a)又は(b) を含む、糖鎖を検出するためのキットも提供する。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ
　糖鎖に結合できる光架橋剤、分子プローブ及び糖鎖に結合できる光架橋剤で標識された分子プローブについては、上述した。 The present invention also provides a kit for detecting a sugar chain, comprising the following (a) or (b).
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain The photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
　本発明のキットは、さらに、糖鎖の標準品を含んでもよい。その他、固相（マイクロプレート、プラスチックチューブ、ビーズなど）、分子プローブに特異的に結合した糖鎖を可視化するための試薬（ストレプトアビジン、ビオチン化試薬など）、緩衝液、洗浄液、フレーム、シール、取扱い説明書、ブロッキング剤などを含んでもよい。固相には、糖鎖の標準品が固定されていてもよい。取扱説明書には、キットの使用方法の他、検量線なども記載しておくとよい。 The kit of the present invention may further contain a standard sugar chain. In addition, solid phases (microplates, plastic tubes, beads, etc.), reagents for visualizing sugar chains specifically bound to molecular probes (streptavidin, biotinylation reagents, etc.), buffers, washing solutions, frames, seals, Instruction manuals, blocking agents and the like may also be included. A standard sugar chain may be immobilized on the solid phase. In the instruction manual, in addition to the method of using the kit, a calibration curve and the like may be described.
　本発明は、下記の(a)又は(b)を含む、糖鎖に特異的に結合する物質のスクリーニングキットも提供する。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ
　糖鎖に結合できる光架橋剤、分子プローブ及び糖鎖に結合できる光架橋剤で標識された分子プローブについては、上述した。 The present invention also provides a screening kit for a substance that specifically binds to a sugar chain, including the following (a) or (b).
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain The photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
　本発明のスクリーニングキットは、さらに、糖鎖の標準品を含んでもよい。その他、固相（マイクロプレート、プラスチックチューブ、ビーズなど）、ストレプトアビジン、ビオチン化試薬、緩衝液、洗浄液、フレーム、シール、取扱い説明書、ブロッキング剤などを含んでもよい。固相には、糖鎖の標準品が固定されていてもよい。取扱説明書には、キットの使用方法の他、スクリーニングの判定基準なども記載しておくとよい。 The screening kit of the present invention may further contain a standard sugar chain. In addition, a solid phase (microplate, plastic tube, beads, etc.), streptavidin, biotinylation reagent, buffer solution, washing solution, frame, seal, instruction manual, blocking agent and the like may be included. A standard sugar chain may be immobilized on the solid phase. In addition to the method of using the kit, the instruction manual may include screening criteria.
　本発明は、下記の(a)又は(b)を含む、糖鎖が診断マーカーとなる疾患の検査キットも提供する。
(a)糖鎖に結合できる光架橋剤及び分子プローブ
(b)糖鎖に結合できる光架橋剤で標識された分子プローブ
　糖鎖に結合できる光架橋剤、分子プローブ及び糖鎖に結合できる光架橋剤で標識された分子プローブについては、上述した。 The present invention also provides a test kit for a disease comprising a sugar chain as a diagnostic marker, comprising the following (a) or (b).
(a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
(b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain The photocrosslinking agent capable of binding to a sugar chain, the molecular probe, and the molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain have been described above.
　本発明の検査キットは、さらに、糖鎖の標準品を含んでもよい。その他、固相（マイクロプレート、プラスチックチューブ、ビーズなど）、ストレプトアビジン、ビオチン化試薬、緩衝液、洗浄液、フレーム、シール、取扱い説明書、ブロッキング剤などを含んでもよい。固相には、糖鎖の標準品が固定されていてもよい。取扱説明書には、キットの使用方法の他、疾患の評価及び／又は鑑別基準なども記載しておくとよい。 The test kit of the present invention may further include a standard sugar chain. In addition, a solid phase (microplate, plastic tube, beads, etc.), streptavidin, biotinylation reagent, buffer solution, washing solution, frame, seal, instruction manual, blocking agent and the like may be included. A standard sugar chain may be immobilized on the solid phase. In addition to the method of using the kit, the instruction manual may describe disease evaluation and / or differentiation criteria.
　本発明の利点を以下に列挙する。
１．標的糖鎖を特異的に認識する分子プローブが、糖鎖と結合した状態で不可逆的に架橋されるため、反応後の十分な洗浄が可能となり、より信頼できる特異的な検出が可能となる。（糖鎖と結合する分子プローブは結合力が弱いうえ、硫酸化糖鎖はその負電荷によってさらに反応性を制限するため、硫酸化糖鎖の検出は非常に難しい。）
２．本検出法で使用する光架橋剤は、システインなどのチオール基を含むプローブであれば簡便に標識することができる。従って、検出したい糖鎖をあらかじめ固相化した定量化キットによる操作が可能である。
３．光架橋剤の種類によって生体内負電荷分子に対しても応用が可能である。光架橋剤は、CH基やNH基を認識し架橋反応する。よって、硫酸化糖鎖に限らず、広範な架橋効果が期待できる。
４．操作として簡便な競合法を利用して定量キットなどの普及タイプの開発が容易である。蛍光色素による蛍光強度の測定は非常に高感度であるばかりでなく定量性にも優れているため、構造特異的な分子プローブの開発により、特定の構造を有する糖鎖（例えば、硫酸化糖鎖）のみを定量的に測定することができるようになる。 The advantages of the present invention are listed below.
1. Since the molecular probe that specifically recognizes the target sugar chain is irreversibly cross-linked in a state of being bound to the sugar chain, sufficient washing after the reaction is possible, and more reliable and specific detection is possible. (Molecular probes that bind to sugar chains have weak binding power, and sulfated sugar chains are more difficult to detect because they are more limited by their negative charges.)
2. The photocrosslinking agent used in this detection method can be easily labeled as long as it is a probe containing a thiol group such as cysteine. Therefore, it is possible to operate with a quantification kit in which a sugar chain to be detected is solid-phased in advance.
3. It can be applied to in vivo negatively charged molecules depending on the type of photocrosslinking agent. The photocrosslinking agent recognizes CH groups and NH groups and performs a crosslinking reaction. Therefore, not only sulfated sugar chains but a wide range of crosslinking effects can be expected.
4). It is easy to develop popular types such as quantification kits using a simple competitive method for operation. The measurement of fluorescence intensity with fluorescent dyes is not only extremely sensitive but also excellent in quantification. Therefore, the development of structure-specific molecular probes has led to the development of sugar chains with specific structures (for example, sulfated sugar chains). ) Only can be measured quantitatively.
　以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
（実験材料）
光架橋剤：Thermo，ヘパリン：ナカライテスク，CS-A：生化学工業，CS-C：生化学工業，パラフィン切片：ジェノスタッフ，抗ヘパラン硫酸抗体(10E4)：生化学工業，小腸組織切片：ジェノスタッフ，腎臓組織切片：ジェノスタッフ，ネコ皮膚組織切片：岐阜大学・酒井洋樹准教授提供，肥満細胞腫（ネコ由来）の皮膚組織切片；岐阜大学・酒井洋樹准教授提供
（プローブの作製方法）
HappY（Heparin-associated peptide Y）と命名された分子プローブ（WO/2011/048922）のC末端にCysを付加した状態のペプチド(HappY-Cys)をFmoc法により合成した。HappYのアミノ酸配列を配列番号３に、それをコードするDNAの塩基配列を配列番号２に示す。
（光反応性架橋試薬によるHappY-Cysの標識）
Mts-Atf-LC-biotin 1 mgに対し，DMSO 25 μLを加え溶かした。20.9 mg/mlのHappY-Cys 50μL(=685 nmol)にPBS 267.5 μLを加えたものに，HappY-Cysに対し2倍モル量の光架橋剤（=1.37 μmol，32.5 μL）を加え，暗所，室温で１時間インキュベートして反応させた。未反応の試薬はVivapureカラム（vivascience）で除去した。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
(Experimental material)
Photocrosslinking agent: Thermo, Heparin: Nacalai Tesque, CS-A: Seikagaku, CS-C: Seikagaku, Paraffin section: Geno staff, Anti-heparan sulfate antibody (10E4): Seikagaku, Small intestine tissue section: Geno Staff, kidney tissue section: Geno staff, cat skin tissue section: Gifu University, provided by Associate Professor Hiroki Sakai, Mastocytoma (cat-derived) skin tissue section; Gifu University, Associate Professor Hiroki Sakai (Procedure preparation method)
Happy the (H eparin- a ssociated p e p tide Y) and named molecular probe (WO / 2011/048922) to the C-terminus of a state obtained by adding Cys peptide (Happy-Cys) were synthesized by the Fmoc method. The amino acid sequence of HappY is shown in SEQ ID NO: 3, and the base sequence of the DNA encoding it is shown in SEQ ID NO: 2.
(Labeling HappY-Cys with photoreactive crosslinking reagent)
To 1 mg of Mts-Atf-LC-biotin, 25 μL of DMSO was added and dissolved. 20.9 mg / ml HappY-Cys 50μL (= 685 nmol) plus PBS 267.5 μL, 2 times molar photocrosslinker (= 1.37 μmol, 32.5 μL) to HappY-Cys, and dark place The reaction was incubated at room temperature for 1 hour. Unreacted reagents were removed with a Vivapure column (vivascience).
〔実施例１〕
光架橋剤で標識した分子プローブ（HappY-Cys）によりヘパリン糖鎖を検出した（競合法、FITCにより検出）。
ビオチンを還元末端に導入したヘパリンを含むPBS-tween(0.025%)溶液をストレプトアビジンをコートした96穴プレートに分注して，室温で１時間または4℃で一晩反応させた。PBS-tweenで洗浄して未結合のヘパリンを除去した後，ストレプトアビジンを含む5％BSA/PBS-tweenで余剰なビオチンをブロッキングした。次いで，ビオチンを含む5％BSA/PBS-tweenで余剰なストレプトアビジンのブロッキングを行った。暗所にてマイクロチューブに既知濃度のヘパリン，コンドロイチン硫酸を分注しておき，これに5％BSA/PBS-tweenで希釈した標識化HappY-Cysを加え，混ぜた後，ヘパリンをコートした96穴プレートに分注した。遮光して室温で15分間インキュベートした後，氷上にプレートを置いて，2.5 cmの距離からUV 365 nmを15分間照射した。2M NaClで6回洗浄後，PBS-tweenで3回洗浄した。PBS-tweenにて希釈したフルオレセイン-ストレプトアビジンを分注し，遮光して室温で30分間インキュベートした。2M NaClで6回洗浄後，PBS-tweenで2回洗浄した。PBS-tweenを200 μL/wellずつ分注し，蛍光プレートリーダー（TECAN）にてEx/Em=485/520 nmにおける蛍光強度を測定した。結果を図３に示す。 [Example 1]
The heparin sugar chain was detected by a molecular probe (HappY-Cys) labeled with a photocrosslinking agent (competitive method, detected by FITC).
A PBS-tween (0.025%) solution containing heparin introduced with biotin at the reducing end was dispensed into a 96-well plate coated with streptavidin and allowed to react at room temperature for 1 hour or at 4 ° C. overnight. After washing with PBS-tween to remove unbound heparin, excess biotin was blocked with 5% BSA / PBS-tween containing streptavidin. Next, excess streptavidin was blocked with 5% BSA / PBS-tween containing biotin. Disperse heparin and chondroitin sulfate at known concentrations in a microtube in the dark, add labeled HappY-Cys diluted with 5% BSA / PBS-tween, mix, and then coat heparin 96 Dispense into hole plates. After incubating for 15 minutes at room temperature in the dark, a plate was placed on ice and irradiated with UV 365 nm for 15 minutes from a distance of 2.5 cm. After washing 6 times with 2M NaCl, it was washed 3 times with PBS-tween. Fluorescein-streptavidin diluted with PBS-tween was dispensed and incubated for 30 minutes at room temperature, protected from light. After washing 6 times with 2M NaCl, it was washed twice with PBS-tween. PBS-tween was dispensed at 200 μL / well, and the fluorescence intensity at Ex / Em = 485/520 nm was measured with a fluorescence plate reader (TECAN). The results are shown in FIG.
　各サンプルの相対蛍光強度は，それぞれ測定値から標識化HappY-Cysを含まない反応液のみのwell（ブランク）の蛍光強度を差し引いた値（ΔFI）として示した。その結果，HappY-Cysのみを含み，グリコサミノグリカン（GAG）を含まない溶液を加えたwellでは，9020.8±1221.1が検出された（コントロール）のに対し，ヘパリンとHappY-Cysとを混合した溶液を加えたwellでは，ヘパリンの濃度依存的に相対蛍光強度が低下し，溶液中のヘパリン量を測定することが出来た。一方，HappY-Cysが特異的に結合することの出来ないコンドロイチン硫酸（CS-AおよびCS-C）は，ヘパリンでは有意に定量可能な1000 ngを含む溶液でもコントロールと比較して，相対蛍光強度の有意な低下がないことから，HappY-Cysはヘパリンのみを特異的に定量していることが示された。 The relative fluorescence intensity of each sample was shown as a value (ΔFI) obtained by subtracting the fluorescence intensity of the well (blank) of only the reaction solution not containing labeled HappY-Cys from the measured value. As a result, in the well containing only HappY-Cys and not containing glycosaminoglycan (GAG), 9020.8 ± 1221.1 was detected (control), whereas heparin and HappY-Cys were mixed. In the well to which the solution was added, the relative fluorescence intensity decreased depending on the concentration of heparin, and the amount of heparin in the solution could be measured. On the other hand, chondroitin sulfate (CS-A and CS-C), to which HappY-Cys cannot specifically bind, has a relative fluorescence intensity compared to the control even in a solution containing 1000 ng that can be significantly quantified with heparin. HappY-Cys showed that heparin was specifically quantified.
〔実施例２〕
　細胞内の核を染色するヘマトキシリンと細胞質を染色するエオジンでパラフィン包埋組織切片（組織：小腸）を染色した。図４に示されているように，小腸組織の絨毛を形づくる上皮細胞が検出された。 [Example 2]
Paraffin-embedded tissue sections (tissue: small intestine) were stained with hematoxylin for staining intracellular nuclei and eosin for staining cytoplasm. As shown in FIG. 4, epithelial cells forming the villi of the small intestine tissue were detected.
　この組織切片に対してヘパラン硫酸を検出することが知られている抗ヘパラン硫酸抗体（10E4）を用いて、通常の免疫組織化学染色法に従い、組織染色を行ったところ、従来報告されているように，絨毛の内側（上皮細胞の底部）に存在する基底膜中のヘパラン硫酸が染色された（図４）。 This tissue section was stained with an anti-heparan sulfate antibody (10E4), which is known to detect heparan sulfate, according to the usual immunohistochemical staining method. Then, heparan sulfate in the basement membrane existing inside the villi (bottom of epithelial cells) was stained (FIG. 4).
　一方，同じ組織切片に対して，光反応性架橋試薬を標識したHappY-Cysを用いて，組織切片とHappY-Cysを室温で30分間インキュベーションし，その後氷上で2.5cmの距離から365nmの紫外線ランプを15分間照射して光架橋反応を行った。PBSで洗浄して未架橋のHappY-Cysプローブを除去した後，ストレプトアビジン-HRPにて標識し，DAB呈色反応により検出した。 On the other hand, HappY-Cys labeled with a photoreactive cross-linking reagent was used to incubate the tissue section and HappY-Cys at room temperature for 30 minutes, and then an ultraviolet lamp of 365 nm from a distance of 2.5 cm on ice. Was irradiated for 15 minutes to carry out a photocrosslinking reaction. After washing with PBS to remove uncrosslinked HappY-Cys probe, it was labeled with streptavidin-HRP and detected by DAB color reaction.
　その結果，10E4とは異なる箇所の染色像，すなわち，上皮細胞の基底膜側はほとんど染色されず，むしろ粘膜側がよく染色されている像が得られた（図４）。 As a result, a stained image of a portion different from 10E4, that is, an image in which the basement membrane side of the epithelial cells was hardly stained, but rather the mucosa side was well stained (FIG. 4).
　HappY-Cysがヘパラン硫酸・ヘパリン糖鎖を特異的に認識せず，非特異的に組織に結合しているかどうかを確認するため，組織切片に対してヘパリン・ヘパラン硫酸特異的分解酵素（ヘパリナーゼ，ヘパリチナーゼI，ヘパリチナーゼII）を用いて処理し，糖鎖を除去した後，同様にHappY-Cysを用いて染色を行った。その結果，糖鎖を除去するとHappY-Cysによる染色像は全く得られず，HappY-Cysはヘパラン硫酸・ヘパリン糖鎖を特異的に検出していることが明らかとなった（図４）。 To confirm whether HappY-Cys does not specifically recognize heparan sulfate / heparin sugar chains and binds to tissues non-specifically, heparin / heparan sulfate specific degrading enzyme (heparinase, After treating with heparitinase I and heparitinase II) and removing the sugar chain, staining was similarly performed with HappY-Cys. As a result, when the sugar chain was removed, no stained images with HappY-Cys were obtained, and it was revealed that HappY-Cys specifically detected heparan sulfate and heparin sugar chains (FIG. 4).
　以上より，光反応性架橋試薬を標識した分子プローブを利用して，パラフィン包埋組織切片のグリコサミノグリカン糖鎖を検出することができた。また，HappY-Cysを用いた場合，検出された糖鎖は，従来の抗体で検出される糖鎖とは異なる構造を有する糖鎖が特異的に検出されることが明らかとなった。 From the above, it was possible to detect glycosaminoglycan sugar chains in paraffin-embedded tissue sections using a molecular probe labeled with a photoreactive crosslinking reagent. In addition, when HappY-Cys was used, it was clarified that the detected sugar chain specifically detected was a sugar chain having a structure different from that detected by conventional antibodies.
〔実施例３〕
　細胞内の核を染色するヘマトキシリンと細胞質を染色するエオジンでパラフィン包埋組織切片（組織：腎臓）を染色した。図５に示されているように，腎臓組織の腎小体が検出された。 Example 3
Paraffin-embedded tissue sections (tissue: kidney) were stained with hematoxylin that stains intracellular nuclei and eosin that stains cytoplasm. As shown in FIG. 5, renal bodies of the kidney tissue were detected.
　この組織切片に対してヘパラン硫酸を検出することが知られている抗ヘパラン硫酸抗体（10E4）を用いて、通常の免疫組織化学染色法に従い、組織染色を行ったところ、従来報告されているように，基底膜中のヘパラン硫酸が染色された（図５）。 This tissue section was stained with an anti-heparan sulfate antibody (10E4), which is known to detect heparan sulfate, according to the usual immunohistochemical staining method. In addition, heparan sulfate in the basement membrane was stained (FIG. 5).
　一方，同じ組織切片に対して，光反応性架橋試薬を標識したHappY-Cysを用いて，組織切片とHappY-Cysを室温で30分間インキュベーションし，その後氷上で2.5cmの距離から365nmの紫外線ランプを15分間照射して光架橋反応を行った。PBSで洗浄して未架橋のHappY-Cysプローブを除去した後，ストレプトアビジン-HRPにて標識し，DAB呈色反応により検出した。 On the other hand, HappY-Cys labeled with a photoreactive cross-linking reagent was used to incubate the tissue section and HappY-Cys at room temperature for 30 minutes, and then an ultraviolet lamp of 365 nm from a distance of 2.5 cm on ice. Was irradiated for 15 minutes to carry out a photocrosslinking reaction. After washing with PBS to remove uncrosslinked HappY-Cys probe, it was labeled with streptavidin-HRP and detected by DAB color reaction.
　その結果，10E4とは異なる箇所の染色像，すなわち，ボーマン嚢の内腔表面のみが染色された像が得られた（図５）。 As a result, a stained image at a location different from 10E4, that is, an image in which only the lumen surface of Bowman's sac was stained (FIG. 5) was obtained.
〔実施例４〕
　皮膚組織切片（ネコ由来）と光反応性架橋試薬を標識したHappYを室温で30分間インキュベーションし，その後氷上で2.5cmの距離から365nmの紫外線ランプ（40ワット）を15分間照射して光架橋反応を行った。1 M NaClとPBSで洗浄して未架橋のHappYプローブを除去した後，ストレプトアビジン-HRPにて標識し，DAB呈色反応により糖鎖を検出した。 Example 4
Skin tissue section (cat-derived) and HappY labeled with photoreactive cross-linking reagent were incubated at room temperature for 30 minutes, and then irradiated with a 365-nm UV lamp (40 watts) from ice at a distance of 2.5 cm for 15 minutes on ice. Went. After washing with 1 M NaCl and PBS to remove uncrosslinked HappY probe, it was labeled with streptavidin-HRP, and sugar chains were detected by DAB color reaction.
　その結果，皮膚表皮層（ケラチノサイトと呼ばれる角化細胞よりなる層）がよく染色された像が得られた（図６上）。また，肥満細胞の一部も染色された。 As a result, an image was obtained in which the skin epidermis layer (layer consisting of keratinocytes called keratinocytes) was well stained (upper part of FIG. 6). Some of the mast cells were also stained.
　HappYがヘパラン硫酸・ヘパリン糖鎖を特異的に認識しているかどうかを確認するため，組織切片をヘパリン・ヘパラン硫酸特異的分解酵素（ヘパリナーゼ，ヘパリチナーゼI，ヘパリチナーゼII）を用いて処理し，糖鎖除去した後，同様にHappYプローブを用いて染色を行った。その結果，糖鎖除去によりHappYによる鮮明な染色性が失われ，HappYはヘパラン硫酸・ヘパリン糖鎖を特異的に検出していることが明らかとなった（図６中央）。 To confirm whether HappY specifically recognizes heparan sulfate / heparin sugar chains, tissue sections were treated with heparin / heparan sulfate specific degrading enzymes (heparinase, heparitinase I, heparitinase II), and sugar chains After removal, staining was similarly performed using a HappY probe. As a result, it was clarified that HappY specifically detected heparan sulfate and heparin sugar chains by the removal of sugar chains, and HappY clearly detected heparin sulfate and heparin sugar chains (center of FIG. 6).
　上述と同じ組織切片に対して，ヘパラン硫酸を検出することが知られ，広く使用されている抗ヘパラン硫酸抗体（10E4）を用いて組織染色を行うと，従来報告されているように，組織内の肥満細胞のヘパリンが染色された（図６下）。HappYプローブよりも多くの肥満細胞が染色されるのが特徴的であった。 It is known to detect heparan sulfate in the same tissue section as described above, and when tissue staining is performed using a widely used anti-heparan sulfate antibody (10E4), as previously reported, Mast cell heparin was stained (FIG. 6 bottom). It was characteristic that more mast cells were stained than the HappY probe.
　以上より，光反応性架橋試薬で標識した分子プローブを利用して，皮膚におけるグリコサミノグリカン糖鎖（ヘパラン硫酸）を検出することができた。また，HappYを用いた場合，検出された糖鎖は，従来の抗体で検出される糖鎖とは異なる構造を有する糖鎖が特異的に検出されることが明らかとなった。 From the above, it was possible to detect glycosaminoglycan sugar chains (heparan sulfate) in the skin using a molecular probe labeled with a photoreactive crosslinking reagent. When HappY was used, it was clarified that the detected sugar chain specifically detects a sugar chain having a structure different from that of a sugar chain detected by a conventional antibody.
〔実施例５〕
　肥満細胞より構成される肥満細胞腫（ネコ由来）の皮膚組織切片を用いて，まず，肥満細胞の分布を調べるために，常法としてもちいられるトルイジンブルー染色を行った。肥満細胞腫は，老齢のイヌやネコにおいて皮膚の悪性腫瘍として一般的である。トルイジンブルー染色の結果，肥満細胞が観察組織中で一面に存在していることを確認した（図７上）。 Example 5
To examine the distribution of mast cells, we first performed toluidine blue staining to examine the distribution of mast cells using mastocytoma (cat-derived) skin tissue sections composed of mast cells. Mastocytoma is a common skin malignancy in old dogs and cats. As a result of toluidine blue staining, it was confirmed that mast cells were present in one plane in the observed tissue (FIG. 7 upper).
　皮膚組織切片（ネコ肥満細胞腫由来）と光反応性架橋試薬を標識したHappYを室温で30分間インキュベーションし，その後氷上で2.5cmの距離から365nmの紫外線ランプ（40ワット）を15分間照射して光架橋反応を行った。1 M NaClとPBSで洗浄して未架橋のHappYプローブを除去した後，ストレプトアビジン-HRPにて標識し，DAB呈色反応により糖鎖を検出した。 Skin tissue section (derived from cat mastocytoma) and HappY labeled with photoreactive cross-linking reagent were incubated at room temperature for 30 minutes, and then irradiated with a 365-nm UV lamp (40 watts) from 2.5 cm distance for 15 minutes on ice. A photocrosslinking reaction was performed. After washing with 1M NaCl and PBS to remove the uncrosslinked HappY probe, it was labeled with streptavidin-HRP and the sugar chain was detected by DAB color reaction.
　その結果，ネコ肥満細胞腫に存在する肥満細胞は，HappYプローブによって，まったく認識されることなく，染色像が見られないことが確認された（図７中央）。肥満細胞は本来HappYが認識するヘパリンを多く含む細胞であり，この結果は，肥満細胞腫における肥満細胞中のヘパリンが特殊な構造を有していることを示唆している。 As a result, it was confirmed that the mast cells present in the cat mastocytoma were not recognized at all by the HappY probe, and no stained image was seen (center of FIG. 7). Mast cells are cells that originally contain a lot of heparin recognized by HappY, and this result suggests that heparin in mast cells in mastocytoma has a special structure.
　上述と同じ組織切片に対して，ヘパラン硫酸を検出することが知られ，広く使用されている抗ヘパラン硫酸抗体（10E4）を用いて組織染色を行うと，正常皮膚組織と同様に，組織内の肥満細胞のヘパリンが染色された（図７下）。トルイジンブルー染色とほぼ同じ染色像が見られる。 It is known to detect heparan sulfate in the same tissue section as described above, and when tissue staining is performed using a widely used anti-heparan sulfate antibody (10E4), as in normal skin tissue, Mast cell heparin was stained (bottom of FIG. 7). Stained image is almost the same as Toluidine Blue staining.
　以上より，肥満細胞腫における肥満細胞が生合成するヘパリンは，HappYによって認識されない特殊な構造をもつヘパリンであることが推察され，これを利用して，皮膚における肥満細胞腫の診断に応用することができると考える。例えば、HappYプローブによる染色をトルイジンブルー染色と組み合わせることで，通常の肥満細胞か，腫瘍化した肥満細胞かを見分けられる可能性がある。 From the above, it is speculated that the heparin biosynthesized by mast cells in mastocytoma is a heparin with a special structure that is not recognized by HappY, and should be used for diagnosis of mastocytoma in the skin. I think you can. For example, by combining staining with a HappY probe with toluidine blue staining, it may be possible to distinguish between normal mast cells and tumorized mast cells.
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.
　特定の病気に関係する糖鎖に対するプローブと本発明の検出（定量も含む）方法の併用により、そのプローブを診断マーカーとして病気の診断に利用する際の簡便な糖鎖の検出（定量も含む）が可能となる。 By using a probe for a sugar chain related to a specific disease in combination with the detection (including quantification) method of the present invention, simple sugar chain detection (including quantification) when using the probe as a diagnostic marker for diagnosis of the disease Is possible.
＜配列番号１＞
配列番号１は、HappYにCysを付加したペプチドのアミノ酸配列(RTRGSTREFRTGC)を示す。このペプチドは、グリコサミノグリカン(ヘパリン、ヘパラン硫酸)に特異的に結合することができ、かつ遊離のチオール基を有する。
＜配列番号２＞
配列番号２は、グリコサミノグリカン（ヘパリン、ヘパラン硫酸）を特異的に認識するペプチド、HappY（Heparin-associated peptide Y）をコードするDNAの塩基配列(CGGACGCGTGGGTCGACCCGGGAATTCCGGACCGGT)を示す。
＜配列番号３＞
配列番号３は、グリコサミノグリカン（ヘパリン、ヘパラン硫酸）を特異的に認識するペプチド、HappY（Heparin-associated peptide Y）のアミノ酸配列(RTRGSTREFRTG)を示す。 <SEQ ID NO: 1>
SEQ ID NO: 1 shows the amino acid sequence (RTRGSTREFRTGC) of a peptide obtained by adding Cys to HappY. This peptide can specifically bind to glycosaminoglycans (heparin, heparan sulfate) and has a free thiol group.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows glycosaminoglycan (heparin, heparan sulfate) which specifically recognize the peptide, HappY (H eparin- a ssociated p e p tide Y) nucleotide sequence of DNA encoding a (ShijijieishijishijitijijiGTCGACCCGGGAATTCCGGACCGGT).
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the glycosaminoglycan (heparin, heparan sulfate) specifically recognize peptides, the amino acid sequence of HappY (H eparin- a ssociated p e p tide Y) a (RTRGSTREFRTG).

Claims (12)

  1. 糖鎖に特異的に結合できる分子プローブを用いて糖鎖を検出する方法であって、糖鎖に結合できる光架橋剤で分子プローブを標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む前記方法。 A method of detecting a sugar chain using a molecular probe that can specifically bind to a sugar chain, wherein a molecular probe labeled with a photocrosslinking agent that can bind to a sugar chain is brought into contact with a sample, and then irradiated with light. And crosslinking the sugar chain specifically bound to the molecular probe.
  2. さらに、前記分子プローブに特異的に結合した糖鎖を可視化することを含む、請求項１記載の方法。 The method according to claim 1, further comprising visualizing a sugar chain specifically bound to the molecular probe.
  3. 検出の対象となる糖鎖が固定されている固相の上で、光を照射する請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein light is irradiated on a solid phase on which a sugar chain to be detected is fixed.
  4. 競合法で糖鎖が定量的に検出される請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the sugar chain is quantitatively detected by a competitive method.
  5. 染色により糖鎖が可視化される請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the sugar chain is visualized by staining.
  6. 糖鎖に結合できる光架橋剤で標識した被験物質を糖鎖と接触させ、その後、光を照射して、前記被験物質に特異的に結合した糖鎖を架橋することを含む、糖鎖に特異的に結合する物質のスクリーニング法。 Specific to a sugar chain, comprising contacting a test substance labeled with a photocrosslinking agent capable of binding to a sugar chain with a sugar chain, and then irradiating light to crosslink the sugar chain specifically bound to the test substance. For screening substances that bind chemically.
  7. 糖鎖に特異的に結合することができる分子プローブを糖鎖に結合できる光架橋剤で標識したものを試料と接触させ、その後、光を照射して、前記分子プローブに特異的に結合した糖鎖を架橋することを含む、糖鎖が診断マーカーとなる疾患の検査法。 A molecular probe that can specifically bind to a sugar chain is contacted with a sample labeled with a photocrosslinking agent that can bind to a sugar chain, and then irradiated with light to specifically bind to the molecular probe. A method for testing a disease in which a sugar chain serves as a diagnostic marker, comprising cross-linking a chain.
  8. 下記の(a)又は(b)を含む、糖鎖を検出するためのキット。
    (a)糖鎖に結合できる光架橋剤及び分子プローブ
    (b)糖鎖に結合できる光架橋剤で標識された分子プローブ     A kit for detecting a sugar chain, comprising the following (a) or (b).
    (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
    (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  9. さらに、検出の対象となる糖鎖が固定された固相を含む請求項８記載のキット。 The kit according to claim 8, further comprising a solid phase on which a sugar chain to be detected is fixed.
  10. さらに、分子プローブに特異的に結合した糖鎖を可視化するための試薬を含む請求項８又は９記載のキット。 Furthermore, the kit of Claim 8 or 9 containing the reagent for visualizing the sugar_chain | carbohydrate specifically couple | bonded with the molecular probe.
  11. 下記の(a)又は(b)を含む、糖鎖に特異的に結合する物質のスクリーニングキット。
    (a)糖鎖に結合できる光架橋剤及び分子プローブ
    (b)糖鎖に結合できる光架橋剤で標識された分子プローブ     A screening kit for a substance that specifically binds to a sugar chain, comprising the following (a) or (b):
    (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
    (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
  12. 下記の(a)又は(b)を含む、糖鎖が診断マーカーとなる疾患の検査キット。
    (a)糖鎖に結合できる光架橋剤及び分子プローブ
    (b)糖鎖に結合できる光架橋剤で標識された分子プローブ     A test kit for a disease comprising a sugar chain as a diagnostic marker, comprising the following (a) or (b):
    (a) Photocrosslinking agent and molecular probe capable of binding to sugar chain
    (b) Molecular probe labeled with a photocrosslinking agent capable of binding to a sugar chain
PCT/JP2012/072574 2011-09-08 2012-09-05 Sugar chain detection method using photocrosslinking agent and molecular probe WO2013035727A1 (en)

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Publication number Priority date Publication date Assignee Title
WO2011048922A1 (en) * 2009-10-23 2011-04-28 国立大学法人岐阜大学 Method for detection of glycosaminoglycan, and molecular probe for use in the method

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Publication number Priority date Publication date Assignee Title
WO2011048922A1 (en) * 2009-10-23 2011-04-28 国立大学法人岐阜大学 Method for detection of glycosaminoglycan, and molecular probe for use in the method

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DAVID A. ET AL.: "Electrostatic Potential of the Acetylcholine Binding Sites in the Nicotinic Receptor Probed by Reactions of Binding-Site Cysteines with Charged Methanethiosulfonates", BIOCHEMISTRY, vol. 33, 1994, pages 6840 - 6849, XP055199565, DOI: doi:10.1021/bi00188a013 *
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