WO2011048922A1 - Method for detection of glycosaminoglycan, and molecular probe for use in the method - Google Patents

Method for detection of glycosaminoglycan, and molecular probe for use in the method Download PDF

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Publication number
WO2011048922A1
WO2011048922A1 PCT/JP2010/067033 JP2010067033W WO2011048922A1 WO 2011048922 A1 WO2011048922 A1 WO 2011048922A1 JP 2010067033 W JP2010067033 W JP 2010067033W WO 2011048922 A1 WO2011048922 A1 WO 2011048922A1
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Prior art keywords
glycosaminoglycan
molecular probe
thiol group
substance
free thiol
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PCT/JP2010/067033
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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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Priority to JP2011537189A priority Critical patent/JP5723285B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • 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

Definitions

  • the present invention relates to a method for detecting glycosaminoglycan and a molecular probe therefor, and more particularly to a method for detecting glycosaminoglycan using a fluorescent labeling reagent and a molecular probe therefor.
  • glycosaminoglycan sugar chains such as heparan sulfate and chondroitin sulfate, which are attracting attention as functional regulators in vivo, are negatively charged due to the acidic sugar (uronic acid) and sulfate groups that they make up. It is known as an in vivo polymer having Such a negative charge molecule in a living body is a non-specific interaction with a negatively charged molecule in a general detection method using a primary antibody and a labeled secondary antibody that specifically recognize this molecule. Therefore, reliable detection is difficult.
  • Non-patent Document 1 There are an increasing number of academic paper reports on the development of probes that specifically recognize glycosaminoglycan sugar chains (Non-patent Document 1). However, in these conventional techniques, the probes to be screened must be labeled one by one, and there is a limitation that labels that do not affect sulfated sugar chains are used.
  • N- (9-acridinyl) ylmaleimide NAM
  • NAM fluorescent labeling reagent
  • An object of the present invention is to provide a molecular probe capable of solving the following problems of the prior art and a method for detecting glycosaminoglycan using the same.
  • a detection error is caused by nonspecific binding of the detection reagent to the sulfated sugar chain. 2. It is difficult to detect the complex when the binding force between the target molecule and the probe is very small. 3. There is a need to label the probe in advance during screening. 4). There is a risk of deactivation of the probe due to the labeling operation and inhibition of binding force to the target substance.
  • the present inventors have to a solid phase heparin sugar chains are fixed, as a molecular probe, the heparin oligosaccharides which specifically recognize the peptide molecules (amino acid sequence: RTRGSTREFRTG) (SEQ ID NO: 3)) (HappY: H eparin- a ssociated p e p tide Y , August 19th, 2008, 28th Annual Meeting of the Glycologic Society of Japan (Tsukuba) Oral Presentations and Abstracts p.79) with the addition of a cysteine residue with a free thiol group
  • the fluorescent labeling reagent N- (9-acridinyl) maleimide (NAM) is added and reacted with the free thiol group in the molecular probe to detect fluorescence
  • NAM fluorescent labeling reagent N- (9-acridinyl) maleimide
  • the gist of the present invention is as follows.
  • N- (9-acridinyl) maleimide is added to generate A method for detecting glycosaminoglycans, comprising measuring measured fluorescence.
  • the detection method according to (1) wherein the molecular probe and the sample are contacted in the presence of glycosaminoglycan immobilized on a solid phase.
  • the detection method according to (1) wherein glycosaminoglycan in the sample is immobilized on a solid phase and then contacted with the molecular probe.
  • a substance that specifically binds to glycosaminoglycan comprising contacting N- (9-acridinyl) maleimide after contacting glycosaminoglycan with a test substance and measuring the generated fluorescence. Screening method. (5) N- (9-acridinyl) maleimide is added after contacting a sample from a subject with a molecular probe that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group And a method for testing a disease in which glycosaminoglycan is a diagnostic marker, comprising measuring the generated fluorescence.
  • a glycosaminoglycan detection kit comprising a molecular probe that can specifically bind to glycosaminoglycan and includes a substance having a free thiol group and N- (9-acridinyl) maleimide.
  • the kit according to (6) further comprising glycosaminoglycan.
  • Specific binding to glycosaminoglycan which can specifically bind to glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide Screening kit for substances to be used.
  • Glycosaminoglycan that can specifically bind to glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide serves as a diagnostic marker Disease testing kit.
  • a molecular probe comprising a substance capable of specifically binding to glycosaminoglycan and having a free thiol group, the detection method according to (1), the screening method according to (4), or ( 5) The molecular probe used in the test method according to 5).
  • the molecular probe according to (12), wherein the substance capable of specifically binding to glycosaminoglycan and having a free thiol group is the following peptide (a) or (b): (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC). (b) a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
  • a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
  • a molecular probe that binds to a sugar chain generally has a weak binding force, and the sulfated sugar chain is further limited in its reactivity due to its negative charge. Therefore, it is very difficult to detect a sulfated sugar chain such as heparan sulfate. Therefore, the method for detecting a sulfated sugar chain of the present invention using a molecular probe into which a free thiol group has been introduced is a very simple and quick method because the detection principle is simple and only a short time is required. Therefore, it is possible to prevent separation of the target and the probe during the reaction due to the weak binding force.
  • the measurement of fluorescence intensity with a fluorescent labeling reagent is not only highly sensitive but also excellent in quantification, so by developing a structure-specific molecular probe, only sulfated sugar chains with a specific structure can be obtained.
  • a ripple effect is expected by the present invention, such as being able to measure quantitatively.
  • the detection result by the probe of the immobilized heparin sugar chain is shown (Example 1).
  • a calibration curve (10-3000 pmol) using L-cysteine is shown (Example 2).
  • a calibration curve (10-300 pmol) using L-cysteine is shown (Example 2).
  • the results of measurement of probe binding amount (constant heparin sugar chain amount) are shown (Example 3).
  • the measurement result of the binding amount of biotinylated heparin (constant probe amount) is shown (Example 4).
  • N- (9-acridinyl) maleimide is added, A method for detecting glycosaminoglycans comprising measuring the generated fluorescence is provided.
  • 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 in a bound state with 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 glycosaminoglycan to be detected may be present in a free state or may be in a state bound to other substances such as a core protein. Good.
  • the molecular probe used in the detection method of the present invention may be any one that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group, and specifically recognizes glycosaminoglycan.
  • the substance to be introduced may include those having a free thiol group introduced as necessary.
  • substances that specifically recognize glycosaminoglycans 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)
  • ⁇ CS-56 anti-chondroitin sulfate antibody; Avnur Z. and Geiger, B., Exp. Cell Res. 158, 321-332, 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.
  • a free thiol group When a free thiol group is introduced into a substance that specifically recognizes glycosaminoglycan, binding should be avoided so as not to interfere with the structure-specific binding force of the substance that specifically recognizes glycosaminoglycan. It may be introduced at a site that does not interfere (for example, when the substance that specifically recognizes glycosaminoglycan is a peptide or protein, 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, fluorescence is generated by the reaction with the fluorescent labeling reagent N- (9-acridinyl) maleimide, so that glycosaminoglycan bound to the molecular probe can be detected. it can. If the molecular probe is a protein or peptide and already contains a free thiol group, fluorescence detection can be performed using this thiol group, so there is no need to label the probe or introduce cysteine in advance. In that case, there is no need to consider the secondary effects associated with the labeling operation.
  • 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 glycosaminoglycan to be detected and the molecular probe are contacted so that the mass ratio is 1: 0.1 to 1: 100, preferably 1: 1 to 1:50, more preferably 1: 5 to 1:20. It is good to let them.
  • a solution in which the molecular probe is dissolved may be added to the sample.
  • the molecular probe is 0.01 to 100 nmol, preferably 0.05 to 50 nmol, more preferably 1 to 100 ⁇ L of the solution. ⁇ 10 It should be used after dissolving in the amount of nmol. Depending on the physical properties of the sample and the content and type of glycosaminoglycan, the amount to be used can be adjusted appropriately by diluting to an appropriate concentration with an appropriate solution such as phosphate buffered saline as necessary. 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 glycosaminoglycan (eg, heparin sugar chain) to be detected, but this has already been confirmed.
  • the molecular probe is brought into contact with the sample, 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 0.5 to
  • the binding reaction between the glycosaminoglycan in the sample and the molecular probe may be performed for 3 hours.
  • washing is performed to remove contaminants such as a free molecular probe (not bound to glycosaminoglycan) and a non-specifically bound molecular probe bound to glycosaminoglycan, and then N- ( 9-Acridinyl) maleimide (N- (9-acridinyl) maleimide (NAM)) is added. Washing may be performed 1 to 6 times with a salt (eg, NaCl) solution of about 0.1 to 2M, and further 1 to 5 times with a buffer solution (eg, phosphate buffer or Tris buffer).
  • a salt eg, NaCl
  • a buffer solution eg, phosphate buffer or Tris buffer
  • NAM is a compound represented by the following structural formula.
  • NAM itself is non-fluorescent, but reacts with SH compounds to emit very strong fluorescence (excitation wavelength: 365 nm, fluorescence wavelength: 435 nm) (Bunseki Kagaku (Japan Analyst), Vol. 22, 451-452 (1973)). NAM has a synthetic method described in Agric. Biol. Chem., 42 (4), 793-798, 1978, but is also commercially available.
  • NAM should be sufficiently saturated, and the ratio of NAM to molecular probe should be 1: 1 to 100: 1, preferably 1: 1 to 50: 1, more preferably 2: 1 to 10: 1. It is good to add so that.
  • NAM should be added after dissolving in a solution such as borate buffer.
  • NAM may be used after being dissolved in an amount of 0.05 to 2 ⁇ mM, preferably 0.1 to 1 ⁇ mM, more preferably 0.2 to 0.5 ⁇ mM with respect to 100 ⁇ L of the solution.
  • the boric acid concentration in the boric acid buffer is suitably 10-100 ⁇ mM, preferably 10-50 ⁇ mm, more preferably 20-50 ⁇ mM.
  • the reaction between NAM and the molecular probe is carried out under conditions of pH 3 to 10, preferably pH 7 to 10, more preferably pH 8 to 9 at 10 to 35 ° C., preferably 15 to 30 ° C., more preferably 20 in a light-shielded state.
  • the reaction may be performed at -25 ° C for 3-60 minutes, preferably 5-30 minutes, more preferably 5-10 minutes.
  • the detection method of the present invention is characterized by the use of a molecular probe having a free thiol group, which allows negatively charged molecules to react with NAM that emits strong blue fluorescence when reacted with a free thiol group. Fluorescence can be detected only depending on the probe without being affected. In addition, the time required for fluorescence detection after probe binding is as fast as 10 minutes, so even molecules with very weak binding force can be detected effectively, and even if the probe and target molecules are measured during fluorescence measurement. Even if is dissociated, it can be detected as the amount of bound probe, so that the amount of binding can be measured more precisely.
  • the detection method of the present invention can be used by either a competitive method or a non-competitive method.
  • the molecular probe is competed by competition between the glycosaminoglycan immobilized on the solid phase and the glycosaminoglycan in the sample.
  • a molecular probe bound to glycosaminoglycan with a high salt concentration (for example, 1 to 2 M) buffer solution for example, a phosphate buffer solution containing high concentration NaCl. Can be dissociated and washed, so that the immobilized glycosaminoglycan can be used any number of times.
  • glycosaminoglycan in a sample After fixing glycosaminoglycan in a sample to a solid phase, it is preferable to contact with a molecular probe, add NAM, and then measure the generated fluorescence. In this case, glycosaminoglycan in the sample can be directly detected. That is, a calibration curve is created with a molecular probe, and the amount of glycosaminoglycan in the sample can be determined from the amount of fluorescence.
  • glycosaminoglycans When immobilizing glycosaminoglycan in a sample on a solid phase, either proteoglycan purified using antibody affinity column chromatography or the like, or treated with sodium borohydride under alkaline conditions, glycosamino Glycans are released and purified by anion exchange column chromatography, and then immobilized on a solid phase by labeling such as biotinylation.
  • Examples of the solid phase include, but are not limited to, microplate wells, plastic tubes, beads, and the like.
  • Biotin-labeled glycosaminoglycan can be immobilized by coating the solid surface with streptavidin. Biotin labeling of glycosaminoglycan can be performed using a commercially available biotinylation reagent.
  • the glycosaminoglycan detection method using the fluorescent labeling reagent NAM can be applied to screening of substances that specifically bind to glycosaminoglycan, examination of diseases in which glycosaminoglycan is a diagnostic marker, and the like.
  • the present invention specifically binds to glycosaminoglycan, which comprises contacting N- (9-acridinyl) maleimide after contacting glycosaminoglycan with a test substance and measuring the generated fluorescence.
  • the screening method of the substance to be provided 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.
  • the substance obtained by the screening method of the present invention has a free thiol group.
  • the present invention adds N- (9-acridinyl) maleimide after contacting a sample with a molecular probe containing a substance having a free thiol group that can specifically bind to glycosaminoglycan. And a method for testing a disease in which glycosaminoglycan is a diagnostic marker, comprising measuring the generated fluorescence.
  • 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 joint disease.
  • 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 morbidity can be determined by comparing the amount of glycosaminoglycan in a sample from a subject (eg, serum, plasma, pleural effusion, ascites, urine, joint fluid, etc. collected from the subject) with a healthy subject. it can.
  • a subject eg, serum, plasma, pleural effusion, ascites, urine, joint fluid, etc. collected from the subject
  • the present invention also provides a glycosaminoglycan detection kit comprising a molecular probe containing a substance having a free thiol group that can specifically bind to glycosaminoglycan and N- (9-acridinyl) maleimide. To do.
  • the kit of the present invention may further contain glycosaminoglycan (standard product).
  • glycosaminoglycan standard product
  • a solid phase microplate, plastic tube, beads, etc.
  • streptavidin streptavidin
  • biotinylation reagent buffer solution
  • washing solution washing solution
  • frame, seal instruction manual
  • blocking agent blocking agent
  • Glycosaminoglycan standard product
  • a calibration curve and the like may be described.
  • the present invention specifically relates to glycosaminoglycans, including molecular probes that can bind specifically to glycosaminoglycans and include a substance having a free thiol group and N- (9-acridinyl) maleimide.
  • a screening kit for binding substances is also provided.
  • the kit of the present invention may further contain glycosaminoglycan (standard product).
  • glycosaminoglycan standard product
  • a solid phase microplate, plastic tube, beads, etc.
  • streptavidin streptavidin
  • biotinylation reagent buffer solution
  • washing solution washing solution
  • frame, seal instruction manual
  • blocking agent blocking agent
  • Glycosaminoglycan standard product
  • a calibration curve and the like may be described.
  • a glycosaminoglycan can be specifically bound to a glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide.
  • a disease testing kit is also provided.
  • the kit of the present invention may further contain glycosaminoglycan (standard product).
  • glycosaminoglycan standard product
  • a solid phase microplate, plastic tube, beads, etc.
  • streptavidin streptavidin
  • biotinylation reagent buffer solution
  • washing solution washing solution
  • frame, seal instruction manual
  • blocking agent blocking agent
  • the instruction manual may describe disease evaluation and / or differentiation criteria.
  • the present invention also provides a molecular probe comprising a substance that can specifically bind to glycosaminoglycan and has a free thiol group.
  • the molecular probe of the present invention can be used in the detection method, screening method and test method of the present invention.
  • Examples of the substance capable of specifically binding to glycosaminoglycan and having a free thiol group include the following peptides (a) and (b).
  • a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
  • It consists of amino acids in which one or several (about 2, 3, 4, 5, 6) amino acids are deleted, substituted, or added in the amino acid of SEQ ID NO: 1, and specifically binds to glycosaminoglycan And a peptide having a free thiol group.
  • the present inventor has confirmed that heparin can be bound if the first, third and seventh arginine (R) counted from the N-terminus are conserved.
  • -A peptide consisting of an amino acid sequence in which cysteine is added to the C-terminal of a continuous sequence consisting of amino acids 1 to 7 in the amino acid sequence of SEQ ID NO: 1-1st to 8th amino acids in the amino acid sequence of SEQ ID NO: 1
  • a peptide consisting of an amino acid sequence in which cysteine is added to the C terminus of a continuous sequence consisting of: from an amino acid sequence in which cysteine is added to the C terminus of a continuous sequence consisting of the first to ninth amino acids in the amino acid sequence of SEQ ID NO: 1 From the 1st to the 11th in the amino acid sequence of the peptide / SEQ ID NO: 1 consisting of the amino acid sequence consisting of the amino acid sequence in which cysteine is added to the C-terminal of the continuous sequence consisting of the 1st to 10th amino acids in the amino acid sequence of the SEQ ID NO: 1 System at the C-terminus of a contiguous sequence of amino acids Peptides of the
  • the present invention provides the above peptide (a) or (b).
  • the peptide of the present invention can be used as a molecular probe for the detection method, screening method and test method of the present invention.
  • a substance that specifically recognizes a target molecule can be used as a fluorescence detection probe simply by introducing a free thiol group such as a cysteine residue, thus affecting the structure of a known probe. Can be used without 2.
  • the detection limit of the molecular probe by NAM used in the detection method of the present invention is as high as about several pmol. 3.
  • NAM used as a fluorescent labeling reagent does not emit fluorescence unless it binds to a target substance having a free thiol group, it does not interfere with measurement values. 5.
  • the probe is labeled with a substance such as HRP, biotin, or FITC to detect the state of a complex bound to the target substance. The binding ability of the original probe to the target substance may be lost by the operation.
  • the detection method of the present invention since it is not necessary to label the molecular probe in advance, it is possible to prevent a phenomenon such as deactivation of the probe due to the labeling operation or inhibition of binding of the probe to the target substance by the labeling,
  • the present invention can be applied not only to sulfated sugar chains but also to in vivo negatively charged molecules that have been difficult to detect.
  • Example 1 Detection of immobilized heparin sugar chain by probe Experimental method Immobilization of heparin sugar chain Addition of ammonium formate and sodium cyanoborohydride to heparin sugar chain derived from porcine small intestine, and the reaction was repeated twice at 70 ° C for 2 days to reduce the reducing end of the sugar chain. An amino group was introduced into. The reaction solution was desalted and then biotin was introduced specifically for the amino group present at the reducing end of the sugar chain. The reaction solution was desalted and the target biotinylated sugar chain was purified by anion exchange column chromatography. This purified sugar chain was applied to a plastic plate coated with streptavidin, reacted at 4 ° C. overnight, and then washed thoroughly by adding phosphate buffered saline to obtain an immobilized heparin sugar chain.
  • probe etc. 4.23 nmol of probe was dissolved in 100 ⁇ l of phosphate buffered saline containing 2% bovine serum albumin and added to the immobilized heparin sugar chain. Similarly, 145 pmol of antithrombin III (AT-III), which is known to bind to heparin, was added.
  • AT-III antithrombin III
  • Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 ⁇ l of 300 mM NaCl per well, then use 2 ⁇ l of phosphate buffered saline with 200 ⁇ l per well). Wash), 0.2 mM NAM (N- (9-acridinyl) 100 ⁇ l of 50 mM borate buffer solution (pH 8.8) containing maleimide) was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm.
  • NAM emits strong blue fluorescence when it reacts with free thiol groups (SH groups), and the reaction proceeds quantitatively in aqueous solution at pH 3-10.
  • Probes with free thiol groups bound to immobilized heparin can be detected by NAM in this experimental system with a significant difference compared to a control with only solution containing bovine serum albumin that does not bind heparin. (Fig. 1).
  • Example 2 Calibration curve using L-cysteine Experimental method L-cysteine was dispensed stepwise from 1 pmol to 10 nmol in 96-well plates, and 50 mM borate buffer (pH 8.8) 100 containing 0.2 mM NAM (N- (9-acridinyl) maleimide). ⁇ l was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm. A calibration curve was prepared by plotting the fluorescence intensity against the amount of each L-cysteine.
  • Example 3 Measurement of probe binding amount (constant heparin sugar chain amount) Experimental method 1. Immobilization of heparin sugar chain A heparin sugar chain was immobilized in the same manner as in Example 1 "Detection of immobilized heparin sugar chain by probe”.
  • Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 ⁇ l of 300 mM NaCl per well, then use 2 ⁇ l of phosphate buffered saline with 200 ⁇ l per well). Washed twice), 100 ⁇ l of 50 mM borate buffer (pH 8.8) containing 0.2 mM NAM (N- (9-acridinyl) maleimide) was added and allowed to react for 20 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm.
  • Example 4 Measurement of binding amount of biotinylated heparin (probe amount constant) Experimental method 1. Immobilization of heparin sugar chain A biotinylated heparin sugar chain was purified in the same manner as in Example 1 “Detection of immobilized heparin sugar chain with probe” to prepare an aqueous solution. 0.5 ⁇ l, 1 ⁇ l, 2.5 ⁇ l, and 5 ⁇ l of this solution are diluted with phosphate buffered saline, each is applied to a plastic plate coated with streptavidin, reacted at 4 ° C. overnight, and then phosphate buffered. Saline was added and washed thoroughly to obtain immobilized heparin sugar chains.
  • probe 4.23 nmol of probe was dissolved in 100 ⁇ l of a solution containing 2% bovine serum albumin and added to the immobilized heparin sugar chain.
  • Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 ⁇ l of 300 mM NaCl per well, then use 2 ⁇ l of phosphate buffered saline with 200 ⁇ l per well). Wash), 0.2 mM NAM (N- (9-acridinyl) 100 ⁇ l of 50 mM borate buffer solution (pH 8.8) containing maleimide) was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm.
  • a probe that specifically recognizes the structure of a sulfated sugar chain related to a specific disease is developed based on the present invention. Furthermore, when the probe is used for disease diagnosis using a diagnostic marker, the sulfated sugar chain can be easily detected by the detection method of the present invention.
  • SEQ ID NO: 1 shows the amino acid sequence (RTRGSTREFRTGC) of a substance (peptide) that can specifically bind to glycosaminoglycan (heparin, heparan sulfate) and has a free thiol group.
  • SEQ ID NO: 2 shows the amino acid sequence (RTRGSTREFRTGC) of a substance (peptide) that can specifically bind to glycosaminoglycan (heparin, heparan sulfate) and has a free thiol group.
  • ⁇ SEQ ID NO: 2> SEQ ID NO: 2 glycosaminoglycan (heparin, heparan sulfate) specifically recognize peptides
  • HappY H eparin- a ssociated p e p tide Y
  • shows the base sequence of the DNA encoding CGGACGCGTGGGTCGACCCGGGAATTCCGGACCGGT).
  • SEQ ID NO: 3 is a peptide that specifically recognizes glycosaminoglycan (heparin, heparan sulfate), HappY ( H eparin- a ssociated p e p tide Y ) shows the amino acid sequence (RTRGSTREFRTG).

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Abstract

Disclosed are: a molecular probe which can overcome the disadvantages of the conventional techniques; and a method for detecting glycosaminoglycan using the molecular probe. Specifically disclosed are: a method for detecting glycosaminoglycan, which comprises bringing a molecular probe that comprises a substance capable of binding to glycosaminoglycan specifically and having a free thiol group into contact with a sample, adding N-(9-acridinyl)maleimide to the resulting mixture to generate a fluorescence, and measuring the fluorescence; a glycosaminoglycan detection kit which comprises a molecular probe that comprises a substance capable of binding to glycosaminoglycan specifically and having a free thiol group and N-(9-acridinyl)maleimide; a method for screening for a substance capable of binding to glycosaminoglycan specifically, and a kit for use in the method; and a method for diagnosing a disease for which glycosaminoglycan can be used as a diagnosis marker, and a kit, a molecular probe and a peptide for use in the method.

Description

グリコサミノグリカンの検出法及びそのための分子プローブDetection method of glycosaminoglycan and molecular probe therefor
　本発明は、グリコサミノグリカンの検出法及びそのための分子プローブに関し、より詳細には、蛍光標識試薬によるグリコサミノグリカンの検出法及びそのための分子プローブに関する。 The present invention relates to a method for detecting glycosaminoglycan and a molecular probe therefor, and more particularly to a method for detecting glycosaminoglycan using a fluorescent labeling reagent and a molecular probe therefor.
　近年、生体内機能制御因子として注目されているヘパラン硫酸やコンドロイチン硫酸などのグリコサミノグリカン糖鎖（硫酸化糖鎖）は、構成する酸性糖（ウロン酸）や硫酸基のために、負電荷を有する生体内高分子として知られている。このような生体内負電荷分子は、特異的にこれを認識する一次抗体と標識化二次抗体を用いた一般的な検出法では、標識物質が非特異的に負電荷分子と相互作用してしまうなどするために、信頼性のある検出が困難である。 In recent years, glycosaminoglycan sugar chains (sulfated sugar chains) such as heparan sulfate and chondroitin sulfate, which are attracting attention as functional regulators in vivo, are negatively charged due to the acidic sugar (uronic acid) and sulfate groups that they make up. It is known as an in vivo polymer having Such a negative charge molecule in a living body is a non-specific interaction with a negatively charged molecule in a general detection method using a primary antibody and a labeled secondary antibody that specifically recognize this molecule. Therefore, reliable detection is difficult.
　グリコサミノグリカン糖鎖を特異的に認識するプローブの開発研究に関する学術論文報告も増加している（非特許文献１）。しかし、これらの従来技術ではスクリーニングしたいプローブをひとつひとつ標識しなければならず、また、硫酸化糖鎖に対して影響を及ぼさない標識を使用するという制限が存在した。 There are an increasing number of academic paper reports on the development of probes that specifically recognize glycosaminoglycan sugar chains (Non-patent Document 1). However, in these conventional techniques, the probes to be screened must be labeled one by one, and there is a limitation that labels that do not affect sulfated sugar chains are used.
　また、蛍光標識試薬であるN-(9-acridinyl) maleimide（NAM）をSH化合物の高感度の検出に用いたことが報告されている（非特許文献２～４）が、NAMを硫酸化糖鎖の検出に利用したという報告はなされていない。 It has also been reported that N- (9-acridinyl) ylmaleimide (NAM), a fluorescent labeling reagent, was used for highly sensitive detection of SH compounds (Non-Patent Documents 2 to 4). There has been no report that it was used for strand detection.
　本発明は、従来技術が持つ以下の問題点を解決できる分子プローブとそれを用いたグリコサミノグリカンの検出法を提供することを目的とする。 An object of the present invention is to provide a molecular probe capable of solving the following problems of the prior art and a method for detecting glycosaminoglycan using the same.
１．硫酸化糖鎖に非特異的に検出試薬が結合することによる検出誤差を生じる。
２．標的分子とプローブの結合力が微小な場合の複合体の検出が困難である。
３．スクリーニング時にプローブをあらかじめ標識する必要性がある。
４．標識操作によるプローブの失活や標的物質への結合力を阻害するリスクがある。 1. A detection error is caused by nonspecific binding of the detection reagent to the sulfated sugar chain.
2. It is difficult to detect the complex when the binding force between the target molecule and the probe is very small.
3. There is a need to label the probe in advance during screening.
4). There is a risk of deactivation of the probe due to the labeling operation and inhibition of binding force to the target substance.
　本発明者は、ヘパリン糖鎖が固着した固相に、分子プローブとして、ヘパリン糖鎖を特異的に認識するペプチド分子（アミノ酸配列：RTRGSTREFRTG）（配列番号３））（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）に遊離のチオール基を持つシステイン残基を導入したものを添加して、ヘパリン糖鎖と分子プローブの結合反応を行わせた後、蛍光標識試薬であるN-(9-acridinyl) maleimide（NAM）を加えて分子プローブ中の遊離のチオール基と反応させ、蛍光検出器により、365 nmで励起し、435 nmの蛍光を検出することによって、ヘパリン糖鎖を選択的に検出することに成功し、本発明を完成させるに到った。 The present inventors have to a solid phase heparin sugar chains are fixed, as a molecular probe, the heparin oligosaccharides which specifically recognize the peptide molecules (amino acid sequence: RTRGSTREFRTG) (SEQ ID NO: 3)) (HappY: H eparin- a ssociated p e p tide Y , August 19th, 2008, 28th Annual Meeting of the Glycologic Society of Japan (Tsukuba) Oral Presentations and Abstracts p.79) with the addition of a cysteine residue with a free thiol group After the binding reaction of heparin sugar chain and molecular probe, the fluorescent labeling reagent N- (9-acridinyl) maleimide (NAM) is added and reacted with the free thiol group in the molecular probe to detect fluorescence By exciting with a vessel at 365 nm and detecting fluorescence at 435 nm, he succeeded in selectively detecting heparin sugar chains and completed the present invention.
　本発明の要旨は以下の通りである。 The gist of the present invention is as follows.
（１）グリコサミノグリカン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物質を含む分子プローブと試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンの検出法。
（２）固相に固定されているグリコサミノグリカンの存在下で、前記分子プローブと前記試料を接触させる（１）記載の検出法。
（３）前記試料中のグリコサミノグリカンを固相に固定した後、前記分子プローブと接触させる（１）記載の検出法。
（４）グリコサミノグリカンを被験物質と接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンに特異的に結合する物質のスクリーニング法。
（５）グリコサミノグリカン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物質を含む分子プローブと被験者からの試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査法。
（６）グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカン検出キット。
（７）さらに、グリコサミノグリカンを含む（６）記載のキット。
（８）グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカンに特異的に結合する物質のスクリーニングキット。
（９）さらに、グリコサミノグリカンを含む（８）記載のキット。
（１０）グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査キット。
（１１）さらに、グリコサミノグリカンを含む（１０）記載のキット。
（１２）グリコサミノグリカン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物質を含む分子プローブであって、（１）記載の検出法、（４）記載のスクリーニング方法又は（５）記載の検査法に使用される前記分子プローブ。
（１３）グリコサミノグリカン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物質が、以下の(a)又は(b)のペプチドである（１２）記載の分子プローブ。
(a)配列番号１のアミノ酸配列（RTRGSTREFRTGC）からなるペプチド。
(b) 配列番号１のアミノ酸において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、グリコサミノグリカン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ペプチド。
（１４）以下の(a)又は(b)のペプチド。
(a)配列番号１のアミノ酸配列（RTRGSTREFRTGC）からなるペプチド。
(b) 配列番号１のアミノ酸において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、グリコサミノグリカン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ペプチド。 (1) After contacting a sample with a molecular probe that can bind specifically to glycosaminoglycan and contains a substance having a free thiol group, N- (9-acridinyl) maleimide is added to generate A method for detecting glycosaminoglycans, comprising measuring measured fluorescence.
(2) The detection method according to (1), wherein the molecular probe and the sample are contacted in the presence of glycosaminoglycan immobilized on a solid phase.
(3) The detection method according to (1), wherein glycosaminoglycan in the sample is immobilized on a solid phase and then contacted with the molecular probe.
(4) A substance that specifically binds to glycosaminoglycan, comprising contacting N- (9-acridinyl) maleimide after contacting glycosaminoglycan with a test substance and measuring the generated fluorescence. Screening method.
(5) N- (9-acridinyl) maleimide is added after contacting a sample from a subject with a molecular probe that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group And a method for testing a disease in which glycosaminoglycan is a diagnostic marker, comprising measuring the generated fluorescence.
(6) A glycosaminoglycan detection kit comprising a molecular probe that can specifically bind to glycosaminoglycan and includes a substance having a free thiol group and N- (9-acridinyl) maleimide.
(7) The kit according to (6), further comprising glycosaminoglycan.
(8) Specific binding to glycosaminoglycan, which can specifically bind to glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide Screening kit for substances to be used.
(9) The kit according to (8), further comprising glycosaminoglycan.
(10) Glycosaminoglycan that can specifically bind to glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide serves as a diagnostic marker Disease testing kit.
(11) The kit according to (10), further comprising glycosaminoglycan.
(12) A molecular probe comprising a substance capable of specifically binding to glycosaminoglycan and having a free thiol group, the detection method according to (1), the screening method according to (4), or ( 5) The molecular probe used in the test method according to 5).
(13) The molecular probe according to (12), wherein the substance capable of specifically binding to glycosaminoglycan and having a free thiol group is the following peptide (a) or (b):
(a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
(b) a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
(14) The following peptide (a) or (b):
(a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
(b) a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
　糖鎖と結合する分子プローブは一般に結合力が弱いうえ、硫酸化糖鎖はその負電荷によってさらに反応性に制限があるため、ヘパラン硫酸のような硫酸化糖鎖の検出は非常に難しい。そこで、遊離のチオール基を導入した分子プローブを用いた本発明の硫酸化糖鎖の検出法は、検出原理が単純であるがゆえに短時間の操作で済み、非常に簡便かつ迅速な方法であり、ゆえに結合力の弱さに由来する反応途中での標的とプローブの剥離を防ぐことができる。また、蛍光標識試薬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など、本発明により波及効果が期待される。 A molecular probe that binds to a sugar chain generally has a weak binding force, and the sulfated sugar chain is further limited in its reactivity due to its negative charge. Therefore, it is very difficult to detect a sulfated sugar chain such as heparan sulfate. Therefore, the method for detecting a sulfated sugar chain of the present invention using a molecular probe into which a free thiol group has been introduced is a very simple and quick method because the detection principle is simple and only a short time is required. Therefore, it is possible to prevent separation of the target and the probe during the reaction due to the weak binding force. In addition, the measurement of fluorescence intensity with a fluorescent labeling reagent is not only highly sensitive but also excellent in quantification, so by developing a structure-specific molecular probe, only sulfated sugar chains with a specific structure can be obtained. A ripple effect is expected by the present invention, such as being able to measure quantitatively.
　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2009‐243995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。 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2009-243995, which is the basis of the priority of the present application.
固定化ヘパリン糖鎖のプローブによる検出結果を示す（実施例１）。The detection result by the probe of the immobilized heparin sugar chain is shown (Example 1). L-システインを用いた検量線（10-3000 pmol）を示す（実施例２）。A calibration curve (10-3000 pmol) using L-cysteine is shown (Example 2). L-システインを用いた検量線（10-300 pmol）を示す（実施例２）。A calibration curve (10-300 pmol) using L-cysteine is shown (Example 2). プローブの結合量の測定（ヘパリン糖鎖量一定）結果を示す（実施例３）。The results of measurement of probe binding amount (constant heparin sugar chain amount) are shown (Example 3). ビオチン化ヘパリンの結合量の測定（プローブ量一定）結果を示す（実施例４）。The measurement result of the binding amount of biotinylated heparin (constant probe amount) is shown (Example 4).
　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、グリコサミノグリカン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物質を含む分子プローブと試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンの検出法を提供する。 In the present invention, after contacting a sample with a molecular probe that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group, N- (9-acridinyl) maleimide is added, A method for detecting glycosaminoglycans comprising measuring the generated fluorescence is provided.
　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 in a bound state with 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 glycosaminoglycan to be detected may be present in a free state or may be in a state bound to other substances such as a core protein. Good.
　本発明の検出方法に用いる分子プローブは、グリコサミノグリカン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物質を含むものであればよく、グリコサミノグリカン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したものなどを例示することができる。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。 The molecular probe used in the detection method of the present invention may be any one that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group, and specifically recognizes glycosaminoglycan. Examples of the substance to be introduced may include those having a free thiol group introduced as necessary. Examples of substances that specifically recognize glycosaminoglycans include the following.
・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） 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)
・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。 ・ CS-56 (anti-chondroitin sulfate antibody; Avnur Z. and Geiger, B., Exp. Cell Res. 158, 321-332, 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).
・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。 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、グリコサミノグリカン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、グリコサミノグリカン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 glycosaminoglycan, binding should be avoided so as not to interfere with the structure-specific binding force of the substance that specifically recognizes glycosaminoglycan. It may be introduced at a site that does not interfere (for example, when the substance that specifically recognizes glycosaminoglycan is a peptide or protein, 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、蛍光標識試薬のN-(9-アクリジニル)マレイミドとの反応により、蛍光が生じるので、分子プローブと結合したグリコサミノグリカンを検出することができる。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蛍光検出できるため、あらかじめプローブを標識したり、システインを導入する必要がなく、その場合、標識操作に伴う二次的な影響は考慮する必要がない。 If the molecular probe has a free thiol group, fluorescence is generated by the reaction with the fluorescent labeling reagent N- (9-acridinyl) maleimide, so that glycosaminoglycan bound to the molecular probe can be detected. it can. If the molecular probe is a protein or peptide and already contains a free thiol group, fluorescence detection can be performed using this thiol group, so there is no need to label the probe or introduce cysteine in advance. In that case, there is no need to consider the secondary effects associated with the labeling operation.
　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.
　検出の対象となるグリコサミノグリカンと分子プローブは、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 glycosaminoglycan to be detected and the molecular probe are contacted so that the mass ratio is 1: 0.1 to 1: 100, preferably 1: 1 to 1:50, more preferably 1: 5 to 1:20. It is good to let them.
　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、その物性やグリコサミノグリカン含有量・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グリコサミノグリカン（例えば、ヘパリン糖鎖）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、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. The molecular probe is 0.01 to 100 nmol, preferably 0.05 to 50 nmol, more preferably 1 to 100 μL of the solution. ~ 10   It should be used after dissolving in the amount of nmol. Depending on the physical properties of the sample and the content and type of glycosaminoglycan, the amount to be used can be adjusted appropriately by diluting to an appropriate concentration with an appropriate solution such as phosphate buffered saline as necessary. 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 glycosaminoglycan (eg, heparin sugar chain) to be detected, but this has already been confirmed.
　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、試料中のグリコサミノグリカンと分子プローブとの結合反応を行わせるとよい。 The molecular probe is brought into contact with the sample, 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 0.5 to The binding reaction between the glycosaminoglycan in the sample and the molecular probe may be performed for 3 hours.
　その後、必要により、洗浄を行い、遊離の（グリコサミノグリカンと結合していない）分子プローブ、非特異的にグリコサミノグリカンに結合した分子プローブなどの夾雑物を除去した後、N-(9-アクリジニル)マレイミド（N-(9-acridinyl) maleimide（NAM））を添加する。洗浄は、0.1～2M程度の塩（例えば、NaCl）溶液で１～6回、さらに、緩衝液（例えば、リン酸緩衝液、トリス緩衝液）で１～5回行なうとよい。 Thereafter, if necessary, washing is performed to remove contaminants such as a free molecular probe (not bound to glycosaminoglycan) and a non-specifically bound molecular probe bound to glycosaminoglycan, and then N- ( 9-Acridinyl) maleimide (N- (9-acridinyl) maleimide (NAM)) is added. Washing may be performed 1 to 6 times with a salt (eg, NaCl) solution of about 0.1 to 2M, and further 1 to 5 times with a buffer solution (eg, phosphate buffer or Tris buffer).
　NAMは、以下の構造式で表される化合物である。 NAM is a compound represented by the following structural formula.
Figure JPOXMLDOC01-appb-C000001
 　NAMは、それ自体無蛍光であるが、SH化合物と反応して、非常に強い蛍光（励起波長：365  nm、蛍光波長：435 nm）を発する（Bunseki Kagaku (Japan Analyst), Vol. 22,
451-452(1973)）。NAMは、Agric. Biol. Chem., 42(4), 793-798, 1978に合成法が記載されているが、市販もされおり、入手可能である。
Figure JPOXMLDOC01-appb-C000001
 NAM itself is non-fluorescent, but reacts with SH compounds to emit very strong fluorescence (excitation wavelength: 365 nm, fluorescence wavelength: 435 nm) (Bunseki Kagaku (Japan Analyst), Vol. 22,
451-452 (1973)). NAM has a synthetic method described in Agric. Biol. Chem., 42 (4), 793-798, 1978, but is also commercially available.
　NAMは十分飽和量を加えるとよく、NAMと分子プローブの比が、1：1～100：1、好ましくは1：1～50：1、より好ましくは2：1～10：1の質量比となるように添加するとよい。 NAM should be sufficiently saturated, and the ratio of NAM to molecular probe should be 1: 1 to 100: 1, preferably 1: 1 to 50: 1, more preferably 2: 1 to 10: 1. It is good to add so that.
　NAMは、硼酸緩衝液などの溶液に溶解して、添加するとよい。NAMは、溶液100μLに対して、0.05～2 mM、好ましくは0.1～1 mM、より好ましくは0.2～0.5 mMの量で溶解して、使用するとよい。硼酸緩衝液の硼酸濃度は、10～100 mMが適当であり、10～50 mMが好ましく、20～50 mMがより好ましい。 NAM should be added after dissolving in a solution such as borate buffer. NAM may be used after being dissolved in an amount of 0.05 to 2 μmM, preferably 0.1 to 1 μmM, more preferably 0.2 to 0.5 μmM with respect to 100 μL of the solution. The boric acid concentration in the boric acid buffer is suitably 10-100 μmM, preferably 10-50 μmm, more preferably 20-50 μmM.
　NAMと分子プローブとの反応は、遮光した状態で、pH3～10、好ましくはpH7～10、より好ましくはpH8～9の条件で、10～35℃、好ましくは15～30℃、より好ましくは20～25℃で、3～60分間、好ましくは5～30分間、より好ましくは5～10分間、行うとよい。 The reaction between NAM and the molecular probe is carried out under conditions of pH 3 to 10, preferably pH 7 to 10, more preferably pH 8 to 9 at 10 to 35 ° C., preferably 15 to 30 ° C., more preferably 20 in a light-shielded state. The reaction may be performed at -25 ° C for 3-60 minutes, preferably 5-30 minutes, more preferably 5-10 minutes.
　NAMと分子プローブとの反応後、発生した蛍光を測定する。蛍光の測定は、蛍光検出器により、355-365 nmで励起し、435-460 nmの蛍光を検出するとよい。 Measure the fluorescence generated after the reaction between NAM and molecular probe. For the measurement of fluorescence, it is preferable to detect fluorescence at 435 to 460 nm by exciting with a fluorescence detector at 355 to 365 nm.
　本発明の検出法では、分子プローブとして遊離のチオール基を有するものを使用することが特徴であり、これにより、遊離のチオール基と反応すると強い青色蛍光を発するNAMを介して、負電荷分子に影響されることなく、プローブにのみ応じて蛍光を検出することができる。また、プローブ結合後から蛍光検出までの所要時間は、10分程度と迅速なため、非常に弱い結合力の分子同士であっても、効果的に検出できるうえ、たとえ蛍光測定時にプローブと標的分子が解離していても、結合していたプローブ量としての検出が可能であるので、結合量をより厳密に測定できる。 The detection method of the present invention is characterized by the use of a molecular probe having a free thiol group, which allows negatively charged molecules to react with NAM that emits strong blue fluorescence when reacted with a free thiol group. Fluorescence can be detected only depending on the probe without being affected. In addition, the time required for fluorescence detection after probe binding is as fast as 10 minutes, so even molecules with very weak binding force can be detected effectively, and even if the probe and target molecules are measured during fluorescence measurement. Even if is dissociated, it can be detected as the amount of bound probe, so that the amount of binding can be measured more precisely.
　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。 The detection method of the present invention can be used by either a competitive method or a non-competitive method.
　競合法で用いる場合には、固相に固定されているグリコサミノグリカンの存在下で、分子プローブと試料を接触させた後、NAMを添加し、発生した蛍光を測定するとよい。固相に固定するグリコサミノグリカンは、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、固相に固定されたグリコサミノグリカンと試料中のグリコサミノグリカンとの競合により、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グリコサミノグリカンに作用させた後、遊離の分子プローブと、試料中のグリコサミノグリカン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グリコサミノグリカン量を求めることができる。本発明の分子プローブのメリットの一つとして、高塩濃度（例えば、1～2 M）の緩衝液（例えば、高濃度のNaClを含むリン酸緩衝液）でグリコサミノグリカンと結合した分子プローブを解離させ、洗浄することができるので、何度でも固定化したグリコサミノグリカンを使用できる。 When used in the competitive method, it is advisable to measure the generated fluorescence by adding NAM after contacting the sample with a molecular probe in the presence of glycosaminoglycan immobilized on a solid phase. The glycosaminoglycan immobilized on the solid phase is sufficient if it is immobilized at a fixed amount, even if it is not a known amount. In the case of the competitive method, the molecular probe is competed by competition between the glycosaminoglycan immobilized on the solid phase and the glycosaminoglycan in the sample. After allowing the sample and the molecular probe to act on the immobilized glycosaminoglycan, the free molecular probe and the molecular probe bound to the glycosaminoglycan in the sample are removed by washing. A calibration curve is created with a molecular probe, and the amount of glycosaminoglycan in the sample can be determined from the amount of decrease in fluorescence due to competition. As one of the merits of the molecular probe of the present invention, a molecular probe bound to glycosaminoglycan with a high salt concentration (for example, 1 to 2 M) buffer solution (for example, a phosphate buffer solution containing high concentration NaCl). Can be dissociated and washed, so that the immobilized glycosaminoglycan can be used any number of times.
　非競合法で用いる場合には、試料中のグリコサミノグリカンを固相に固定した後、分子プローブと接触させ、その後、NAMを添加し、発生した蛍光を測定するとよい。この場合、試料中のグリコサミノグリカン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グリコサミノグリカン量を求めることができる。 When using in a non-competitive method, after fixing glycosaminoglycan in a sample to a solid phase, it is preferable to contact with a molecular probe, add NAM, and then measure the generated fluorescence. In this case, glycosaminoglycan in the sample can be directly detected. That is, a calibration curve is created with a molecular probe, and the amount of glycosaminoglycan in the sample can be determined from the amount of fluorescence.
　試料中のグリコサミノグリカンを固相に固定する際に，プロテオグリカンとして抗体アフィニティーカラムクロマトグラフィーなどを利用して精製したものを用いるか，または，アルカリ性下で水素化ホウ素ナトリウム処理してグリコサミノグリカンを遊離し，陰イオン交換カラムクロマトグラフィーにより精製した後，ビオチン化などの標識化によって固相に固定するとよい。 When immobilizing glycosaminoglycan in a sample on a solid phase, either proteoglycan purified using antibody affinity column chromatography or the like, or treated with sodium borohydride under alkaline conditions, glycosamino Glycans are released and purified by anion exchange column chromatography, and then immobilized on a solid phase by labeling such as biotinylation.
　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、ビオチン標識したグリコサミノグリカンを固定できる。グリコサミノグリカン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. Biotin-labeled glycosaminoglycan can be immobilized by coating the solid surface with streptavidin. Biotin labeling of glycosaminoglycan can be performed using a commercially available biotinylation reagent.
　蛍光標識試薬NAMを利用するグリコサミノグリカン検出法は、グリコサミノグリカンに特異的に結合する物質のスクリーニング、グリコサミノグリカン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 glycosaminoglycan detection method using the fluorescent labeling reagent NAM can be applied to screening of substances that specifically bind to glycosaminoglycan, examination of diseases in which glycosaminoglycan is a diagnostic marker, and the like.
　従って、本発明は、グリコサミノグリカンを被験物質と接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカン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。 Accordingly, the present invention specifically binds to glycosaminoglycan, which comprises contacting N- (9-acridinyl) maleimide after contacting glycosaminoglycan with a test substance and measuring the generated fluorescence. The screening method of the substance to be provided 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。本発明のスクリーニング法により得られる物質は、遊離のチオール基を有するものとなる。 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. The substance obtained by the screening method of the present invention has a free thiol group.
　また、本発明は、グリコサミノグリカン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物質を含む分子プローブと試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査法を提供する。 Further, the present invention adds N- (9-acridinyl) maleimide after contacting a sample with a molecular probe containing a substance having a free thiol group that can specifically bind to glycosaminoglycan. And a method for testing a disease in which glycosaminoglycan is a diagnostic marker, comprising measuring the generated fluorescence.
　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）中のグリコサミノグリカン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 joint disease. 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 morbidity can be determined by comparing the amount of glycosaminoglycan in a sample from a subject (eg, serum, plasma, pleural effusion, ascites, urine, joint fluid, etc. collected from the subject) with a healthy subject. it can.
　本発明は、グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカン検出キットも提供する。 The present invention also provides a glycosaminoglycan detection kit comprising a molecular probe containing a substance having a free thiol group that can specifically bind to glycosaminoglycan and N- (9-acridinyl) maleimide. To do.
　本発明のキットは、さらに、グリコサミノグリカン（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、グリコサミノグリカン（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 glycosaminoglycan (standard product). 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. Glycosaminoglycan (standard product) 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、グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカン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。 The present invention specifically relates to glycosaminoglycans, including molecular probes that can bind specifically to glycosaminoglycans and include a substance having a free thiol group and N- (9-acridinyl) maleimide. A screening kit for binding substances is also provided.
　本発明のキットは、さらに、グリコサミノグリカン（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、グリコサミノグリカン（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 glycosaminoglycan (standard product). 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. Glycosaminoglycan (standard product) 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、グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査キットも提供する。 In the present invention, a glycosaminoglycan can be specifically bound to a glycosaminoglycan and includes a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide. A disease testing kit is also provided.
　本発明のキットは、さらに、グリコサミノグリカン（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、グリコサミノグリカン（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 kit of the present invention may further contain glycosaminoglycan (standard product). 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. Glycosaminoglycan (standard product) 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.
　また、本発明は、グリコサミノグリカン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物質を含む分子プローブを提供する。本発明の分子プローブは、本発明の検出法、スクリーニング法及び検査法に使用することができる。グリコサミノグリカン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物質としては、以下の(a)又は(b)のペプチドを例示することができる。 The present invention also provides a molecular probe comprising a substance that can specifically bind to glycosaminoglycan and has a free thiol group. The molecular probe of the present invention can be used in the detection method, screening method and test method of the present invention. Examples of the substance capable of specifically binding to glycosaminoglycan and having a free thiol group include the following peptides (a) and (b).
(a)配列番号１のアミノ酸配列（RTRGSTREFRTGC）からなるペプチド。
(b) 配列番号１のアミノ酸において１若しくは数個（２、３、４、５、６個程度）のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、グリコサミノグリカン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ペプチド。 (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
(b) It consists of amino acids in which one or several (about 2, 3, 4, 5, 6) amino acids are deleted, substituted, or added in the amino acid of SEQ ID NO: 1, and specifically binds to glycosaminoglycan And a peptide having a free thiol group.
　本発明者は、N末端から数えて１番目、３番目及び７番目のアルギニン(R)が保存されていれば、ヘパリンと結合できることを確認している。 The present inventor has confirmed that heparin can be bound if the first, third and seventh arginine (R) counted from the N-terminus are conserved.
　従って、(b)のペプチドとしては、以下のものを例示することができる。 Therefore, the following can be exemplified as the peptide of (b).
・配列番号１のアミノ酸配列における１番目から７番目までのアミノ酸からなる連続した配列のC末端にシステインを付加したアミノ酸配列からなるペプチド
・配列番号１のアミノ酸配列における１番目から８番目までのアミノ酸からなる連続した配列のC末端にシステインを付加したアミノ酸配列からなるペプチド
・配列番号１のアミノ酸配列における１番目から９番目までのアミノ酸からなる連続した配列のC末端にシステインを付加したアミノ酸配列からなるペプチド
・配列番号１のアミノ酸配列における１番目から１０番目までのアミノ酸からなる連続した配列のC末端にシステインを付加したアミノ酸配列からなるペプチド
・配列番号１のアミノ酸配列における１番目から１１番目までのアミノ酸からなる連続した配列のC末端にシステインを付加したアミノ酸配列からなるペプチド
　(a)及び(b)のペプチドは、公知の化学合成法、遺伝子工学的手法により、製造することができる。 -A peptide consisting of an amino acid sequence in which cysteine is added to the C-terminal of a continuous sequence consisting of amino acids 1 to 7 in the amino acid sequence of SEQ ID NO: 1-1st to 8th amino acids in the amino acid sequence of SEQ ID NO: 1 A peptide consisting of an amino acid sequence in which cysteine is added to the C terminus of a continuous sequence consisting of: from an amino acid sequence in which cysteine is added to the C terminus of a continuous sequence consisting of the first to ninth amino acids in the amino acid sequence of SEQ ID NO: 1 From the 1st to the 11th in the amino acid sequence of the peptide / SEQ ID NO: 1 consisting of the amino acid sequence consisting of the amino acid sequence in which cysteine is added to the C-terminal of the continuous sequence consisting of the 1st to 10th amino acids in the amino acid sequence of the SEQ ID NO: 1 System at the C-terminus of a contiguous sequence of amino acids Peptides of the peptide consisting of the amino acid sequence obtained by adding the emission (a) and (b), known methods of chemical synthesis, by genetic engineering techniques, can be produced.
　さらに、本発明は、上記の(a)又は(b)のペプチドを提供する。本発明のペプチドは、本発明の検出法、スクリーニング法及び検査法などの分子プローブとして使用することができる。 Furthermore, the present invention provides the above peptide (a) or (b). The peptide of the present invention can be used as a molecular probe for the detection method, screening method and test method of the present invention.
　本発明の利点を以下に列挙する。 The advantages of the present invention are listed below.
１．標的分子を特異的に認識する物質（分子プローブ）に、システイン残基のような遊離のチオール基を導入するだけで蛍光検出プローブとして使用できるようになるため、既知のプローブの構造に影響を与えることなく利用できる。
２．本発明の検出法で使用するNAMによる分子プローブの検出限界は、数pmol程度と非常に感度が高い。
３．プローブ結合から蛍光による検出までの操作手順が少なく、簡便かつ迅速な検出が可能である。このことは、ELISA法やウェスタンブロッティング法のような一般的な免疫生化学的方法では、操作の過程でプローブが標的である硫酸化糖鎖から剥離してしまう可能性を最小限に抑える効果がある。
４．蛍光標識試薬として使用するNAMは、遊離のチオール基を有する対象物質と結合しなければ蛍光を発しないため、測定値を干渉することがない。
５．一般的に，標的物質を可視化する方法としてプローブを使用する場合に、プローブをHRPやビオチン、FITCなどの物質によって標識をして、標的物質と結合した複合体の状態を検出するが、しばしば標識操作により、本来のプローブの標的物質への結合能が失われてしまうことがある。しかし、本発明の検出法では、分子プローブをあらかじめ標識する必要がないため、標識操作によるプローブの失活や、標識によるプローブの標的物質への結合を阻害するなどの現象を防ぐことができ、硫酸化糖鎖に限らず、これまで検出が困難であった生体内負電荷分子に対しても応用が可能である。 1. A substance that specifically recognizes a target molecule (molecular probe) can be used as a fluorescence detection probe simply by introducing a free thiol group such as a cysteine residue, thus affecting the structure of a known probe. Can be used without
2. The detection limit of the molecular probe by NAM used in the detection method of the present invention is as high as about several pmol.
3. There are few operation procedures from probe binding to fluorescence detection, and simple and rapid detection is possible. This means that in general immunobiochemical methods such as ELISA and Western blotting, the effect of minimizing the possibility of the probe detaching from the target sulfated glycan during the operation is effective. is there.
4). Since NAM used as a fluorescent labeling reagent does not emit fluorescence unless it binds to a target substance having a free thiol group, it does not interfere with measurement values.
5. In general, when a probe is used as a method for visualizing a target substance, the probe is labeled with a substance such as HRP, biotin, or FITC to detect the state of a complex bound to the target substance. The binding ability of the original probe to the target substance may be lost by the operation. However, in the detection method of the present invention, since it is not necessary to label the molecular probe in advance, it is possible to prevent a phenomenon such as deactivation of the probe due to the labeling operation or inhibition of binding of the probe to the target substance by the labeling, The present invention can be applied not only to sulfated sugar chains but also to in vivo negatively charged molecules that have been difficult to detect.
　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。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
（実験材料）
ブタ小腸由来ヘパリン糖鎖：ナカライテスク，Acros Organics（アメリカ）；アンチトロンビンIII：Hyphen BioMed（フランス）；NAM：東京化成工業；マウス脳由来cDNAライブラリー：Spring Bioscience（アメリカ）
（プローブの作製方法）
　アンチトロンビンIII（AT-III）アフィニティーカラムを作製した後，ブタ小腸由来ヘパリンを供して，AT-III結合型ヘパリン糖鎖を精製し，得られたヘパリンをビオチン化した後にストレプトアビジンプレートに固定化した。この固定化糖鎖を標的として，マウス脳由来cDNAライブラリーを導入したファージディスプレイ法（2008年8月19日第28回日本糖質学会年会（つくば）要旨集p.79）により，これと相互作用するペプチドをスクリーニングし，得られた分子をプローブとした。得られた分子は、HappY（Heparin-associated
peptide Y）と命名され、そ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。 (Experimental material)
Porcine small intestine derived heparin sugar chain: Nacalai Tesque, Acros Organics (USA); Antithrombin III: Hyphen BioMed (France); NAM: Tokyo Chemical Industry; Mouse brain-derived cDNA library: Spring Bioscience (USA)
(Probing method)
After preparing an antithrombin III (AT-III) affinity column, subject it to porcine small intestine-derived heparin, purify the AT-III-linked heparin sugar chain, biotinylate the resulting heparin, and then immobilize it on the streptavidin plate did. Using this immobilized sugar chain as a target, a phage display method in which a mouse brain-derived cDNA library was introduced (August 19, 2008, The 28th Annual Meeting of the Carbohydrate Society of Japan (Tsukuba) p. 79) The interacting peptides were screened and the resulting molecules were used as probes. The resulting molecule is HappY ( H eparin- a ssociated
p e p tide Y ), the amino acid sequence of which is shown in SEQ ID NO: 3, and the base sequence of the DNA encoding it is shown in SEQ ID NO: 2.
〔実施例１〕固定化ヘパリン糖鎖のプローブによる検出
実験方法
ヘパリン糖鎖の固定化
　ブタ小腸由来のヘパリン糖鎖に対してギ酸アンモニウムと水素化シアノホウ素ナトリウムを加え，70℃で２日間の反応を２度繰り返し，糖鎖の還元末端にアミノ基を導入した。反応液は脱塩操作を行った後，糖鎖の還元末端に存在するアミノ基特異的にビオチンを導入した。反応液は脱塩操作を行った後，陰イオン交換カラムクロマトグラフィーにより，目的のビオチン化糖鎖を精製した。この精製糖鎖をストレプトアビジンがコーティングされたプラスチックプレートに供し，４℃で一晩反応させた後，リン酸緩衝生理食塩水を添加して十分に洗浄し，固定化ヘパリン糖鎖とした。 [Example 1] Detection of immobilized heparin sugar chain by probe
Experimental method Immobilization of heparin sugar chain Addition of ammonium formate and sodium cyanoborohydride to heparin sugar chain derived from porcine small intestine, and the reaction was repeated twice at 70 ° C for 2 days to reduce the reducing end of the sugar chain. An amino group was introduced into. The reaction solution was desalted and then biotin was introduced specifically for the amino group present at the reducing end of the sugar chain. The reaction solution was desalted and the target biotinylated sugar chain was purified by anion exchange column chromatography. This purified sugar chain was applied to a plastic plate coated with streptavidin, reacted at 4 ° C. overnight, and then washed thoroughly by adding phosphate buffered saline to obtain an immobilized heparin sugar chain.
プローブ等の添加
　２％ウシ血清アルブミンを含むリン酸緩衝生理食塩水100 μlにプローブを4.23 nmol加えて溶解し，固定化ヘパリン糖鎖に添加した。また，ヘパリンとの結合が既知のアンチトロンビンIII（AT-III）145 pmolを同様に添加した。 Addition of probe, etc. 4.23 nmol of probe was dissolved in 100 μl of phosphate buffered saline containing 2% bovine serum albumin and added to the immobilized heparin sugar chain. Similarly, 145 pmol of antithrombin III (AT-III), which is known to bind to heparin, was added.
蛍光強度の測定
　25℃で3.5時間反応させた後，素早く洗浄し（300 mM NaClを1ウェル当たり200μl使用して4回洗浄した後，リン酸緩衝生理食塩水を1ウェル当たり200μl使用して2回洗浄），0.2 mM NAM（N-(9-acridinyl)
maleimide）を含む50 mMホウ酸緩衝液（pH 8.8）100 μlを加えて，遮光した状態で10分間反応させた。蛍光プレートリーダーを用いて，励起波長：355 nm，蛍光波長：460 nmで蛍光強度を測定した。 Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 µl of 300 mM NaCl per well, then use 2 µl of phosphate buffered saline with 200 µl per well). Wash), 0.2 mM NAM (N- (9-acridinyl)
100 μl of 50 mM borate buffer solution (pH 8.8) containing maleimide) was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm.
結果
　NAMは遊離のチオール基（SH基）と反応すると強い青色蛍光を発し，pH 3 - 10の水溶液中で反応が定量的に進行する。固定化ヘパリンに結合した遊離のチオール基をもつプローブは，この実験系において，ヘパリンと結合能がないウシ血清アルブミンを含む溶液のみのコントロールと比較して，有意な差でNAMにより検出することができた（図１）。 As a result, NAM emits strong blue fluorescence when it reacts with free thiol groups (SH groups), and the reaction proceeds quantitatively in aqueous solution at pH 3-10. Probes with free thiol groups bound to immobilized heparin can be detected by NAM in this experimental system with a significant difference compared to a control with only solution containing bovine serum albumin that does not bind heparin. (Fig. 1).
〔実施例２〕L-システインを用いた検量線
実験方法
　L-システインを1 pmolから10 nmolまで段階的に96穴プレートにそれぞれ分注し， 0.2 mM NAM（N-(9-acridinyl) maleimide）を含む50 mMホウ酸緩衝液（pH 8.8）100 μlを加えて，遮光した状態で10分間反応させた。蛍光プレートリーダーを用いて，励起波長：365 nm，蛍光波長：435 nmで蛍光強度を測定した。それぞれのL-システインの量に対する蛍光強度をプロットし，検量線を作成した。 [Example 2] Calibration curve using L-cysteine
Experimental method L-cysteine was dispensed stepwise from 1 pmol to 10 nmol in 96-well plates, and 50 mM borate buffer (pH 8.8) 100 containing 0.2 mM NAM (N- (9-acridinyl) maleimide). μl was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm. A calibration curve was prepared by plotting the fluorescence intensity against the amount of each L-cysteine.
結果
　L-システインを10 pmolから3 nmol含む溶液における蛍光強度のプロットを元に，最小二乗法により算出した近似直線は，寄与率（R2）が0.998となり，濃度依存的な蛍光強度の増加を示した（図２）。この傾向は，10 pmolから300 pmolの場合（図３）と比較してもほぼ同様の結果であったことから，本法によりNAMを用いて遊離のチオール基が検出できる範囲は，10 pmol～3 nmolである。 Results Based on the plot of fluorescence intensity in a solution containing 10 pmol to 3 nmol of L-cysteine, the approximate straight line calculated by the least square method has a contribution ratio (R 2 ) of 0.998, indicating a concentration-dependent increase in fluorescence intensity. Shown (FIG. 2). This tendency was almost the same as that in the case of 10 pmol to 300 pmol (Fig. 3), so the range in which free thiol groups can be detected using NAM by this method is 10 pmol to 3 nmol.
〔実施例３〕プローブの結合量の測定（ヘパリン糖鎖量一定）
実験方法
１．ヘパリン糖鎖の固定化
　実施例１「固定化ヘパリン糖鎖のプローブによる検出」と同様にヘパリン糖鎖を固定した。 [Example 3] Measurement of probe binding amount (constant heparin sugar chain amount)
Experimental method 1. Immobilization of heparin sugar chain A heparin sugar chain was immobilized in the same manner as in Example 1 "Detection of immobilized heparin sugar chain by probe".
２．プローブ等の添加
　２％ウシ血清アルブミンを含む溶液100 μlにプローブを42.3 pmol，126 pmol, 423 pmol，1.26 nmol加えてそれぞれ溶解し，固定化ヘパリン糖鎖に添加した。 2. Addition of probe, etc. 42.3 pmol, 126 pmol, 423 pmol and 1.26 nmol were added to 100 μl of a solution containing 2% bovine serum albumin, respectively, and dissolved in the immobilized heparin sugar chain.
３．蛍光強度の測定
　25℃で3.5時間反応させた後，素早く洗浄し（300 mM NaClを1ウェル当たり200μl使用して4回洗浄した後，リン酸緩衝生理食塩水を1ウェル当たり200μl使用して2回洗浄），0.2 mM NAM（N-(9-acridinyl) maleimide）を含む50 mMホウ酸緩衝液（pH 8.8）100 μlを加えて，遮光した状態で20分間反応させた。蛍光プレートリーダーを用いて，励起波長：365 nm，蛍光波長：435 nmで蛍光強度を測定した。 3. Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 µl of 300 mM NaCl per well, then use 2 µl of phosphate buffered saline with 200 µl per well). Washed twice), 100 μl of 50 mM borate buffer (pH 8.8) containing 0.2 mM NAM (N- (9-acridinyl) maleimide) was added and allowed to react for 20 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm.
結果result
　ヘパリン糖鎖を一定量固定化したプレートに対して，42.3 pmolから1.26 nmolのプローブを添加した場合において，量依存的かつ直線的な蛍光強度の増加がみられた（図４）。これにより，検出された蛍光強度は，ヘパリン糖鎖に特異的に結合したプローブに由来していることが明らかとなった。 When a probe from 42.3 μpmol to 1.26 nmol was added to a plate with a fixed amount of heparin sugar chain immobilized, a dose-dependent and linear increase in fluorescence intensity was observed (FIG. 4). This revealed that the detected fluorescence intensity was derived from a probe that was specifically bound to the heparin sugar chain.
〔実施例４〕ビオチン化ヘパリンの結合量の測定（プローブ量一定）
実験方法
１．ヘパリン糖鎖の固定化
　実施例１「固定化ヘパリン糖鎖のプローブによる検出」と同様にビオチン化ヘパリン糖鎖を精製し，水溶液とした。この溶液0.5 μl，1 μl，2.5 μl，5 μlをリン酸緩衝生理食塩水で希釈して，ストレプトアビジンがコーティングされたプラスチックプレートにそれぞれ供し，４℃で一晩反応させた後，リン酸緩衝生理食塩水を添加して十分に洗浄し，固定化ヘパリン糖鎖とした。 [Example 4] Measurement of binding amount of biotinylated heparin (probe amount constant)
Experimental method 1. Immobilization of heparin sugar chain A biotinylated heparin sugar chain was purified in the same manner as in Example 1 “Detection of immobilized heparin sugar chain with probe” to prepare an aqueous solution. 0.5 μl, 1 μl, 2.5 μl, and 5 μl of this solution are diluted with phosphate buffered saline, each is applied to a plastic plate coated with streptavidin, reacted at 4 ° C. overnight, and then phosphate buffered. Saline was added and washed thoroughly to obtain immobilized heparin sugar chains.
２．プローブの添加
　２％ウシ血清アルブミンを含む溶液100 μlにプローブを4.23 nmol加えて溶解し，固定化ヘパリン糖鎖に添加した。 2. Addition of probe 4.23 nmol of probe was dissolved in 100 μl of a solution containing 2% bovine serum albumin and added to the immobilized heparin sugar chain.
３．蛍光強度の測定
　25℃で3.5時間反応させた後，素早く洗浄し（300 mM NaClを1ウェル当たり200μl使用して4回洗浄した後，リン酸緩衝生理食塩水を1ウェル当たり200μl使用して2回洗浄），0.2 mM NAM（N-(9-acridinyl)
maleimide）を含む50 mMホウ酸緩衝液（pH 8.8）100 μlを加えて，遮光した状態で10分間反応させた。蛍光プレートリーダーを用いて，励起波長：365 nm，蛍光波長：435 nmで蛍光強度を測定した。 3. Fluorescence intensity measurement After reacting at 25 ° C for 3.5 hours, wash quickly (wash 4 times with 200 µl of 300 mM NaCl per well, then use 2 µl of phosphate buffered saline with 200 µl per well). Wash), 0.2 mM NAM (N- (9-acridinyl)
100 μl of 50 mM borate buffer solution (pH 8.8) containing maleimide) was added and allowed to react for 10 minutes in the dark. Using a fluorescence plate reader, the fluorescence intensity was measured at an excitation wavelength of 365 nm and a fluorescence wavelength of 435 nm.
結果
　0.5 μlビオチン化ヘパリン水溶液を用いて糖鎖を固定化したものに比べて，1 μlビオチン化ヘパリン水溶液を用いた場合に，蛍光強度の増加がみられた（図５）。このことから，試料中の糖鎖をビオチン化しプレートに固定化することにより，本法での定量が可能であることを示した。また，2.5 μl以上のビオチン化ヘパリン水溶液で飽和していた。 Results The fluorescence intensity increased when 1 μl biotinylated heparin aqueous solution was used compared to the case where sugar chains were immobilized using 0.5 μl biotinylated heparin aqueous solution (FIG. 5). From this, it was shown that the quantification by this method is possible by biotinylating the sugar chain in the sample and immobilizing it on the plate. It was saturated with more than 2.5 μl of biotinylated heparin aqueous solution.
　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硫酸化糖鎖の構造を特異的に認識するプローブが本発明に基づいて開発される。さらに、診断マーカーによる病気診断にそのプローブを利用する際に、本発明の検出法により簡便にその硫酸化糖鎖を検出することができるようになる。 A probe that specifically recognizes the structure of a sulfated sugar chain related to a specific disease is developed based on the present invention. Furthermore, when the probe is used for disease diagnosis using a diagnostic marker, the sulfated sugar chain can be easily detected by the detection method of the present invention.
＜配列番号１＞
配列番号１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物質（ペプチド）のアミノ酸配列(RTRGSTREFRTGC)を示す。
＜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 substance (peptide) that can specifically bind to glycosaminoglycan (heparin, heparan sulfate) and has a free thiol group.
<SEQ ID NO: 2>
SEQ ID NO: 2, glycosaminoglycan (heparin, heparan sulfate) specifically recognize peptides, HappY (H eparin- a ssociated p e p tide
Y ) shows the base sequence of the DNA encoding (CGGACGCGTGGGTCGACCCGGGAATTCCGGACCGGT).
<SEQ ID NO: 3>
SEQ ID NO: 3 is a peptide that specifically recognizes glycosaminoglycan (heparin, heparan sulfate), HappY ( H eparin- a ssociated p e p tide
Y ) shows the amino acid sequence (RTRGSTREFRTG).

Claims (14)

  1. グリコサミノグリカン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物質を含む分子プローブと試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンの検出法。 After contacting the sample with a molecular probe that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group, N- (9-acridinyl) maleimide is added, and the generated fluorescence is A method for detecting glycosaminoglycan, comprising measuring.
  2. 固相に固定されているグリコサミノグリカンの存在下で、前記分子プローブを前記試料と接触させる請求項１記載の検出法。 The detection method according to claim 1, wherein the molecular probe is brought into contact with the sample in the presence of glycosaminoglycan immobilized on a solid phase.
  3. 前記試料中のグリコサミノグリカンを固相に固定した後、前記分子プローブと接触させる請求項１記載の検出法。 The detection method according to claim 1, wherein the glycosaminoglycan in the sample is immobilized on a solid phase and then contacted with the molecular probe.
  4. グリコサミノグリカンを被験物質と接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンに特異的に結合する物質のスクリーニング法。 A method for screening a substance that specifically binds to glycosaminoglycan, comprising bringing N- (9-acridinyl) maleimide into contact with a glycosaminoglycan and then measuring the generated fluorescence.
  5. グリコサミノグリカン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物質を含む分子プローブと被験者からの試料を接触させた後、N-(9-アクリジニル)マレイミドを添加し、発生した蛍光を測定することを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査法。 After contacting a sample from a subject with a molecular probe that can specifically bind to glycosaminoglycan and contains a substance having a free thiol group, N- (9-acridinyl) maleimide is added and generated A method for testing a disease in which glycosaminoglycan serves as a diagnostic marker, comprising measuring the measured fluorescence.
  6. グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカン検出キット。 A glycosaminoglycan detection kit comprising a molecular probe that can specifically bind to a glycosaminoglycan and includes a substance having a free thiol group and N- (9-acridinyl) maleimide.
  7. さらに、グリコサミノグリカンを含む請求項６記載のキット。 Furthermore, the kit of Claim 6 containing glycosaminoglycan.
  8. グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカンに特異的に結合する物質のスクリーニングキット。 Of a substance that can specifically bind to glycosaminoglycan and specifically binds to glycosaminoglycan, including a molecular probe containing a substance having a free thiol group and N- (9-acridinyl) maleimide Screening kit.
  9. さらに、グリコサミノグリカンを含む請求項８記載のキット。 The kit according to claim 8, further comprising glycosaminoglycan.
  10. グリコサミノグリカン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物質を含む分子プローブ及びN-(9-アクリジニル)マレイミドを含む、グリコサミノグリカンが診断マーカーとなる疾患の検査キット。 Testing for diseases in which glycosaminoglycan is a diagnostic marker, including molecular probes that can bind specifically to glycosaminoglycan and contain a substance with a free thiol group and N- (9-acridinyl) maleimide kit.
  11. さらに、グリコサミノグリカンを含む請求項１０記載のキット。 The kit according to claim 10, further comprising glycosaminoglycan.
  12. グリコサミノグリカン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物質を含む分子プローブであって、請求項１記載の検出法、請求項４記載のスクリーニング方法又は請求項５記載の検査法に使用される前記分子プローブ。 A molecular probe comprising a substance capable of specifically binding to glycosaminoglycan and having a free thiol group, comprising the detection method according to claim 1, the screening method according to claim 4, or the claim 5. The molecular probe used in the inspection method.
  13. グリコサミノグリカン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物質が、以下の(a)又は(b)のペプチドである請求項１２記載の分子プローブ。
    (a)配列番号１のアミノ酸配列（RTRGSTREFRTGC）からなるペプチド。
    (b) 配列番号１のアミノ酸において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、グリコサミノグリカン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ペプチド。     The molecular probe according to claim 12, wherein the substance capable of specifically binding to glycosaminoglycan and having a free thiol group is the following peptide (a) or (b).
    (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
    (b) a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
  14. 以下の(a)又は(b)のペプチド。
    (a)配列番号１のアミノ酸配列（RTRGSTREFRTGC）からなるペプチド。
    (b) 配列番号１のアミノ酸において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、グリコサミノグリカン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ペプチド。     The following peptide (a) or (b).
    (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RTRGSTREFRTGC).
    (b) a peptide comprising an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid of SEQ ID NO: 1 and capable of specifically binding to glycosaminoglycan and having a free thiol group .
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WO2013035727A1 (en) * 2011-09-08 2013-03-14 国立大学法人岐阜大学 Sugar chain detection method using photocrosslinking agent and molecular probe
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