WO2018174044A1 - 糖鎖を有する物質とレクチンとの複合体形成方法 - Google Patents
糖鎖を有する物質とレクチンとの複合体形成方法 Download PDFInfo
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- WO2018174044A1 WO2018174044A1 PCT/JP2018/010966 JP2018010966W WO2018174044A1 WO 2018174044 A1 WO2018174044 A1 WO 2018174044A1 JP 2018010966 W JP2018010966 W JP 2018010966W WO 2018174044 A1 WO2018174044 A1 WO 2018174044A1
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13B—PRODUCTION OF SUCROSE; APPARATUS SPECIALLY ADAPTED THEREFOR
- C13B50/00—Sugar products, e.g. powdered, lump or liquid sugar; Working-up of sugar
- C13B50/002—Addition of chemicals or other foodstuffs
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
- G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
Definitions
- the present invention relates to a method for forming a complex of a substance having a sugar chain and a lectin.
- a sugar chain is a compound in which the same monosaccharide molecule or two or more types of monosaccharide molecules are linked in a chain via a glycosidic bond.
- Sugar chains are present mainly on the cell surface in the form of complex carbohydrates bound to proteins and lipids in vivo. It is involved in important physiological functions in the body, such as cell growth, bacterial and viral infection, nerve growth, inflammation, and immunity.
- sugar chains added to substances having sugar chains such as glycoproteins existing in the living body are usually constant. However, sugar chains may change in a disease-specific manner, and such sugar chains are used as diagnostic biomarkers.
- sugar chains and glycoproteins whose sugar chains change with canceration of cells and progression of cancer are known, and these are known as tumor markers.
- Tumor markers with sugar chains include ⁇ -fetoprotein (AFP: liver cancer marker), prostate specific antigen (prostate specific antigen, PSA: prostate cancer marker), carcinoembryonic antigen, CEA: marker for colorectal cancer, etc.
- AFP liver cancer marker
- PSA prostate specific antigen
- CEA carcinoembryonic antigen
- CA19-9 carbohydrate antigen 19-9, CA 19-9: pancreatic cancer marker, digestive cancer marker
- podocalyxin and the like are known.
- a method for detecting and analyzing sugar chains a method using a lectin is generally used.
- a lectin is a generic term for substances that have the property of specifically recognizing and binding sugar chains among proteins or glycoproteins present in plants, animals, microorganisms, etc., excluding enzymes and antibodies. Since a lectin has high specificity for a sugar chain, a substance having a sugar chain can be specifically detected using the lectin.
- a method for detecting a sugar chain or a substance having a sugar chain using a lectin a surface plasmon resonance method, a lectin electrophoresis method (for example, capillary electrophoresis), a lectin column method (for example, a lectin affinity chromatography), a lectin microarray method
- Immunological measurement methods such as a sandwich method using a lectin immobilized on an insoluble carrier or a biotin-labeled lectin are known.
- the binding power to sugar chains is very weak compared to the binding power of antibodies to sugar chains.
- the dissociation constant of the antigen-antibody reaction is about 10 ⁇ 9 to 10 ⁇ 7 , but the dissociation constant between the lectin and the sugar chain is only about 10 ⁇ 7 to 10 ⁇ 3 (Jun Hirabayashi, et al, Chem. Soc. Rev., vol.442, pp.4443-4458, 2013, etc.). Therefore, it is difficult to form a strong complex such as an antigen-antibody reaction between a lectin and a sugar chain.
- Patent Document 1 a complex of an affinity molecule having affinity for an analyte such as an antibody or a lectin and a charge carrier molecule is formed in the presence of a polyanionic polymer.
- An antigen-antibody reaction is carried out in the presence of a polyanionic polymer, but there is no specific disclosure about the reaction between a substance having a sugar chain and a lectin. Therefore, it is not clear from the disclosure of Patent Document 1 whether the polyanionic polymer has any influence on the interaction between sugar chain and lectin.
- an object of the present invention is to provide a method for increasing the amount of a complex of a substance having a sugar chain and a lectin.
- the present inventors have conducted a complex formation reaction between a sugar chain and a lectin and have a water-soluble polysaccharide or N-acetylglucosamine that does not have N-acetylglucosamine.
- a water-soluble compound having a non-polysaccharide the amount of the complex of a substance having a sugar chain and a lectin is increased, and the above-mentioned problems can be solved, and the present invention has been completed. .
- the present invention has the following configuration.
- a sample containing a substance having a sugar chain in the presence of a water-soluble polysaccharide not having N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine and a substance having the sugar chain A method of forming a complex between a substance having a sugar chain and the lectin, wherein the lectin has affinity for the sugar chain.
- a water-soluble compound having a water-soluble polysaccharide not having N-acetylglucosamine or a polysaccharide having no N-acetylglucosamine is one having no sugar chain to which the lectin has affinity (1) ) Method.
- the water-soluble polysaccharide not having N-acetylglucosamine or the water-soluble compound having a polysaccharide not having N-acetylglucosamine is dextran sulfate or a salt thereof, or chondroitin sulfate c or a salt thereof (1) ) Or the method according to (2).
- a substance having a sugar chain wherein the water-soluble polysaccharide not having N-acetylglucosamine or the water-soluble compound having a polysaccharide not having N-acetylglucosamine is dextran sulfate or a salt thereof, or chondroitin sulfate c or a salt thereof Is an ⁇ (2, 3) sugar chain free prostate specific antigen, and the lectin is canine endlectin.
- a substance having a sugar chain and a sugar chain of the substance having the sugar chain comprising a water-soluble polysaccharide having no N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine
- a complex formation accelerator with a lectin having an affinity for (6)
- a water-soluble compound having a water-soluble polysaccharide not having N-acetylglucosamine or a polysaccharide not having N-acetylglucosamine is one in which the lectin does not have a sugar chain having an affinity (5) ).
- the water-soluble polysaccharide not having N-acetylglucosamine or the water-soluble compound having a polysaccharide not having N-acetylglucosamine is dextran sulfate or a salt thereof, or chondroitin sulfate c or a salt thereof (5) ) Or (9) The complex formation promoter according to 6.
- the complex formation promoter according to any one of (5), (6), and (7) selected from the following (i) to (ii): (i) The water-soluble polysaccharide having no N-acetylglucosamine is dextran sulfate or a salt thereof, the substance having a sugar chain is ⁇ -fetoprotein-L3 (AFP-L3), and the lectin is lentil lectin.
- AFP-L3 ⁇ -fetoprotein-L3
- a water-soluble polysaccharide having no N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine is dextran sulfate or a salt thereof, or chondroitin sulfate c or a salt thereof, and has a sugar chain
- the substance is ⁇ (2, 3) sugar chain free prostate specific antigen, and the lectin is canine endlectin.
- a method for measuring a substance having a sugar chain wherein a complex of a substance having a sugar chain and a lectin is formed by the method described in (1) above, and then the amount of the complex is measured.
- the amount of the complex of a substance having a sugar chain and a lectin is increased. Therefore, by using the complex formation method of the present invention, a substance having a sugar chain in the presence of a water-soluble polysaccharide not having N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine is used. If it measures, the highly sensitive measurement of the substance which has a sugar chain can be performed.
- the complex formation method of the present invention can be used for all reactions, detection, measurement, sugar chain analysis, etc. using the affinity of lectins for sugar chains. Further, there are effects such as enhancing the sensitivity of detection and measurement, enabling high-accuracy analysis of sugar chains, and efficiently separating substances having sugar chains.
- Example 2 It is a schematic diagram of the flow path in the chip of the microchip used in Example 2 and Example 3.
- 2 is a graph showing the relationship between the concentration of c sodium chondroitin sulfate obtained in Example 2 and the peak area of the complex 1 fraction. It is a graph which shows the relationship between the density
- Complex formation method of the present invention is performed in the presence of a water-soluble polysaccharide having no N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine.
- a method for forming a complex of a substance having a sugar chain and the lectin, wherein a sample containing the substance having a sugar chain is brought into contact with a lectin having affinity for the sugar chain of the substance having the sugar chain is performed in the presence of a water-soluble polysaccharide having no N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine.
- a lectin having affinity for a sugar chain of a substance having a sugar chain may hereinafter be simply referred to as “lectin”.
- Water-soluble polysaccharides that do not have N-acetylglucosamine are generally referred to as polysaccharides, and do not have N-acetylglucosamine as a constituent sugar. A water-soluble thing is mentioned.
- a homopolysaccharide simple polysaccharide in which one type of monosaccharide is polymerized by glycoside bonds
- a heteropolysaccharide complex polysaccharide in which multiple types of monosaccharides are polymerized by glycoside bonds
- water-soluble compounds that do not have N-acetylglucosamine.
- Water-soluble homopolysaccharides that do not have N-acetylglucosamine as a constituent sugar include dextran, agarose, carrageenan, dextran sulfate, carboxymethyldextran, fucoidan, funolan, chitosan, and these Derivatives and the like.
- water-soluble heteropolysaccharides that do not have N-acetylglucosamine as a constituent sugar
- examples of water-soluble heteropolysaccharides that do not have N-acetylglucosamine as a constituent sugar include porphyran, glucomannan, alginic acid, xanthan gum, and derivatives thereof.
- water-soluble sugar alcohols that do not have N-acetylglucosamine as a constituent sugar include erythritol, xylitol, and derivatives thereof.
- water-soluble polysaccharides not having N-acetylglucosamine water-soluble homopolysaccharides are preferable, dextran sulfate and its derivatives are more preferable, and dextran sulfate is particularly preferable.
- the water-soluble polysaccharide having no N-acetylglucosamine according to the present invention includes, for example, alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, ammonium salt and triethylamine salt.
- alkali metal salts such as lithium salt, sodium salt and potassium salt
- alkaline earth metal salts such as calcium salt and magnesium salt
- ammonium salt and triethylamine salt such as dimethylamine salts may be used.
- the preferred salt varies depending on the type of polysaccharide or the compound having a polysaccharide, but a sodium salt is preferred, and sodium dextran sulfate is more preferred.
- the molecular weight of the water-soluble polysaccharide having no N-acetylglucosamine according to the present invention is several hundred to several million, preferably about 1,000 to 1,000,000, more preferably about 5,000 to 500,000.
- Water-soluble compound having polysaccharide without N-acetylglucosamine is a so-called complex carbohydrate in which protein or lipid is bound to polysaccharide.
- As a constituent sugar of the polysaccharide portion water-soluble ones having no N-acetylglucosamine can be mentioned.
- complex carbohydrates such as proteoglycan in which a protein is bound to glycosaminoglycan, and water-soluble ones that do not have N-acetylglucosamine as a constituent sugar of the glycosaminoglycan part are mentioned.
- water-soluble proteoglycans examples include chondroitin sulfate a, chondroitin sulfate b, chondroitin sulfate c, chondroitin sulfate d, chondroitin sulfate e, and derivatives thereof.
- chondroitin sulfate c and derivatives thereof are preferable, and chondroitin sulfate c is particularly preferable.
- Examples of the water-soluble compound having a polysaccharide having no N-acetylglucosamine according to the present invention include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt.
- alkali metal salts such as lithium salt, sodium salt and potassium salt
- alkaline earth metal salts such as calcium salt and magnesium salt
- ammonium salt organic amine salts such as triethylamine salt and dimethylamine salt may be used.
- the preferred salt varies depending on the type of polysaccharide or the compound having a polysaccharide, but a sodium salt is preferred.
- a sodium salt is preferred.
- sodium chondroitin sulfate is preferable, and c sodium chondroitin sulfate is more preferable.
- the molecular weight of the water-soluble compound having a polysaccharide not containing N-acetylglucosamine according to the present invention is several hundred to several million, preferably about 1,000 to 1,000,000, more preferably about 5,000 to 500,000.
- the water-soluble polysaccharide having no N-acetylglucosamine according to the present invention or the water-soluble compound having a polysaccharide not having N-acetylglucosamine is, for example, “contacting a substance having a sugar chain with a lectin in the presence thereof.
- a substance having a sugar chain with a lectin in the presence thereof.
- a water-soluble compound having no N-acetylglucosamine or a water-soluble compound having a polysaccharide not having N-acetylglucosamine may be collectively referred to as “the polysaccharide according to the present invention” hereinafter.
- Substance having a sugar chain is not particularly limited as long as it is a substance having a “sugar chain having a sugar chain structure to which a lectin has affinity (bonds)”.
- the substance having a sugar chain include tumor markers such as AFP, PSA, CA19-9, CA125, PIVKA-II, podocalyxin, ⁇ 1-globulin, ⁇ 2-globulin, ⁇ -globulin, ⁇ -globulin, fetuin and the like.
- Serum proteins fiber proteins such as collagen, gonadotropins, thyroid stimulating hormone, human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), hormones such as thyroid stimulating hormone (TSH), Ribonuclease, Takaamylase, ⁇ -glutamyltransferase ( ⁇ -GTP), enzymes such as alkaline phosphatase, cholinesterase, lysozyme, intercellular substances such as fibronectin, proteoglycan, ⁇ 1-acid glycoprotein, ⁇ 1-antitrypsin, ⁇ 2-macroglobulin , ⁇ 2-HS glycoprotein, transferrin, Ptoglobin, ceruloplasmin, carcinoembryonic antigen (CEA), immunoglobulin (IgG, IgM, IgA, IgD, IgE), complement components (C1, C1q, C1r, C1s, C4, C2, C3, C5, C6, C7
- AFP, PSA and podocalyxin are preferable, and AFP and PSA are more preferable.
- the substance having a sugar chain according to the present invention includes a “sugar chain” having a sugar chain structure in which the lectin has an affinity.
- a sugar chain structure in which the lectin has an affinity For example, amylose, amylopectin, cellulose, glycogen and the like can be mentioned.
- Lewis X (Gal ⁇ 1-4 [Fuc ⁇ 1-3] GlcNac), which has been confirmed to be expressed in undifferentiated stem cells such as mouse ES cells, and fragments of sugar chains released from glycoproteins, etc. are also included in the sugar chain according to the present invention. Included in the substance.
- Lewis X (Gal ⁇ 1-4 [Fuc ⁇ 1-3] GlcNac) is used.
- sugar chain related to the “substance having a sugar chain” according to the present invention examples include those having a sugar chain structure in which the lectin has an affinity.
- sugar chain possessed by the substance having the sugar chain described above can be mentioned.
- sugar chains observed in the blood of cancer patients and the like can be mentioned.
- AFP is a hepatocellular carcinoma marker
- the blood concentration is high not only in hepatocellular carcinoma but also in chronic hepatitis and cirrhosis.
- AFP has a sugar chain (Fuc ⁇ 1 ⁇ 6GlcNac) with ⁇ -L-fucose residue or N-acetylglucosamine residue (bisecting N-acetylglucosamine) added to the sugar chain. It has been confirmed that (AFP-L3) is frequently observed.
- PSA is known as a prostate cancer marker. Most PSA observed in normal serum has an N-type sugar chain in which the terminal sialic acid residue of the sugar chain is ⁇ (2, ⁇ 6) linked to the second galactose residue from the end of the sugar chain. PSA. However, in the serum of prostate cancer patients, PSA has a sugar chain (Sia ⁇ 2 ⁇ 3Gal) in which the terminal sialic acid residue of the sugar chain is ⁇ (2, 3) linked to the second galactose residue from the end of the sugar chain. (Hereinafter referred to as “ ⁇ (2,3) sugar chain PSA”) is known to increase (Tajiri M. et al., 2008, vol. 18, pp. 2-8).
- the sugar chain (Sia ⁇ 2 ⁇ 3Gal) in which the terminal sialic acid residue of the sugar chain is ⁇ (2, 3) linked to the second galactose residue from the end of the sugar chain specifically refers to the following structure.
- glycoprotein sugar chains known as cell undifferentiation markers and differentiation markers are also exemplified as substances having a sugar chain according to the present invention.
- Examples include (Fuc ⁇ 1-2Gal ⁇ 1-3GlcNac) and (Fuc ⁇ 1-2Gal ⁇ 1-3GalNac), which are sugar chains that have recently been found to exist on the cell surface of pluripotent stem cells such as ES cells and iPS cells. . Moreover, podocalyxin, a complex carbohydrate, has been identified as an undifferentiated sugar chain marker having the sugar chain structure (WO2013 / 065302).
- Sample containing a substance having a sugar chain for example, blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, ascites, fecal suspension, Ascites, tissue extracts, tissue sections, tissue biopsy samples, organs, etc., biological samples such as humans and animals, or prepared from these biological samples, viruses, bacteria, microorganisms such as cells, or from these What was prepared is mentioned.
- blood, serum, or plasma is preferable, and serum is more preferable.
- a cell extract, an extract derived from the above microorganism, or one prepared from the extract can also be used as a sample containing a substance having a sugar chain according to the present invention.
- the above-described biologically-derived sample and microorganism culture solution can also be used as a sample containing a substance having a sugar chain according to the present invention.
- the sample containing the substance having a sugar chain according to the present invention may be diluted with water or an appropriate buffer.
- Examples of water used for this purpose include purified water such as distilled water and deionized water.
- Buffers include, for example, Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, Tris-HCl buffer, MES buffer, HEPES buffer, borate buffer, phosphate buffer Liquid, veronal buffer, Good buffer, and the like.
- the buffer concentration of these buffers is appropriately selected from the range of usually 5 to 1,000 mM, preferably 5 to 300 mM.
- the pH is not particularly limited, and examples thereof include a range of 5 to 9.
- sample containing a substance having a sugar chain and “substance having a sugar chain” may be used without distinction. That is, in the present specification, the simple description “sample containing a substance having a sugar chain” may include the meaning of “substance having a sugar chain”.
- the lectin used in the present invention may be any lectin that has affinity for a sugar chain of a substance having a sugar chain that is a target for forming a complex, recognizes the sugar chain, and binds to the sugar chain.
- a lectin having such properties may be arbitrarily selected from known lectins. Among these, a lectin that specifically binds to the sugar chain is preferable.
- lectins are not limited, and lectins derived from animals, plants, fungal bacteria, viruses, and the like may be used. Moreover, it may be a naturally derived lectin, a recombinant product, or a commercially available lectin.
- the lectin include, for example, lotus bean lectin (LTL), pea lectin (PSA), lentil lectin (Lens culimaris agglutinin: LCA), gorse lectin (UEA-1), gonococcal lectin (AOL), and yellow pea lectin ( AAL), mushroom lectin (ABA), jacarin lectin (Jacalin), peanut lectin (PNA), nodafuji lectin (WFA), sennin clectin (ACA), osage orange lectin (MPA), edible snail lectin (HPA), Hairy vetch lectin (VVA), Doricos bean lectin (DBA), soybean lectin (SBA), bandilla pea lectin (GSL-I, GSL-IA4, GSL-II, GSL-IB4), winged bean lectin (PTL-I), kidney bean Lectin (PHA-E, PHA-L), Mura
- BC2LCN which is a lectin that recognizes (Fuc ⁇ 1-2Gal ⁇ 1-3GlcNac) or (Fuc ⁇ 1-2Gal ⁇ 1-3GalNac), which is a sugar chain of podocalyxin known as an undifferentiated marker of pluripotent stem cells (Tateno H, et al., J. Biol. Chem., vol. 286, No. 23, pp. 20345-20353, 2011).
- BC2LCN is a lectin corresponding to the N-terminal domain of BC2L-C protein derived from Gram-negative bacteria ( Burkholderia cenocepacia ) (GenBank Accession No. YP_002232818).
- the lectin used in the present invention is preferably LCA, MAA, or BC2LCN, and more preferably LCA or MAA.
- Table 1 shows examples of the lectins used in the present invention, the origin of each lectin (Origin), and examples of the sugar chain structure (Specificity) with which the lectin has an affinity.
- the lectin may be labeled with a detectable labeling substance.
- labeling substances used for labeling lectins include fluorescent dyes (fluorescein isothiocyanate (FITC), Cy5, Alexa Fluor 647, etc.), enzymes (horseradish-derived peroxidase), coenzymes, chemiluminescent substances, radioactive substances ( 32P, 14C, 125I, 3H, 131I, etc.), and labeling substances such as biotin. Further, the labeling substance may be directly bound to the lectin or may be bound via an appropriate spacer.
- fluorescent dyes fluorescein isothiocyanate (FITC), Cy5, Alexa Fluor 647, etc.
- enzymes horseradish-derived peroxidase
- coenzymes chemiluminescent substances
- radioactive substances 32P, 14C, 125I, 3H, 131I, etc.
- labeling substances such as biotin.
- the labeling substance may be directly bound to the lectin or may be bound via an appropriate spacer.
- the lectin may be labeled by a known labeling method according to the type of labeling substance.
- the lectin may be immobilized on a solid phase such as an insoluble carrier.
- a solid phase such as an insoluble carrier.
- an insoluble carrier used as the solid phase any carrier that is usually used in this field can be used.
- the shape is usually used in this field such as particles (latex particles, beads, magnetic beads, etc.), tubes, carbon nanotubes, chips, disk-like pieces, fine particles, thin films, microtubes, plates, microplates, filters, etc. Any material may be used, and the material is not particularly limited.
- microchips, microarrays, macroarrays, and microtiter plates produced by immobilizing lectins on a substrate can be used in the present invention.
- Examples of the method for immobilizing the lectin on the insoluble carrier include known immobilization methods according to the type of the insoluble carrier to be used.
- the complex formation method of the present invention may be any method that can finally obtain a “complex of a substance having a sugar chain and a lectin containing the polysaccharide according to the present invention”. .
- a method of contacting a sample containing a substance having a sugar chain with a lectin having affinity for the sugar chain of the substance having a sugar chain in the presence of the polysaccharide according to the present invention A method of obtaining a “solution of a complex of a substance having a sugar chain and a lectin” containing the polysaccharide according to the invention is preferable.
- a sample containing a substance having a sugar chain is mixed with a polysaccharide according to the present invention or a solution containing a polysaccharide according to the present invention to obtain a mixed solution, and then the obtained mixed solution is A method of reacting by mixing with a lectin having affinity for a sugar chain of a substance having a sugar chain or a solution containing the lectin, (Ii) A lectin having affinity for a sugar chain of a substance having a sugar chain or a solution containing the lectin is mixed with the polysaccharide according to the present invention or the solution containing the polysaccharide according to the present invention to obtain a mixed solution.
- a method of mixing and reacting the obtained liquid mixture with a sample containing a substance having a sugar chain (Iii) A sample containing a substance having a sugar chain is mixed with the polysaccharide according to the present invention or the solution containing the polysaccharide according to the present invention to obtain a mixed solution, and then the obtained mixed solution is mixed with the sugar.
- a method of mixing and reacting with a solution containing a lectin having affinity for a sugar chain of a substance having a chain and the polysaccharide according to the present invention (Iv) A reaction solution obtained after reacting a sample containing a substance having a sugar chain with a lectin having affinity for the sugar chain of the substance having a sugar chain or a solution containing the lectin. Or the like, or a solution containing the polysaccharide according to the present invention to obtain a mixed solution.
- a sugar chain of a substance having a sugar chain reacts with a lectin in the presence of the polysaccharide according to the present invention to form a complex of the substance having a sugar chain and the lectin. To do.
- reaction refers to a complex formation reaction between a sugar chain-containing substance and a lectin sugar chain.
- the methods (i) to (iii) are preferable, and the method (i) or (iii) is more preferable.
- the polysaccharide according to the present invention may be contained in any of the test solutions used.
- the solution containing the polysaccharide according to the present invention may be used by being set in one of the cells into which the reagent of the automatic analyzer is placed. Then, in the measurement using an automatic analyzer, a substance having a sugar chain and a lectin are brought into contact with each other in the presence of the polysaccharide of the present invention, and the sugar chain of the substance having a sugar chain and the lectin may be reacted. .
- Examples of the solvent used in the solution containing the polysaccharide according to the present invention include water or a buffer solution.
- Examples of the solvent used in the solution containing lectin include a buffer solution.
- Examples of the solvent used in the solution containing the polysaccharide and lectin according to the present invention include a buffer solution.
- water used for the solution containing the polysaccharide according to the present invention examples include purified water such as distilled water and deionized water.
- the buffer solution used in the solution containing the polysaccharide according to the present invention includes, for example, Tris buffer solution and phosphate buffer.
- Solution veronal buffer, borate buffer, Good buffer, Tris-HCl buffer, MES buffer, HEPES buffer, borate buffer, phosphate buffer, veronal buffer, Good buffer, etc.
- a complex formation reaction between a substance having a sugar chain and a lectin is performed using a commercially available kit or a measuring instrument such as an automatic analyzer, a buffer solution attached to the kit, a buffer solution dedicated to the automatic analyzer, etc. (For example, HBS-EP buffer for Biacore TM described later) may be used.
- the buffering agent concentration of these buffers is appropriately selected from the range of usually 5 to 1,000 mM, preferably 5 to 300 mM.
- the pH is not particularly limited, and examples thereof include a range of 5 to 9.
- buffers include reagents usually used in this field, for example, buffers, reaction accelerators, proteins, salts, stabilizers such as surfactants, preservatives, etc., coexisting reagents, etc. It may be included that does not inhibit the stability of the sugar chain and does not inhibit the reaction between the sugar chain and the lectin. Also, the concentration may be appropriately selected from the concentration range usually used in this field.
- the concentration of the polysaccharide according to the present invention in the solution containing the polysaccharide according to the present invention, the concentration of the lectin in the solution containing the lectin, and the substance having a sugar chain in the sample containing the substance having a sugar chain is such that the concentration in the reaction solution when the lectin is brought into contact with the substance having a sugar chain in the presence of the polysaccharide according to the present invention is within the target concentration range. Good.
- the concentration of the polysaccharide according to the present invention in the solution containing the polysaccharide according to the present invention is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to It is 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)%.
- the concentration of the lectin in the solution containing the lectin is 1 ⁇ g / mL to 30 mg / mL, preferably 10 ⁇ g / mL to 20 mg / mL, more preferably 1 to 10 mg / mL.
- the concentration of the substance having a sugar chain in the sample containing the substance having a sugar chain is 1 pg / mL to 1 mg / mL, preferably 10 pg / mL to 100 ⁇ g / mL, more preferably 1 ng to 50 ⁇ g / mL.
- the concentration of the polysaccharide according to the present invention in the reaction solution when the lectin is brought into contact with the substance having a sugar chain in the presence of the polysaccharide according to the present invention is reacted.
- 0.01 (w / v)% to 50 (w / v)% preferably 0.1 (w / v) % To 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)%.
- the concentration of the polysaccharide according to the present invention in the microchannel is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 1 (w / v)% to 15 (w / v)%, still more preferably 4 to 10 (w / v)%.
- the concentration of lectin in the reaction solution is 1 ⁇ g / mL to 30 mg / mL, preferably 10 ⁇ g / mL to 20 mg / mL, more preferably 1 to 10 mg / mL.
- the concentration of the substance having a sugar chain in the reaction solution is 1 pg / mL to 1 mg / mL, preferably 10 pg / mL to 100 ⁇ g / mL, more preferably 1 ng to 50 ⁇ g / mL.
- the polysaccharide according to the present invention in the finally obtained “solution of a complex of a substance having a sugar chain and a lectin containing the polysaccharide according to the present invention”.
- concentration of the saccharide, the concentration of the lectin, and the concentration of the substance having a sugar chain may be within the above-described target concentration ranges.
- Conditions for contacting and reacting a sample containing a substance having a sugar chain with a lectin and conditions for bringing a sample containing a substance having a sugar chain into contact with a lectin in the presence of the polysaccharide according to the present invention Any condition may be used as long as the sugar chain of the substance having a sugar chain sufficiently reacts with the lectin to form a complex of the substance having the sugar chain and the lectin.
- the temperature at the time of reaction between a sample containing a substance having a sugar chain and a lectin is not particularly limited as long as the reaction between the sugar chain of the substance having a sugar chain and the lectin is not suppressed, and is 10 to 50 ° C. The preferred range is 20 to 40 ° C.
- the reaction time varies depending on the lectin used and the reaction conditions such as pH and temperature. For example, the reaction may be performed for 1 to 60 minutes, preferably about 1 to 15 minutes.
- the method for selecting polysaccharides and lectins according to the present invention used in the complex forming method of the present invention is as follows. That is, the polysaccharide according to the present invention used in the complex forming method of the present invention is selected from those having no sugar chain structure to which the lectin used in the complex forming method of the present invention has an affinity. That is, when a complex of a substance having a certain sugar chain and a lectin is formed, a lectin that has affinity for the sugar chain of the substance having the sugar chain and recognizes and binds to the sugar chain is selected. And as the polysaccharide which concerns on this invention, the thing which does not have the sugar chain structure which this selected lectin has affinity is selected.
- the polysaccharide according to the present invention is the lectin described in Table 1.
- a sugar chain according to the present invention is selected which does not have a sugar chain structure (Specificity) having affinity.
- this mutant sugar chain has an affinity.
- LCA can be used as the lectin.
- the polysaccharide according to the present invention to be coexisted when AFP-L3 and LCA are contacted (at the time of reaction) the one that does not have a sugar chain (Fuc ⁇ 1 ⁇ 6GlcNAc, ⁇ -Man) having an affinity for LCA is selected. .
- polysaccharides according to the present invention include dextran sulfate, chondroitin sulfate, or salts thereof (for example, dextran sulfate sodium, chondroitin sulfate c sodium) and the like.
- Dextran sulfate has a structure in which a part of dextran in which glucose is polymerized by 1,6 bonds is sulfated.
- Chondroitin sulfate has a structure in which sulfate is bound to a sugar chain in which D-glucuronic acid (GlcUA) and N-acetyl-D-galactosamine (GalNAc) are repeated.
- GlcUA D-glucuronic acid
- GalNAc N-acetyl-D-galactosamine
- MAA when a complex of PSA having a mutated sugar chain (Sia ⁇ 2 ⁇ 3Gal) and a lectin is formed, MAA can be used as a lectin having affinity for the sugar chain (Sia ⁇ 2 ⁇ 3Gal), for example.
- the polysaccharide according to the present invention that coexists in the reaction of PSA and MAA is selected so that MAA does not have a sugar chain (Sia ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 4GlcNAc) with affinity.
- polysaccharides according to the present invention include dextran sulfate, chondroitin sulfate, or salts thereof (for example, dextran sulfate sodium, chondroitin sulfate c sodium, etc.).
- the polysaccharide according to the present invention coexisting when pluripotent stem cells and BC2LCN are contacted (at the time of reaction) does not have a sugar chain (Fuc ⁇ 1-2Gal ⁇ 1-3GlcNac) having affinity for BC2LCN, and (Fuc ⁇ 1 -2Gal ⁇ 1-3GalNac) is also selected.
- polysaccharides according to the present invention include dextran sulfate, chondroitin sulfate, or salts thereof (for example, dextran sulfate sodium, chondroitin sulfate c sodium, etc.).
- the concentration of AFP-L3 in the reaction solution is 1 pg / mL to 1 mg / mL
- the concentration of c sodium chondroitin sulfate is 0.01 (w / v)% to 50 (w / v)%
- the concentration of LCA is 1 ⁇ g / mL 30 mg / mL.
- the concentration of ⁇ (2, 3) sugar chain free PSA in the reaction solution is 1 pg / mL to 1 mg / mL
- the concentration of c sodium chondroitin sulfate is 0.01 (w / v)% to 50 (w / v)%
- the concentration of MAA is 1 ⁇ g / mL to 30 mg / mL.
- (v) Serum samples containing 1 pg / mL to 1 mg / mL AFP-L3, LCA from 1 ⁇ g / mL to 30 mg / mL, and 0.01 (w / v)% to 50 (w / v)% dextran Mix with a buffer solution such as 5 to 1000 mM Tris-HCl buffer (pH 5 to 9) containing sodium sulfate or c sodium chondroitin sulfate, and react at 10 to 50 ° C. for 1 to 60 minutes.
- a buffer solution such as 5 to 1000 mM Tris-HCl buffer (pH 5 to 9) containing sodium sulfate or c sodium chondroitin sulfate
- the concentration of AFP-L3 in the reaction solution is 1 pg / mL to 1 mg / mL
- the concentration of sodium dextran sulfate or chondroitin sulfate c is 0.01 (w / v)% to 50 (w / v)%
- the concentration of LCA is 1 ⁇ g / mL to 30 mg / mL.
- the concentration of ⁇ (2, 3) sugar chain free PSA in the reaction solution is 1 pg / mL to 1 mg / mL, and the concentration of dextran sulfate sodium or chondroitin sulfate c sodium is 0.01 (w / v)% to 50 (w / v)
- the concentration of MAA is 1 ⁇ g / mL to 30 mg / mL.
- Serum samples containing 1 pg / mL to 1 mg / mL AFP-L3 contain 0.01 (w / v)% to 50 (w / v)% dextran sulfate sodium or chondroitin sulfate c sodium Mix with a buffer solution such as 5-1000 mM HBS-EP buffer (pH 5-9) to obtain a mixture.
- the obtained mixed solution is brought into contact with LCA immobilized on a solid phase such as a sensor chip and reacted at 10 to 50 ° C. for 1 to 60 minutes.
- the concentration of AFP-L3 in the reaction solution is 1 pg / mL to 1 mg / mL
- the concentration of sodium dextran sulfate or chondroitin sulfate c is 0.01 (w / v)% to 50 (w / v)%
- the concentration of LCA is 1 ⁇ g / mL to 30 mg / mL.
- a mixed solution is obtained by mixing with a buffer solution such as 5-1000 mM HBS-EP buffer solution (pH 5-9) containing c sodium chondroitin sulfate.
- the obtained mixed solution is brought into contact with MAA immobilized on a solid phase such as a sensor chip, and reacted at 10 to 50 ° C. for 1 to 60 minutes.
- the concentration of ⁇ (2, 3) sugar chain free PSA in the reaction solution is 1 pg / mL to 1 mg / mL, and the concentration of dextran sulfate sodium or chondroitin sulfate c sodium is 0.01 (w / v)% to 50 (w / v)
- the concentration of MAA is 1 ⁇ g / mL to 30 mg / mL.
- the amount of the complex of a glycine and a substance having a sugar chain is increased by the complex forming method of the present invention is not clear, for example, a substance having a sugar chain and a lectin in the presence of the polysaccharide according to the present invention ,
- the complex once formed is stabilized and does not re-separate into a substance having a sugar chain and the lectin, or the amount to be separated is reduced, and as a result, in the absence of the polysaccharide according to the present invention. It is conceivable that the amount of the complex increases as compared with the case where the reaction is carried out.
- the presence of the polysaccharide according to the present invention increases the affinity of the substance having a lectin sugar chain for the sugar chain, and therefore, the substance having a sugar chain in the absence of the polysaccharide according to the present invention It is conceivable that the amount of the complex increases when the lectin is brought into contact with a substance having a sugar chain in the presence of the polysaccharide according to the present invention, rather than when the lectin is brought into contact.
- the complex-forming method of the present invention can be used for all measurement methods, analysis methods, detection methods and the like that utilize the affinity of lectins for sugar chains.
- Examples of the measurement method, analysis method, or detection method using the affinity of lectin for sugar chains described above include surface plasmon resonance method, capillary electrophoresis, lectin affinity chromatography, lectin microarray method, ELISA, tissue staining, electrophoresis Method, flow cytometry, and the like.
- the surface plasmon resonance method or capillary electrophoresis is preferable.
- each component such as the polysaccharide according to the present invention used in each method, a substance having a sugar chain, a sample containing a substance having a sugar chain, and a lectin, a solvent used in a solution of each component, The concentration of each component in the solution, reaction conditions, and the like are as described in the above description of the complex forming method of the present invention.
- the solution containing the complex until the target reaction or the target measurement / detection ends (for example, until the measurement / detection ends when the measurement / detection of the complex is the target). It is preferable that the polysaccharide according to the present invention coexists therein.
- the kind of the solution and the concentration of the polysaccharide in the solution are as described above.
- washing is performed using a washing solution containing the polysaccharide according to the present invention. It is preferable to carry out the treatment. In addition, it is preferable to measure the amount of the complex in the presence of the polysaccharide according to the present invention.
- SPR surface plasmon resonance
- Examples of methods for applying the complex forming method of the present invention to the surface plasmon resonance method include the following methods.
- a lectin having affinity for a sugar chain of a substance having a sugar chain to be measured is immobilized on the sensor chip.
- a solution containing the sample to be measured and the polysaccharide according to the present invention may be prepared and flowed from the flow path of the surface plasmon resonance spectrometer to the surface of the sensor chip on which the lectin is immobilized.
- any method can be used as long as the solution is finally obtained.
- examples thereof include a method of adding the polysaccharide according to the present invention or a solution thereof to the sample, and a method of dissolving the sample and the polysaccharide according to the present invention in water or a suitable buffer solution.
- a “solution containing the sample to be measured and the polysaccharide according to the present invention” related to other measurement methods described later can also be obtained by the same method.
- the measurement by the surface plasmon resonance method may be performed under the optimum conditions for the measurement according to the instruction manual attached to the surface plasmon resonance spectrometer.
- the concentration of the polysaccharide according to the present invention in the solution containing the substance having a sugar chain and the polysaccharide according to the present invention flowing from the flow path of the apparatus is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)%.
- the complex formation method of the present invention When the complex formation method of the present invention is carried out, more complex of a substance having a sugar chain and a lectin can be obtained as compared with the conventional surface plasmon resonance method. Therefore, by applying the complex formation method of the present invention to the conventional surface plasmon resonance method and confirming or analyzing the lectin bound to the substance having the sugar chain, more information on the structure of the sugar chain possessed by the substance can be obtained. Accurate and highly sensitive.
- Biacore TM method is a typical analysis system for surface plasmon resonance.
- the Biacore method is generally simply referred to as Biacore.
- Biacore may refer to a Biacore device used in a Biacore analysis system.
- the LCA is fixed on the via core dedicated chip (sensor chip) by a conventional method.
- a buffer solution for example, HBS-EP, which is a running buffer dedicated to Biacore
- HBS-EP running buffer dedicated to Biacore
- This solution is sent to the sensor chip for reaction at, for example, 10 to 50 ° C., preferably 20 to 40 ° C., and a flow rate of 10 to 50 ⁇ L / min.
- the same measurement is performed using a buffer, a buffer containing AFP, a buffer containing AFP and the polysaccharide according to the present invention, and the background value is adjusted based on the obtained value.
- the amount of AFP-L3 can be obtained by a conventional method.
- the amount of AFP-L3 may be determined using a calibration curve obtained by conducting measurement with Biacore in the same manner using AFP-L3 with a known concentration in advance.
- a complex of a substance having a sugar chain and a lectin having affinity for the sugar chain of the substance having a sugar chain is formed, and the resulting complex is subjected to capillary electrophoresis.
- the amount of the substance having a sugar chain can be measured.
- Examples of methods for applying the complex forming method of the present invention to capillary electrophoresis include the following [Method 1] and [Method 2].
- Method 1 “The sample to be measured is reacted with an antibody that specifically binds to the substance having the sugar chain to be measured to obtain an antigen-antibody complex of the substance having the sugar chain and the antibody.
- the substance according to the present invention is subjected to capillary electrophoresis in the presence of a lectin having affinity for the sugar chain of the polysaccharide and the substance having the sugar chain, and the substance having the sugar chain has an affinity for the sugar chain of the lectin
- the substance having a sugar chain is measured by separating and measuring based on the degree of ".”
- an antibody that specifically binds to a substance having a sugar chain one kind of antibody that specifically binds to a substance having a sugar chain may be used, or it specifically binds to a substance having a sugar chain, but they are different from each other. Two or more antibodies that recognize the epitope may be used.
- the antibody that specifically binds to the substance having a sugar chain includes an antibody that specifically binds to the protein portion (core protein) of the glycoprotein.
- the antibody may be labeled with a detectable labeling substance.
- either antibody may be labeled with a charge carrier molecule such as an anionic substance.
- Charged carrier molecules include nucleic acid chains such as DNA.
- nucleic acid chain used for such a purpose any nucleic acid chain that is usually used in capillary electrophoresis may be used, and the method for binding the nucleic acid to an antibody may be a method known per se.
- the antibody may be labeled with both a charge carrier molecule and a detectable labeling substance.
- Capillary electrophoresis may be electrophoresis performed on a capillary chip ((micro) chip capillary electrophoresis).
- the electrophoresis solution, electrophoresis conditions, operation method, reaction conditions, etc. used for capillary electrophoresis according to the present invention may be the same as those known per se except that the capillary electrophoresis is performed in the presence of the polysaccharide according to the present invention. .
- the polysaccharide and lectin according to the present invention may be contained in advance in the electrophoresis solution serving as the mobile phase. Then, the antigen-antibody complex of the substance having a sugar chain and the antibody and the lectin are reacted in the presence of the polysaccharide according to the present invention in the flow path of the fully automatic measuring apparatus, and then capillary electrophoresis is performed. Good. Otherwise, capillary electrophoresis may be performed according to the method described in the instruction manual under the conditions described in the instruction manual attached to the apparatus.
- Examples of a fully automatic measuring apparatus for capillary electrophoresis include Mutus Wako i30 (manufactured by Wako Pure Chemical Industries, Ltd.).
- the concentration of the polysaccharide according to the present invention in the electrophoresis solution is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 1 (w / v)% to 15 (w / v)%, still more preferably 4 to 10 (w / v)%.
- the concentration of the lectin in the electrophoresis solution is higher than the amount at which the sugar chain of the substance having a sugar chain and the lectin can be completely bound during the separation by capillary electrophoresis. It is desirable. That is, the concentration of the lectin is 1 ⁇ g / mL to 30 mg / mL, preferably 10 ⁇ g / mL to 20 mg / mL, more preferably 1 to 10 mg / mL.
- the concentration of the polysaccharide according to the present invention in the solution is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% -25 (w / v)%, more preferably 1 (w / v)% to 15 (w / v)%, still more preferably 4 to 10 (w / v)%.
- the concentration (concentration in the microchannel) is 1 ⁇ g / mL to 30 mg / mL, preferably 10 ⁇ g / mL to 20 mg / mL, more preferably 1 to 10 mg / mL.
- the concentration of the polysaccharide and lectin according to the present invention in the electrophoresis solution, the substance having a sugar chain and the labeled lectin are brought into contact with each other in the presence of the polysaccharide according to the present invention and reacted with the present invention in the reaction solution.
- the concentration of the polysaccharide if necessary, the concentration of the polysaccharide according to the present invention in the solution (concentration in the microchannel) and the concentration of the lectin when separating and detecting the complex
- concentration of the lectin in the solution (concentration in the microchannel) when separating and detecting the complex is the same as in [Method 1] described above.
- PSA can be separated and measured by the difference in sugar chain by performing the complex formation method and capillary electrophoresis method of the present invention.
- the complex formation method of the present invention By applying the complex formation method of the present invention to capillary electrophoresis, it is possible to obtain more complex of lectin and a substance having a sugar chain, as compared with the conventional method.
- the signal value (peak area etc.) of the peak of the detected complex increases. As a result, a substance having a sugar chain with higher sensitivity can be measured.
- PSA such as ⁇ (2,3) sugar chain PSA has a binding type bound to a binding protein such as ⁇ 1-antichymotrypsin and ⁇ 2-macroglobulin, and a free type not bound to the binding protein. .
- a labeling substance for example, a fluorescent substance
- anti-PSA antibody 1 capable of detecting an anti-PSA antibody (anti-PSA antibody 1) that specifically binds to free PSA in a liquid phase.
- PSA binds to fluorescently labeled anti-PSA antibody 1 to form the following two types of complexes.
- ⁇ [Labeled anti-PSA antibody 1- ⁇ (2, 3) sugar chain free PSA] complex • [Labeled anti-PSA antibody 1- ⁇ (2, 3) sugar chain free PSA other than free PSA] complex
- anti-PSA antibody 2 obtained by labeling anti-PSA antibody (anti-PSA antibody 2) that specifically binds to PSA with DNA 2 to 50 ⁇ L of the test solution, electrophoresis buffer, and internal standard substance (for example, fluorescent substance: HiLyte647 [AnaSpec), etc.) are applied at a pressure of 1 to 10 psi for 30 to 60 seconds.
- reaction solution containing the complex 1 and the complex 2 is mixed with MAA (1 ⁇ g / mL to 30 mg / mL) and polysaccharide (0.01 (w / v)%) according to the present invention such as sodium dextran sulfate or c sodium sodium chondroitin sulfate.
- polysaccharide 0.01 (w / v)%) according to the present invention such as sodium dextran sulfate or c sodium sodium chondroitin sulfate.
- electrophoresis buffer mobile phase
- Complex 1 interacts with MAA, but complex 2 does not interact with MAA, so complex 1 migrates slower than complex 2. Therefore, the appearance position of the peak of the labeling substance derived from the labeled anti-PSA antibody 1 to be detected differs between the complex 1 and the complex 2. Therefore, the peak of the complex 1 and the peak of the complex 2 can be known based on the positions of the peaks.
- the amount of ⁇ (2, 3) sugar chain free PSA can be determined based on the peak area and peak height of Complex 1.
- the complex detected is increased by reacting complex 1 and complex 2 with MAA in the presence of the polysaccharide according to the present invention, so that the ⁇ (2,3) sugar is more sensitive. Chain free PSA can be measured.
- anti-PSA antibody 1 an antibody that specifically binds to free PSA
- anti-PSA antibody 2 an antibody that specifically binds to PSA
- anti-PSA antibody 1 an antibody that specifically binds to free PSA
- examples of commercially available anti-PSA antibody 1 include, for example, Anti PSA monoclonal antibody 8A6 (HyTest), Anti PSA monoclonal antibody (PS1) (HyTest), and Anti PSA monoclonal antibody. (Clone 108) (Anogen), Anti-Prostate Specific Antigen Antibody (PS2) (Abcam), Anti-Prostate Specific Antigen Antibody (2H9) (Abcam), and the like.
- PSA antibody 2 may be an antibody that can bind to both free PSA and bound PSA.
- an antibody that specifically binds to the core protein of PSA can be mentioned.
- the commercially available products include Anti PSA monoclonal antibody 5A6 (HyTest), Anti PSA monoclonal antibody 5G6 (HyTest), Anti PSA monoclonal antibody (PS6) (HyTest), Anti-Prostate Specific Antigen antibody (EP1588Y) (Abcam) ), Anti-Prostate Specific Antigen antibody (A67-B / E3) (Abcam), Anti-Prostate Specific Antigen antibody (35H9) (Abcam), Anti-Prostate Specific Antigen antibody (KLK3 / 801) (Abcam), Anti-Prostate Specific Antigen antibody (3E6) (Abcam), Anti-Prostate Specific Antigen antibody (8301) (Abcam), Anti-Prostate Specific Antigen antibody (A5D5) (Abcam), Anti-Prostate Specific Antigen antibody (PSA) 28 / A4) (Ab
- Lectin affinity chromatography is often used as a method for purifying glycoproteins, glycopeptides, sugar chains and the like.
- a sample is passed through a column packed with a packing material in which a lectin having affinity for a sugar chain of a substance having a sugar chain to be measured is immobilized on a solid phase such as agarose. If there is a substance having the sugar chain to be measured in the sample, the substance having the target sugar chain is separated and measured based on the elution delay of the substance having the sugar chain caused by the interaction between the sugar chain and the lectin. To do.
- Examples of a method for applying the complex forming method of the present invention to lectin affinity chromatography include a method of performing normal lectin affinity chromatography using a mobile phase containing the polysaccharide according to the present invention.
- the concentration of the polysaccharide according to the present invention in the mobile phase is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably Is 0.5 (w / v)% to 15 (w / v)%, more preferably 0.5 to 10 (w / v)%.
- the complex formation method of the present invention can be applied to the lectin microarray method developed by National Institute of Advanced Industrial Science and Technology (AIST), Glycomedical Engineering Research Center.
- AIST Advanced Industrial Science and Technology
- a lectin array is an array in which dozens of lectins having different specificities (affinities) are arranged in a spot shape on a glass slide and immobilized.
- Examples of methods for applying the complex forming method of the present invention to the lectin microarray method include the following [Method 1] and [Method 2].
- Method 1 After a solution containing a substance having a sugar chain and the polysaccharide according to the present invention interacts with a lectin microarray, the microarray is fluorescently labeled with an antibody that specifically binds to the substance having a sugar chain React with. Then, by detecting the fluorescence derived from the fluorescent-labeled antibody in the same manner as described above, it is possible to know the lectin to which the substance having a sugar chain is bound, and based on the result, the sugar chain of the substance having a sugar chain Can be analyzed.
- Method 2 A solution containing a labeled substance of a substance having a sugar chain obtained by labeling a substance having a sugar chain with a detectable labeling substance such as a fluorescent substance and the polysaccharide according to the present invention is allowed to interact with a lectin microarray, and then excited light. To generate an evanescent field. Next, by detecting the signal derived from the label of the substance having a sugar chain, the lectin to which the substance having the sugar chain is bound can be known. Based on the result, the sugar chain of the substance having the sugar chain is determined. Can be analyzed.
- the concentration of the polysaccharide according to the present invention in the solution containing the substance having a sugar chain and the polysaccharide according to the present invention is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 ( w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)%.
- Measurement with a lectin microarray may be carried out according to the protocol described in, for example, MICROARRAYMETHODS AND PROTOCOLS (CRC Press), edit by Robert S. Matson, “” Chapter 9: Lectin Microarrays ”, Masao Yamada, p.141, 2009.
- the lectin microarray method is a method that can detect the fluorescence of the lectin array without performing a washing operation.
- it is a sugar chain having a weak affinity for lectins.
- a larger amount of sugar chain-lectin complex is maintained on the microarray. Therefore, information on the sugar chain can be obtained with higher accuracy.
- immunological fluid measurement method When the complex formation method of the present invention is applied to the immunological fluid measurement method (sandwich method), it has a sugar chain in the presence of the polysaccharide according to the present invention. Measurement may be performed according to a known immunological measurement method (sandwich method) except that the substance and lectin are reacted.
- Examples of the method for applying the complex forming method of the present invention to the sandwich method using a solid phase include the following [Method 1] and [Method 2].
- [Method 1] A lectin having affinity for a sugar chain of a substance having a sugar chain to be measured is immobilized on a solid phase.
- a solution containing the sample to be measured and the polysaccharide according to the present invention is brought into contact with the solid phase and reacted.
- the solution containing the polysaccharide according to the present invention may be allowed to react with the solid phase in the order of the sample to be measured, the sample to be measured, and the solution containing the polysaccharide according to the present invention in order. You may make it react by making it contact with the said solid-phase.
- a solution of labeled antibody labeled with a labeling substance capable of detecting an antibody that specifically binds to the substance having the sugar chain to be measured is brought into contact with the solid phase and reacted.
- a labeling substance capable of detecting an antibody that specifically binds to the substance having the sugar chain to be measured is brought into contact with the solid phase and reacted.
- an antibody specific for the core protein may be used.
- the solid phase is washed and unreacted labeled antibody is removed.
- the amount of labeling substance derived from the complex of [lectin-substance with sugar chain-labeled antibody] formed on the solid phase is measured by a measuring method according to the labeling substance of the labeled antibody. Based on the result, a substance having a sugar chain is measured.
- Method 2 An antibody that specifically binds to a substance having a sugar chain to be measured is immobilized on a solid phase.
- the substance having a sugar chain is a glycoprotein
- an antibody specific for the core protein may be used.
- the sample to be measured is brought into contact with the solid phase and reacted.
- the polysaccharide according to the present invention and a labeled lectin labeled with a labeling substance capable of detecting a lectin having affinity for the sugar chain of the substance having the sugar chain to be measured are contained.
- the solution is brought into contact with the solid phase and allowed to react.
- the solution containing the polysaccharide according to the present invention, and the solution containing a labeled lectin labeled with a labeling substance capable of detecting a lectin having affinity for the sugar chain of the substance having a sugar chain are contacted with the solid phase in this order.
- the amount of the labeled substance derived from the [antibody-substance with sugar chain-labeled lectin] complex formed on the solid phase is measured by a method according to the labeled substance of the labeled lectin. Based on the result, a substance having a sugar chain is measured.
- the concentration of the polysaccharide according to the present invention in the reaction solution when the sample to be measured is reacted with the lectin is 0.01 (w / v)% to 50 (w / v). )%, Preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v) )%is there.
- the washing solution used in [Method 1] and [Method 2] for washing the solid phase after each reaction and the labeled antibody solution used in [Method 1] contain the polysaccharide according to the present invention. Preferably it is.
- the concentration of the polysaccharide according to the present invention in the cleaning liquid is as described above.
- the amount of the labeled substance derived from the complex of [antibody-substance with sugar chain-labeled lectin] is preferably measured.
- the concentration of the polysaccharide according to the present invention at the time of measurement may be the same as the concentration in the reaction solution described above.
- the sample to be measured is subjected to gel electrophoresis by a conventional method and then transferred to a membrane such as a PVDF membrane.
- a membrane such as a PVDF membrane.
- the obtained membrane contains a labeled lectin labeled with a labeling substance capable of detecting a lectin having affinity for the sugar chain of the substance having a sugar chain and the polysaccharide according to the present invention. It is immersed in a solution to form a complex of a substance having a sugar chain and a labeled lectin on the membrane. Subsequently, a substance having a sugar chain can be detected and measured with high sensitivity by detecting the labeling substance by a measurement method according to the labeling substance.
- a biotin-labeled lectin obtained by labeling a lectin having affinity for a sugar chain of a substance having a sugar chain with biotin as the labeled lectin is used as follows. First, a sample containing a substance having a sugar chain is transferred to a membrane after gel electrophoresis. The membrane is immersed in a solution containing a biotin-labeled lectin and the polysaccharide according to the present invention to form a complex of a substance having a sugar chain and a biotin-labeled lectin on the membrane. Next, the membrane is immersed in an HRP-labeled avidin solution (which may contain the polysaccharide according to the present invention). Further, the membrane is dipped in a coloring solution to cause color development. By detecting the color development, a substance having a sugar chain is detected and measured.
- the concentration of the polysaccharide according to the present invention in the solution containing the labeled lectin and the polysaccharide according to the present invention or the solution containing the biotin labeled lectin and the polysaccharide according to the present invention used above is 0.01. (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v) %, More preferably 0.5 to 10 (w / v)%.
- the complex formation method of the present invention By applying the complex formation method of the present invention to electrophoresis, the amount of the complex of the substance having a sugar chain formed on the membrane and the labeled lectin increases, so that the signal derived from the labeled lectin of the complex The value increases. As a result, it is possible to detect and measure a substance having a sugar chain with higher sensitivity.
- Tissue sections prepared by a conventional method are labeled lectins obtained by labeling a lectin having affinity for a sugar chain to be detected with a detectable labeling substance such as a fluorescent substance or a radioactive substance, and many of the present invention.
- a detectable labeling substance such as a fluorescent substance or a radioactive substance
- the concentration of the polysaccharide according to the present invention in the solution containing the labeled lectin used for the tissue staining and the polysaccharide according to the present invention is 0.01 (w / v)% to 50 (w / v)%, preferably Is 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)% .
- a lectin labeled with a labeling substance capable of detecting a lectin having affinity for the sugar chain of the target (target) present in the cell membrane is bound to the cell, and a cell separator (cell sorter) is used.
- a cell separator cell sorter
- subpopulations of cells can be fractionated based on the types of sugar chains on the cell surface (on the cell membrane) using the sugar chain specificity of lectins.
- Examples of a method for applying the complex forming method of the present invention to the method include the following methods.
- a solution containing a labeled lectin obtained by labeling a lectin having affinity for a target sugar chain (a sugar chain that the cell is supposed to have on the cell membrane) with a fluorescent substance and the polysaccharide according to the present invention Contact is made to form a complex of the sugar chain on the cell membrane and the fluorescently labeled lectin. Thereafter, cells that have bound to the labeled lectin are separated from cells that have not bound to the labeled lectin by a method of detecting fluorescence using a normal cell sorter.
- the separation liquid for separating cells with a cell sorter may contain the polysaccharide according to the present invention.
- the concentration of the polysaccharide according to the present invention when the cells are brought into contact with the solution containing the fluorescently labeled lectin and the polysaccharide according to the present invention is 0.01 (w / v)% to 50 (w / v)%, preferably Is 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)% .
- the concentration of the polysaccharide according to the present invention in the separation solution is 0.01 (w / v)% to 50 (w / v)%, preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v)%.
- the amount of the complex of cells and lectins having a substance having a sugar chain is increased.
- the amount increases. That is, cells having the target sugar chain can be efficiently recovered.
- a complex of undifferentiated cells and labeled lectin is formed by the complex forming method of the present invention.
- undifferentiated cells and differentiated cells can be efficiently separated by separating undifferentiated cells and differentiated cells with a cell sorter.
- a blood type (subtype) determination method using a lectin Other techniques to which the complex formation reaction of the present invention can be applied include, for example, a blood type (subtype) determination method using a lectin. The determination may be carried out using a blood type determination reagent added with the polysaccharide according to the present invention.
- the method for measuring a substance having a sugar chain of the present invention is “to form a complex of a substance having a sugar chain and a lectin by the complex forming method of the present invention. After that, the method for measuring a substance having a sugar chain, wherein the amount of the complex is measured. " The details of the complex forming method of the present invention in the method for measuring a substance having a sugar chain of the present invention are as described in the above-mentioned “complex forming method of the present invention”.
- the substance having a sugar chain may be measured by measuring the amount of the complex.
- Specific methods for measuring the complex include, for example, the surface plasmon resonance method, capillary electrophoresis, lectin affinity chromatography, lectin microarray method, immunological measurement method such as sandwich method, electrophoresis method and the like. .
- the specific conditions and methods are as described in the explanation of each method described above.
- complex formation promoter of the present invention has an affinity for “a substance having a sugar chain and a sugar chain of the substance having the sugar chain, comprising the polysaccharide of the present invention. It is a complex formation promoter with a lectin having a property. "
- a water-soluble compound having a polysaccharide without glucosamine can be mentioned.
- the preferred embodiments and specific examples thereof are also as described above. Specific examples include dextran sulfate or a salt thereof, chondroitin sulfate c sodium or a salt thereof, and the like.
- the concentration of the polysaccharide according to the present invention in the complex formation accelerator according to the present invention is 0.01 (w / v)% to 50 (w / v) %, Preferably 0.1 (w / v)% to 25 (w / v)%, more preferably 0.5 (w / v)% to 15 (w / v)%, still more preferably 0.5 to 10 (w / v) %.
- the complex formation accelerator of the present invention is a solution
- specific examples of the solvent used in the solution include water or a buffer solution.
- the water used as the solvent is not particularly limited as long as it is water used in this field, and specific examples thereof include purified water such as distilled water and deionized water.
- buffer used as a solvent examples include, for example, Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good buffer, Tris-HCl buffer, MES buffer, HEPES buffer, boron
- examples include an acid buffer, a phosphate buffer, a veronal buffer, and a Good buffer.
- the buffering agent concentration of these buffers is appropriately selected from the range of usually 5 to 1,000 mM, preferably 5 to 300 mM.
- the pH is not particularly limited, and examples thereof include a range of 5 to 9.
- the complex formation promoter of the present invention may contain a lectin.
- a lectin is as described in the explanation relating to the complex forming method of the present invention.
- LCA, MAA, BC2LCN and the like can be mentioned.
- the concentration of the lectin in the complex formation accelerator according to the present invention is 1 ⁇ g / mL to 30 mg / mL, preferably 10 ⁇ g / mL to 20 mg / mL, more preferably 1 to 10 mg / mL.
- the complex formation accelerator of the present invention contains the polysaccharide of the present invention and a lectin
- the polysaccharide of the present invention has an affinity for the lectin as described in the complex formation method of the present invention. It is selected from those that do not have a sugar chain.
- Specific examples of the complex formation accelerator of the present invention containing the polysaccharide of the present invention and lectin include, for example, those having the following compositions.
- the complex formation accelerator of the present invention is a reagent commonly used in this field, such as a buffer, a reaction accelerator, a protein, a salt, a stabilizer such as a surfactant, a preservative, and the like, and coexists. Those that do not inhibit the stability of the reagent or the like and do not inhibit the reaction between the sugar chain and the lectin may be included. Also, the concentration may be appropriately selected from the concentration range usually used in this field.
- the complex formation accelerator of the present invention may be combined with instructions for using the complex formation accelerator.
- the “instructions” are the instruction manuals and attachments of the co-complex formation accelerator of the present invention, in which the characteristics, usage, etc. of the complex formation accelerator of the present invention are substantially described by sentences or diagrams. It means a document or a pamphlet (leaflet).
- Example 1 Detection of AFP-L3 (1) Measuring instrument, etc.
- Measuring instrument Biacore X (manufactured by GE Healthcare UK Ltd.)
- Chip Sensor Chip CM5 (GE Healthcare UK Ltd.)
- Running buffer HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P 20, pH7.4, manufactured by GE Healthcare UK Ltd.)
- AFP-L3 LCA binding fraction of ⁇ -fetoprotein, manufactured by Wako Pure Chemical Industries, Ltd.
- dextran sulfate sodium MW 36,000-50,000, manufactured by Wako Pure Chemical Industries, Ltd.
- chondroitin sulfate c sodium MW 40,000-80,000, manufactured by Wako Pure Chemical Industries, Ltd.
- Sample solution The following sample solutions were prepared using the HBS-EP buffer and the above reagents. ⁇ 28 ⁇ g / mL AFP-L3 solution containing 1 ⁇ M (4w / v%) dextran sulfate sodium ⁇ 28 ⁇ g / mL AFP-L3 solution containing 1 ⁇ M (1w / v%) chondroitin sulfate c ⁇ 28 ⁇ g / mL AFP-L3 solution
- the horizontal axis represents time (s (seconds)), and the vertical axis represents response values (RU, Resonance Unit, resonance unit).
- the size of RU reflects the amount of complex of AFP-L3 and LCA.
- + indicates the results obtained using an AFP-L3 solution containing dextran sulfate sodium
- ⁇ indicates the results obtained using an AFP-L3 solution not containing dextran sulfate sodium.
- ⁇ indicates the results obtained using the AFP-L3 solution containing chondroitin sulfate c-sodium
- ⁇ indicates the results obtained using the AFP-L3 solution not containing chondroitin sulfate c-sodium.
- a 250 bp DNA fragment having an NH 2 group introduced at the 5 ′ end was purified by a conventional method (purified end aminated DNA), and then the NH 2 group introduced into this DNA fragment and sulfosuccinimidyl.
- a succinimide group of 4- (p-maleimidophenyl) butyrate (Sulfo-SMPB) linker (linker having a succinimide group and a maleimide group, manufactured by Pierce) was reacted in a conventional manner. Subsequently, gel filtration treatment was performed to remove unreacted linker, and a 250 bp DNA fragment to which the linker was bound was obtained.
- the obtained linker-bound 250 bp DNA fragment was reacted with an anti-PSA antibody 5G6 Fab ′ fragment prepared according to a conventional method using an anti-human PSA mouse monoclonal antibody (Anti PSA monoclonal antibody 5G6, HyTest).
- the obtained reaction product was purified using a DEAE column to prepare an anti-PSA antibody 5G6 Fab ′ fragment (hereinafter referred to as “DNA-labeled anti-PSA antibody”) to which a 250 bp DNA fragment was bound.
- Anti PSA monoclonal antibody 5G6 used is an antibody having affinity for human PSA and can bind to both bound PSA and free PSA. That is, the antibody can bind to both “ ⁇ (2, 3) sugar chain free PSA” and “free PSA other than ⁇ (2, 3) sugar chain free PSA”. 2) Preparation of fluorescently labeled anti-free PSA antibody Anti-human PSA monoclonal antibody that recognizes a PSA epitope different from Anti PSA monoclonal antibody 5G6 and specifically binds only to free PSA (Anti PSA monoclonal antibody 8A6, HyTest Was processed by a conventional method to obtain an anti-PSA antibody 8A6 Fab ′ fragment.
- HiLyte647 (manufactured by AnaSpec) is introduced into the amino group of the obtained fragment by a conventional method, and HiLyte647-labeled anti-free PSA antibody 8A6 Fab ′ fragment (hereinafter referred to as “fluorescent-labeled anti-free PSA antibody” ).
- microchip capillary electrophoresis Electrophoresis (microchip capillary electrophoresis) Using a fully automatic fluorescence immunoassay device Mutus Wako i30 (manufactured by Wako Pure Chemical Industries, Ltd.), microchip capillary electrophoresis was performed according to the procedure shown below according to the instruction manual of the device.
- the concentration of PSA in the obtained r-free PSA solution was measured and diluted with PBS (-) (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a sample solution prepared to have a concentration of 1 ng / mL PSA protein. . 2 ⁇ L of the obtained sample solution, 1 ⁇ L of 1 ⁇ M fluorescence-labeled anti-free PSA antibody prepared in (1) 2) above, and electrophoresis buffer 1 [5% (w / v) polyethylene glycol (PEG 20000), 3% ( w / v) Contains glycerol, 150 mM NaCl, 0.01% BSA, 75 mM Tris-HCl, 10 mM MES. pH 7.5] 7 ⁇ L was added to a 0.5 mL tube and mixed to prepare a 10 ⁇ L reaction solution.
- the final concentration of the fluorescence-labeled anti-free PSA antibody in this reaction solution is 100 nM.
- Running buffer 2 (contains MAA and chondroitin sulfate c sodium) A 75 mM Tris-HCl buffer (pH 7.5) containing 5.0% (w / v) polyethylene glycol (PEG8000), 3% (w / v) glycerol, 10 mM NaCl, 0.01% BSA was prepared.
- MAA manufactured by VECTOR
- VECTOR has a final concentration (concentration in electrophoresis buffer 2) of 4 mg / mL, and chondroitin sulfate c sodium (manufactured by Wako Pure Chemical Industries, Ltd.) as a polysaccharide according to the present invention.
- the final concentration (w / v) (concentration in running buffer 2) is 0%, 2.8%, 3.4%, 3.5%, 3.6%, 3.8%, 4.1%, 4.3%, 4.5%, respectively.
- the mixture was added and mixed to prepare running buffer 2.
- Running buffer 3 A buffer containing 2% (w / v) polyethylene glycol (PEG 20000), 3% (w / v) glycerol, 0.01% BSA, 125 mM HEPES, 75 mM Tris-HCl (no pH adjustment) was used as running buffer 3 .
- Running buffer 4 The running buffer 4 was 75 mM Tris-HCl buffer (pH 7.5) containing 2% (w / v) polyethylene glycol (PEG 20000), 3% (w / v) glycerol, and 0.01% BSA.
- DNA labeled antibody solution (containing DNA labeled anti-PSA antibody)
- -Fluorescent solution 30 nM HiLyte 647, 20 mM (w / v) 50 mM BisTris (pH 6.0) containing glycerol was used as the fluorescent solution.
- the fluorescent solution is used for adjustments such as position confirmation at the detector of the measuring instrument (Mutus Wako i30).
- Electrophoresis procedure i) Introduction of electrophoresis sample A and electrophoresis sample solution 5.4 ⁇ L of electrophoresis sample A prepared in (2) 1) above is dispensed into a predetermined well (SP well) of a Mutus Wako i30 dedicated microchip. Noted. Subsequently, each test solution prepared in the above (2) 2) was dispensed into each well of the microchip as described below.
- R2 well R2 (FLB) well, R2 (LB) well
- running buffer 2 containing MAA and chondroitin sulfate c sodium
- R3 well 10.0 ⁇ L of running buffer 3 -R4 well: 5.4 ⁇ L of running buffer 4
- C1 well 3.0 ⁇ L of DNA labeled antibody solution
- ⁇ FD well 7.0 ⁇ L of fluorescent solution.
- FIG. 1 A schematic diagram of the microchip used is shown in FIG. 1
- the waste well is used as a waste liquid reservoir (drain well) when the sample solution for each well (R2, R3, R4, C1) and the sample A for electrophoresis are introduced into the analysis channel.
- FIG. 4 shows a schematic diagram of the flow path in the microchip used.
- W indicates a waste well.
- the R3 well side is the cathode and the R2 (LB) well side is the anode.
- the arrangement portion of the electrophoresis sample A and each test solution is shown by color-coded into a dot portion and a white portion (portion without a dot).
- PSA was separated and detected by the following method.
- the immune reaction time for each labeled antibody for capturing free PSA was about 200 seconds.
- the complex formed here is a complex (complex 1) of [fluorescence labeled anti-free PSA antibody-r ⁇ (2, 3) sugar chain free PSA-DNA labeled anti-PSA antibody] and [ Fluorescently labeled anti-free PSA antibody-r ⁇ (2, -3) sugar chain free PSA other than free PSA-DNA labeled anti-PSA antibody] (complex 2).
- R2 (FLB) and R2 (LB) are subjected to isotachophoresis up to the channel cross part, and the complex has passed through the channel cross part. Switched to. Further, in the detection part (capillary part 2 cm downstream from the channel crossing part of R2 (FLB) and R2 (LB)), [fluorescence-labeled anti-free PSA antibody-r-free PSA-DNA-labeled anti-PSA antibody] complex Capillary gel electrophoresis (CE) was performed in the presence of MAA and chondroitin sulfate c sodium until a body peak was detected. The position where CE was performed and the electrophoresis direction of electrophoresis are indicated by “CE” and a dotted line in FIG.
- CE Capillary gel electrophoresis
- complex 1 and complex 2 come into contact with MAA and react in the presence of chondroitin sulfate c-sodium.
- concentration of MAA in the reaction solution is 4 mg / mL
- concentration of chondroitin sulfate c-sodium is 0%, 2.8%, 3.4%, 3.5%, 3.6%, 3.8%, 4.1% 4.3% or 4.5%.
- the detection was performed by measuring the fluorescence intensity of the capillary part 2 cm from the channel cross part of R2 (FLB) and R2 (LB) with a photodiode (Fuji Film Co., Ltd.) with 635 nm laser excitation over time.
- the vertical axis represents the peak area of the complex 1 fraction
- the horizontal axis represents the concentration of chondroitin sulfate c sodium (% (w / v%)) during the reaction of MAA with complex 1 and complex 2.
- the peak area of the complex 1 increased in a concentration-dependent manner with c sodium chondroitin sulfate.
- the amount of the complex 1 is reduced by performing the reaction between the sugar chain and the lectin in the presence of chondroitin sulfate c-sodium, compared to the case where the reaction is performed in the absence of chondroitin sulfate c-sodium. It can be seen that it increases.
- the electrophoresis reagent 2 contains polyethylene glycol (PEG8000; 5%), under conditions where the chondroitin sulfate c-sodium concentration is “0 (w / v)%”, the presence of polyethylene glycol and chondroitin sulfate c-sodium In the absence of this, the substance having a sugar chain reacts with the lectin. As is clear from FIG. 5, when the reaction between a substance having a sugar chain and a lectin was carried out in the presence of polyethylene glycol and in the absence of c sodium chondroitin sulfate, the complex 1 was hardly detected.
- Example 3 Detection of ⁇ (2, 3) sugar chain free PSA-2
- Preparation of sample and sample solution, and electrophoresis (microchip capillary electrophoresis) Except that the following electrophoresis buffer 2 was used, the same electrophoresis reagent, equipment, etc. as used in Example 2 were used, and the same method as in Example 2 was used to [fluorescently labeled anti-free PSA antibody- ⁇ (2 , 3) Sugar chain free PSA-DNA labeled anti-PSA antibody] complex (complex 1) was detected.
- -Electrophoresis buffer 2 (containing polysaccharide according to the present invention)
- a 75 mM Tris-HCl buffer (pH 7.5) containing 5.0% (w / v) polyethylene glycol (PEG8000), 3% (w / v) glycerol, 10 mM NaCl, 0.01% BSA was prepared.
- MAA manufactured by VECTOR
- a final concentration concentration in electrophoresis buffer 2 of 4 mg / mL
- dextran sulfate sodium MW 6,500 to 10,000
- concentration in electrophoresis buffer 2 (w / v) of 0%, 3.0%, 5.0%, 5.5%, 6.0%, 6.7%, 7.0%, 7.7%, 8.1% Then, a mixture was prepared and used as running buffer 2.
- the concentration of MAA in the reaction solution was 4 mg / mL, and the concentration of dextran sulfate sodium (w / v%) was 0%, 3.0%, 5.0%, 5.5%, 6.0%, 6.7%, 7.0%, 7.7%, or 8.1%.
- the vertical axis indicates the peak area of the complex 1 fraction
- the horizontal axis indicates the concentration (% (w / w) of the polysaccharide (dextran sulfate) of the present invention during the reaction of MAA with complex 1 and complex 2. v%)).
- the peak area of Complex 1 increased depending on the concentration of sodium dextran sulfate.
- the amount of the complex 1 is increased by performing the reaction between the sugar chain and the lectin in the presence of dextran sulfate sodium as compared to the case where the reaction is performed in the absence of dextran sulfate sodium. I understand that.
- the electrophoresis reagent 2 contains polyethylene glycol (PEG8000; 5%), under the condition that the sodium dextran sulfate concentration is “0 (w / v)%”, the non-existence of dextran sulfate sodium in the presence of polyethylene glycol. In the presence, the sugar chain reacts with the lectin. As is clear from FIG. 6, when the reaction between a substance having a sugar chain and lectin was carried out in the presence of polyethylene glycol and in the absence of sodium dextran sulfate, complex 1 was hardly detected.
- PEG8000 polyethylene glycol
- the amount of the complex of a substance having a sugar chain and a lectin increases.
- highly sensitive measurement of a substance having a sugar chain can be performed by applying the complex forming method of the present invention to the measurement of a substance having a sugar chain.
- the complex formation method of the present invention can be used for all reactions, detection, measurement, sugar chain analysis, etc. using the affinity of lectins for sugar chains. And the sensitivity of a detection or a measurement can be improved, and a sugar chain can be analyzed with high accuracy.
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Abstract
Description
(1)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物の存在下に、糖鎖を有する物質を含有する試料と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとを接触させる、該糖鎖を有する物質と該レクチンとの複合体形成方法。
(2)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、該レクチンが親和性を有する糖鎖を持たないものである、上記(1)に記載の方法。
(3)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、デキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩である、上記(1)又は(2)に記載の方法。
(4)下記(i)~(ii)から選択される、上記(1)、(2)、(3)のいずれかに記載の方法 (i)N-アセチルグルコサミンを有さない水溶性多糖がデキストラン硫酸又はその塩で、糖鎖を有する物質がαフェトプロテイン-L3(AFP-L3)で、レクチンがレンズマメレクチンである、
(ii)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物がデキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩で、糖鎖を有する物質がα(2, 3)糖鎖遊離型前立腺特異抗原で、レクチンがイヌエンジュレクチンである。
(5)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物を含有してなる、糖鎖を有する物質と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとの複合体形成促進剤。
(6)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、該レクチンが親和性を有する糖鎖を持たないものである、上記(5)に記載の複合体形成促進剤。
(7)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、デキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩である、上記(5)又は(9)6に記載の複合体形成促進剤。
(8)下記(i)~(ii)から選択される、上記(5)、(6)、(7)のいずれかに記載の複合体形成促進剤:
(i)N-アセチルグルコサミンを有さない水溶性多糖がデキストラン硫酸又はその塩で、糖鎖を有する物質がαフェトプロテイン-L3(AFP-L3)で、レクチンがレンズマメレクチンである、
(ii)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、デキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩で、糖鎖を有する物質がα(2, 3)糖鎖遊離型前立腺特異抗原で、レクチンがイヌエンジュレクチンである。
(9)上記(1)に記載の方法で糖鎖を有する物質とレクチンとの複合体を形成させた後、該複合体の量を測定する、糖鎖を有する物質の測定方法。
本発明の複合体形成方法は、「N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物の存在下に、糖鎖を有する物質を含有する試料と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとを接触させる、該糖鎖を有する物質と該レクチンとの複合体形成方法。」である。
本発明に係るN-アセチルグルコサミンを有さない水溶性多糖としては、一般に多糖といわれるものであって、構成糖としてN-アセチルグルコサミンを有さない、水溶性のものが挙げられる。
本発明に係るN-アセチルグルコサミンを有さない多糖を有する水溶性化合物としては、多糖にタンパク質や脂質が結合した所謂複合糖質であって、その多糖部分の構成糖としてN-アセチルグルコサミンを有さない、水溶性のものが挙げられる。
本発明に係る「糖鎖を有する物質」は、「レクチンが親和性を有する(結合する)糖鎖構造を有する糖鎖」を有する物質であればよく、特に限定されない。
(1)フコースが付加された糖鎖:肝臓癌患者で観察される、APF-L3の糖鎖であるFucα1→6GlcNac (コアフコース)(Kobayashi Y. et al., J. Biol. Chem., vol.287, No.41, pp. 33973-33982, October 5, 2012)等、
(2)末端にシアル酸が付加された糖鎖:肝臓癌患者で観察される、PSAの糖鎖であるSiaα2→3Gal(Chikara Oyama et al., Glycobiology, vol.14, No.8, pp.671-679, 2004、Yoneyama T. et al., Biochem. Biophys. Res. Commun., vol.448, No.4, pp.390-396, 2014, Tomokazu Ishikawa et al., J. Urology, vol.196, p.e331)等、
(3)N-アセチルガラクトサミンが付加された糖鎖:肝臓癌患者で観察される、PSAの糖鎖であるGalNacβ1-R(特許第5630757、Takatoshi Kaya, et al., Anal. Chem., vol. 87, No. 3, pp. 1797-1803, 2015)等、
が挙げられる。
本発明に係る糖鎖を有する物質を含有する試料としては、例えば血液、血清、血漿、尿、精液、髄液、唾液、汗、腹水、糞便懸濁液、腹水、組織抽出液、組織切片、組織生検試料、器官等の、ヒトや動物等の生体由来試料、又はこれらの生体由来試料から調製されたもの、ウイルス,細菌,細胞等の微生物又はこれらから調製されたものが挙げられる。
本発明で用いられるレクチンは、複合体を形成する対象となる糖鎖を有する物質の糖鎖に親和性を有し、該糖鎖を認識して該糖鎖に結合するレクチンであればよく、そのような性質を有するレクチンを、既知のレクチンから任意に選択すればよい。中でも、該糖鎖に特異的に結合するレクチンが好ましい。
本発明の複合体形成方法は、最終的に「本発明に係る多糖類を含有する、糖鎖を有する物質とレクチンとの複合体」が得られる方法であればよい。本発明に係る多糖類の存在下に、糖鎖を有する物質を含有する試料と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとを接触させる方法であって、最終的に「本発明に係る多糖類を含有する、糖鎖を有する物質とレクチンとの複合体の溶液」が得られる方法が好ましい。
(i)糖鎖を有する物質を含有する試料を、本発明に係る多糖類又は本発明に係る多糖類を含有する溶液と混合して混合液を得た後、得られた混合液を、該糖鎖を有する物質の糖鎖に親和性を有するレクチン又は該レクチンを含有する溶液と混合して反応させる方法、
(ii)糖鎖を有する物質の糖鎖に親和性を有するレクチン又は該レクチンを含有する溶液を、本発明に係る多糖類又は本発明に係る多糖類を含有する溶液と混合して混合液を得た後、得られた混合液を、糖鎖を有する物質を含有する試料と混合して反応させる方法、
(iii)糖鎖を有する物質を含有する試料を本発明に係る多糖類又は本発明に係る多糖類を含有する溶液と混合して混合液を得た後、得られた混合液を、該糖鎖を有する物質の糖鎖に親和性を有するレクチンと本発明に係る多糖類とを含有する溶液と混合して反応させる方法、
(iv)糖鎖を有する物質を含有する試料を、該糖鎖を有する物質の糖鎖に親和性を有するレクチン又は該レクチンを含有する溶液と混合して反応させた後、得られた反応液を、本発明に係る多糖類又は本発明に係る多糖類を含有する溶液と混合して混合液を得る方法、等が挙げられる。
すなわち、本発明の複合体形成方法に用いられる本発明に係る多糖類は、本発明の複合体形成方法に用いられるレクチンが親和性を有する糖鎖構造を持たないものを選択する。すなわち、ある糖鎖を有する物質とレクチンとの複合体を形成させる場合、該糖鎖を有する物質の糖鎖に親和性を有し、該糖鎖を認識して結合するレクチンを選択する。そして、本発明に係る多糖類としては、選択した該レクチンが親和性を有する糖鎖構造を持たないものを選択する。
または、本発明に係る多糖類が存在することにより、レクチンの糖鎖を有する物質の糖鎖に対する親和性が高くなり、そのため、本発明に係る多糖類の非存在下に糖鎖を有する物質とレクチンを接触させた場合よりも、本発明に係る多糖類の存在下に糖鎖を有する物質とレクチンを接触させた場合の方が、該複合体の量が増加する、ということが考えられる。
表面プラズモン共鳴法は、表面プラズモンが金属/液体界面で励起した場合に起こる、いわゆる表面プラズモン共鳴(Surface Plasmon Resonance = SPR)の光学現象を利用して生体分子間の相互作用を解析(測定)する分子間相互作用解析システムである。
本発明の複合体形成方法により糖鎖を有する物質と糖鎖を有する物質の糖鎖に親和性を有するレクチンとの複合体を形成させ、得られた複合体をキャピラリー電気泳動により分離し、該複合体の量を測定することにより、糖鎖を有する物質の量を測定することができる。
「測定対象の試料を、測定対象の糖鎖を有する物質に特異的に結合する抗体と反応させ、糖鎖を有する物質と抗体との抗原抗体複合体を得る。得られた抗原抗体複合体を、本発明に係る多糖類及び該糖鎖を有する物質の糖鎖に親和性を有するレクチンの存在下にキャピラリー電気泳動に付し、該糖鎖を有する物質を、該レクチンの糖鎖に対する親和性の程度に基づいて分離し、測定することにより、糖鎖を有する物質を測定する。」という方法である。
測定対象の試料と、該糖鎖を有する物質の糖鎖に親和性を有するレクチンを標識物質で標識した標識レクチンとを、上記した本発明の複合体形成方法に従って、本発明に係る多糖類の存在下に接触させ、反応させる。次いで、得られた反応液中の[標識レクチン-糖鎖を有する物質]の複合体を、キャピラリー電気泳動を行って分離する。キャピラリー電気泳動を行う際、本発明に係る多糖類を溶解させた泳動溶液を用いてもよい。そして、[標識レクチン-糖鎖を有する物質]の複合体由来の標識物質の量を測定することにより、試料中の糖鎖を有する物質の量を測定することができる。
・[標識抗PSA抗体1-α(2, 3)糖鎖遊離型PSA]複合体
・[標識抗PSA抗体1-α(2, 3)糖鎖遊離型PSA以外の遊離型PSA]複合体
複合体2:[標識抗PSA抗体1-α(2, 3)糖鎖遊離型PSA以外の遊離型PSA-DNA標識抗PSA抗体2]の複合体
糖タンパク質、糖ペプチド、糖鎖等を精製する方法として、レクチンアフィニティークロマトグラフィーがよく用いられる。
国立研究開発法人産業技術総合研究所糖鎖医工学研究センター等の開発したレクチンマイクロアレイ法に、本発明の複合体形成方法を適用することができる。
糖鎖を有する物質と本発明に係る多糖類とを含有する溶液をレクチンマイクロアレイと相互作用させた後、そのマイクロアレイを、糖鎖を有する物質に特異的に結合する抗体を蛍光標識した蛍光標識抗体と反応させる。次いで、上記と同様の方法で蛍光標識抗体由来の蛍光を検出することにより糖鎖を有する物質が結合したレクチンを知ることができるので、その結果をもとに、糖鎖を有する物質の糖鎖を解析することができる。
糖鎖を有する物質を蛍光物質等の検出可能な標識物質で標識した糖鎖を有する物質の標識物と本発明に係る多糖類とを含有する溶液をレクチンマイクロアレイと相互作用させた後、励起光を照射してエバネッセント場を発生させる。次いで、糖鎖を有する物質の標識物由来のシグナルを検出することにより糖鎖を有する物質が結合したレクチンを知ることができるので、その結果をもとに、糖鎖を有する物質の糖鎖を解析することができる。
本発明の複合体形成方法を免疫学液測定方法(サンドイッチ法)へ応用する場合は、本発明に係る多糖類の存在下に、糖鎖を有する物質とレクチンを反応させる以外は、公知の免疫学的測定方法(サンドイッチ法)に準じた測定を行えばよい。
[方法1]
測定対象である糖鎖を有する物質の糖鎖に親和性を有するレクチンを固相に固定化する。測定対象の試料と本発明に係る多糖類とを含有する溶液を、上記固相と接触させ、反応させる。又は、本発明に係る多糖類を含有する溶液、測定対象の試料の順に上記固相と接触させて反応させてもよく、測定対象の試料、本発明に係る多糖類を含有する溶液の順に、上記固相と接触させて反応させてもよい。
[方法2]
測定対象の糖鎖を有する物質に特異的に結合する抗体を固相に固定化する。糖鎖を有する物質が糖タンパク質の場合には、そのコアタンパク質に特異的な抗体を用いればよい。
固相を洗浄処理し、未反応の標識レクチン等を除去する。
測定対象の試料を常法によりゲル電気泳動後、PVDF膜等のメンブレンに転写する。次いで適宜メンブレンをブロッキング処理した後、得られたメンブレンを、糖鎖を有する物質の糖鎖に親和性を有するレクチンを検出可能な標識物質で標識した標識レクチンと本発明に係る多糖類とを含有する溶液に浸漬させ、メンブレン上で糖鎖を有する物質と標識レクチンとの複合体を形成させる。次いで、標識物質に応じた測定方法で標識物質を検出することにより、糖鎖を有する物質を高感度に検出し、測定することができる。
常法により作製した組織切片を、検出対象である糖鎖に親和性を有するレクチンを蛍光物質や放射性物質等の検出可能な標識物質で標識した標識レクチンと本発明に係る多糖類とを含有する溶液に浸漬させて、組織切片上の糖鎖と標識レクチンとの複合体を形成させることにより、組織切片をより効率よく標識することができる。
細胞膜に存在する検討対象(標的)の糖鎖に親和性を有するレクチンを検出可能な標識物質で標識したレクチンを細胞に結合させ、細胞分離装置(セルソーター)を用いた通常のフローサイトメトリーを実施すれば、レクチンの糖鎖特異性を利用して、細胞表面(細胞膜上)にある糖鎖の種類に基づいて細胞の亜集団を分画することができる。
本発明の糖鎖を有する物質の測定方法とは、「本発明の複合体形成方法で糖鎖を有する物質とレクチンとの複合体を形成させた後、該複合体の量を測定する、糖鎖を有する物質の測定方法。」である。
本発明の糖鎖を有する物質の測定方法における、本発明の複合体形成方法の詳細は、上記した「本発明の複合体形成方法」に記載した通りである。
本発明の複合体形成方法で糖鎖を有する物質とレクチンとの複合体を形成させた後は、該複合体の量を測定することにより、糖鎖を有する物質を測定すればよい。該複合体の具体的な測定方法としては、例えば上記した表面プラズモン共鳴法、キャピラリー電気泳動、レクチンアフィニティークロマトグラフィー、レクチンマイクロアレイ法、サンドイッチ法等の免疫学的測定方法、電気泳動法等が挙げられる。その具体的な条件や方法等は、上記した各方法の説明に記載した通りである
本発明の複合体形成促進剤は、「本発明の多糖類を含有してなる、糖鎖を有する物質と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとの複合体形成促進剤。」である。
具体的には、例えばデキストラン硫酸又はその塩、コンドロイチン硫酸cナトリウム又はその塩等が挙げられる。
本発明の多糖類とレクチンとを含有する本発明の複合体形成促進剤の具体例としては、例えば以下の組成のものが挙げられる。
(1)0.01(w/v)%~50(w/v)%のデキストラン硫酸又はその塩と1μg/mL~30mg/mLのLCAを5~1000mM Tris-HCl緩衝液(pH5~9)等の緩衝液に溶解させた、複合体形成促進剤、
(2)0.01(w/v)%~50(w/v)%のデキストラン硫酸又はその塩と1μg/mL~30mg/mLのMAAを5~1000mM Tris-HCl緩衝液(pH5~9)等の緩衝液に溶解させた、複合体形成促進剤、
(3)0.01(w/v)%~50(w/v)%のコンドロイチン硫酸c又はその塩と1μg/mL~30mg/mLのMAAを含有する5~1000mM Tris-HCl緩衝液(pH5~9)等の緩衝液に溶解させた、複合体形成促進剤。
(1)測定機器等
測定機器: Biacore X (GE Healthcare UK Ltd.製)
チップ: Sensor Chip CM5 (GE Healthcare UK Ltd.製)
ランニングバッファー: HBS-EP バッファー(10mM HEPES, 0.15M NaCl, 3mM EDTA, 0.005 % Surfactant P 20, pH7.4, GE Healthcare UK Ltd.製)
糖鎖を有する物質として、AFP-L3(α-フェトプロテインのLCA結合性分画、和光純薬工業(株)製)を用いた。
HBS-EPバッファー及び上記試薬を用いて、下記のサンプル溶液をそれぞれ調製した。
・1μM(4w/v%)デキストラン硫酸ナトリウムを含有する28μg/mL AFP-L3溶液
・1μM(1w/v%)コンドロイチン硫酸cナトリウムを含有する28μg/mL AFP-L3溶液
・28μg/mL AFP-L3溶液
アミンカップリングキット(GE Healthcare UK Ltd.製)を用いて、LCA(Lens Culinaris Agglutinin、)をSensor Chip CM5(CMセンサーチップ、GE Healthcare UK Ltd.製)のセンサーチップ上に固定化した。
以下の測定は、Biacore X (GE Healthcare UK Ltd.製)を用いて行った。
上記(3)で調製したサンプル溶液60μLを、温度25℃、流速30μL、結合時間120秒間の条件でゆっくりと送液して、LCAが固定化されているセンサーチップに流し、AFP-L3とLCAとを反応させた。送液直後から、経時的にBiacore Xによる測定を行った。次いでHBS-EPバッファーのみを、180秒間(解離時間)送液した。得られた測定結果を、ビアコア専用解析ソフトであるBIAevaluation(Version4.1) を用いて解析して、センサーグラムを得た。
得られたセンサーグラムを図1及び図2に示す。
2)蛍光標識抗遊離型PSA抗体の調製
Anti PSAモノクローナル抗体5G6とは異なるPSAのエピトープを認識し、かつ遊離型PSAのみと特異的に結合する抗ヒトPSAモノクローナル抗体(Anti PSAモノクローナル抗体 8A6、HyTest社製)を常法により処理して、抗PSA抗体8A6 Fab'フラグメントを得た。得られたフラグメントのアミノ基に、常法により蛍光物質HiLyte647(AnaSpec社製)を導入して、HiLyte647標識抗遊離型PSA抗体8A6 Fab'フラグメント(以下、「蛍光標識抗遊離型PSA抗体」と記載する。)を得た。
全自動蛍光免疫測定装置ミュータスワコー i30(和光純薬工業(株)製)を用い、装置の取扱説明書に従い、以下に示した手順にてマイクロチップキャピラリー電気泳動を行った。
Yoneyama T. et al., Biochem. Biophys. Res. Commun., vol. 448, No. 4, pp. 390-396, 2014の「2. Materials and methods (2.7 Forced expression of FLAG-tag-fused S2,3PSA)」に開示された方法に従ってリコンビナント遊離型PSA[以下、「r遊離型PSA」と略記する。r遊離型PSAは、リコンビナントα(2, 3)遊離型PSA(以下、rα(2, 3)遊離型PSAと略記する)と、リコンビナントα(2, 3)遊離型PSA以外の遊離型PSAを含有する。]を取得した。取得したr遊離型PSA溶液中のPSA濃度を測定し、PBS(-)(和光純薬工業(株)製)で希釈して1ng/mL PSAタンパク質濃度となるように調製したサンプル溶液を得た。得られたサンプル溶液を2μL、上記(1)2)で調製した1μM 蛍光標識抗遊離型PSA抗体を1μL、及び泳動緩衝液1〔5% (w/v) ポリエチレングリコール(PEG20000)、3%(w/v) グリセロール、150mM NaCl、0.01% BSA、75mM Tris-HCl、10mM MESを含有する。pH 7.5〕7μLを0.5mLチューブに加えて混合して、10μLの反応液を調製した。
下記の各試液を調製した。
・泳動緩衝液2(MAAとコンドロイチン硫酸cナトリウム含有)
5.0% (w/v) ポリエチレングリコール(PEG8000)、3%(w/v) グリセロール、10mM NaCl、0.01 % BSAを含有する75mM Tris-HClバッファー(pH 7.5)を調製した。これにMAA(VECTOR社製)を終濃度(泳動緩衝液2中の濃度)4mg/mLとなるように、また本発明に係る多糖類としてコンドロイチン硫酸cナトリウム(和光純薬工業(株)製)を、終濃度(w/v)(泳動緩衝液2中の濃度)がそれぞれ0%、2.8%、3.4%、3.5%、3.6%、3.8%、4.1%、4.3%、4.5%となるように添加し、混合したものを調製し、泳動緩衝液2とした。
・泳動緩衝液3
2% (w/v) ポリエチレングリコール(PEG20000)、3%(w/v) グリセロール、0.01 % BSA、125mM HEPES、75mM Tris-HClを含有するバッファー(pH調製なし)を、泳動緩衝液3とした。
・泳動緩衝液4
2% (w/v) ポリエチレングリコール(PEG20000)、3%(w/v) グリセロール、0.01 % BSAを含有する75mM Tris-HClバッファー(pH 7.5)を、泳動緩衝液4とした。
・DNA標識抗体液(DNA標識抗PSA抗体含有)
上記(1)1)で得られたDNA標識抗PSA抗体 100nMを含有するバッファー〔2% (w/v) ポリエチレングリコール(PEG20000) 、0.5mM EDTA(2Na)、3%(w/v) グリセロール、50mM NaCl、0.01 % BSA、75mM BisTris(pH 6.0)を含有する。〕を調製し、DNA標識抗体液とした。
・蛍光液
30nM HiLyte647、20%(w/v) グリセロールを含有する50mM BisTris(pH 6.0)を、蛍光液とした。蛍光液は測定機器(ミュータスワコー i30)の検出部での位置確認等の調整のために用いられる。
i)泳動用試料A及び泳動用試液の導入
上記(2)1)で調製した泳動用試料A 5.4μLを、ミュータスワコー i30専用マイクロチップの所定ウェル(SPウェル)に分注した。次いで、下記のように該マイクロチップの各ウェルに上記(2)2)で調製した各試液を分注した。
・R2ウェル(R2(FLB)ウェル、R2(LB)ウェル):泳動緩衝液2(MAAとコンドロイチン硫酸cナトリウム含有)を10.0μLずつ、
・R3ウェル:泳動緩衝液3を10.0μL、
・R4ウェル:泳動緩衝液4を5.4μL、
・C1ウェル:DNA標識抗体液を3.0μL、
・FDウェル:蛍光液を7.0μL。
使用したマイクロチップのチップ内流路を模式化したものを図4に示す。
得られた電気泳動像(エレクトロフェログラム)より、MAAと反応した[蛍光標識抗遊離型PSA抗体-rα(2, 3)糖鎖遊離型PSA-DNA標識抗PSA抗体]の複合体(複合体1)分画のピーク面積を、i30装置付属の解析用ソフトで求めた。
(1)試料及び試液の調製、及び電気泳動(マイクロチップキャピラリー電気泳動)
下記の泳動緩衝液2を用いる以外は、実施例2で使用したものと同じ泳動用試液、機器等を用い、実施例2と同様の方法で、[蛍光標識抗遊離型PSA抗体-α(2, 3)糖鎖遊離型PSA-DNA標識抗PSA抗体]の複合体(複合体1)の検出を行った。
・泳動緩衝液2(本発明に係る多糖類含有)
5.0% (w/v) ポリエチレングリコール(PEG8000)、3%(w/v) グリセロール、10mM NaCl、0.01 % BSAを含有する75mM Tris-HClバッファー(pH 7.5)を調製した。これにMAA(VECTOR社製)を終濃度(泳動緩衝液2中の濃度)4mg/mLとなるように、また本発明に係る多糖類としてデキストラン硫酸ナトリウム(M.W. 6,500~10,000)(Sigma社製)を、終濃度(泳動緩衝液2中の濃度)(w/v)が0%、3.0%、5.0%、5.5%、6.0%、6.7%、7.0%、7.7%、8.1%となるように添加し、混合したものを調製し、泳動緩衝液2とした。
得られた電気泳動像(エレクトロフェログラム)より、MAAと反応した[蛍光標識抗遊離型PSA抗体-α(2, 3)糖鎖遊離型PSA-DNA標識抗PSA抗体]の複合体(複合体1)分画のピーク面積を、i30装置付属の解析用ソフトで求めた。
Claims (9)
- N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物の存在下に、糖鎖を有する物質を含有する試料と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとを接触させる、該糖鎖を有する物質と該レクチンとの複合体形成方法。
- N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、該レクチンが親和性を有する糖鎖を持たないものである、請求項1に記載の方法。
- N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物がデキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩である、請求項1に記載の方法。
- 下記(1)~(2)から選択される、請求項1に記載の方法:
(1)N-アセチルグルコサミンを有さない水溶性多糖がデキストラン硫酸又はその塩で、糖鎖を有する物質がαフェトプロテイン-L3(AFP-L3)で、レクチンがレンズマメレクチンである、
(2)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物がデキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩で、糖鎖を有する物質がα(2, 3)糖鎖遊離型前立腺特異抗原で、レクチンがイヌエンジュレクチンである。 - N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物を含有してなる、糖鎖を有する物質と該糖鎖を有する物質の糖鎖に親和性を有するレクチンとの複合体形成促進剤。
- N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、該レクチンが親和性を有する糖鎖を持たないものである、請求項5に記載の複合体形成促進剤。
- N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物がデキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩である、請求項5に記載の複合体形成促進剤。
- 下記(1)~(2)から選択される、請求項5に記載の複合体形成促進剤:
(1)N-アセチルグルコサミンを有さない水溶性多糖がデキストラン硫酸又はその塩で、糖鎖を有する物質がαフェトプロテイン-L3(AFP-L3)で、レクチンがレンズマメレクチンである、
(2)N-アセチルグルコサミンを有さない水溶性多糖又はN-アセチルグルコサミンを有さない多糖を有する水溶性化合物が、デキストラン硫酸若しくはその塩、又はコンドロイチン硫酸c若しくはその塩で、糖鎖を有する物質がα(2, 3)糖鎖遊離型前立腺特異抗原で、レクチンがイヌエンジュレクチンである。 - 請求項1に記載の方法で糖鎖を有する物質とレクチンとの複合体を形成させた後、該複合体の量を測定する、糖鎖を有する物質の測定方法。
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JP2021038980A (ja) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp−l3測定方法及びafp−l3測定キット、並びに、これらに用いるブロック化標識レクチン |
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