WO2001061353A1 - Procede de detection d'une substance a analyser - Google Patents

Procede de detection d'une substance a analyser Download PDF

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Publication number
WO2001061353A1
WO2001061353A1 PCT/JP2001/001247 JP0101247W WO0161353A1 WO 2001061353 A1 WO2001061353 A1 WO 2001061353A1 JP 0101247 W JP0101247 W JP 0101247W WO 0161353 A1 WO0161353 A1 WO 0161353A1
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WIPO (PCT)
Prior art keywords
array
sugar
immobilized
target
target substance
Prior art date
Application number
PCT/JP2001/001247
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English (en)
Japanese (ja)
Inventor
Tsuyoshi Miyamura
Tomoe Egashira
Mutsumi Sano
Ikunoshin Kato
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Takara Bio Ltd.
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Publication date
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Priority to AU2001234103A priority Critical patent/AU2001234103A1/en
Publication of WO2001061353A1 publication Critical patent/WO2001061353A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to an array for detecting or quantifying an analyte (ana 1 yte), particularly a sugar, a sugar target or a protein, a method for detecting or quantifying an analyte, a kit for detecting or quantifying an analyte. And a method for producing glycosylamine.
  • the present invention provides an array capable of easily detecting or quantifying an analyte, particularly a saccharide, a sugar target or a protein, a method for detecting or quantifying an analyte using the array,
  • the present invention relates to a kit for detecting or quantifying an analyte (a kit for detecting a conjugate), and a method for producing glycosylamine useful for preparing the array.
  • glycoconjugates such as glycoproteins and glycolipids play a very important role in various in vivo reactions such as cell-cell interaction, cell-extracellular matrix interaction, antigen-antibody reaction or microbial infection. It is becoming clear that it is fulfilling.
  • infectious diseases caused by pathogenic microorganisms are those in which pathogenic microorganisms enter the host orally or nasally, and these microorganisms are present in the digestive system of the host.
  • infectious diseases caused by pathogenic microorganisms viruses, bacteria, molds, yeasts
  • pathogenic microorganisms are those in which pathogenic microorganisms enter the host orally or nasally, and these microorganisms are present in the digestive system of the host.
  • infection begins by selectively attaching an adhesin on the surface of a microorganism to a receptor on the surface of a host cell, and then forming a colony or invading a cell or tissue. Then, some microorganisms, such as bacteria, produce and release toxins as they grow. Furthermore, it is known that this toxin acts on a host cell via a specific receptor on the host cell. In other words, factors that determine the host to be infected by the pathogenic microorganism, as well as the tissues and cells of the host, depend on the phase between the receptor expressed by the host cell and the adhesion factor on the pathogenic microorganism. It will be determined by the specificity of the interaction. Similarly, the combination of the toxin produced by the microorganism and the host cell affected by the toxin is also determined by the specificity of the interaction between the toxin and the receptor on the host cell.
  • V. Ginsberg and colleagues similarly applied Mycoplasma pneumoniae or Cryptococcus' neoforman to carbohydrate receptors immobilized on an insoluble substrate. Reports a method for detecting these microorganisms by measuring the degree of binding of microorganisms.
  • Japanese Patent Publication No. 8-133278 Japanese Patent Publication No. 8-133278.
  • a glycolipid molecule separated on a silica gel thin-layer plate, or a microorganism labeled with a radioactive substance for a carbohydrate receptor such as a glycolipid or glycoprotein molecule adsorbed on a microtiter plate is used.
  • a method for measuring binding is disclosed.
  • Also disclosed is a method for measuring the binding of glycoproteins, glycolipids and sugar chains to substances such as antibodies and lectins by the same method. [Method Enzymology I, Vol. 138, Vol. 5-21-2 page (19987)]. In such binding test methods, when the concentration of the carbohydrate receptor, antibody, lectin, etc.
  • glycosylamine can be effectively used as a very important intermediate in preparing derivatives of monosaccharides and oligosaccharides. That is, the amino group specifically introduced at the 1-position carbon of the reducing terminal residue is rich in reactivity, and is coupled with a compound having an active ester of carboxylic acid, a compound having an isothiocyanate group, etc. under mild conditions. Can be done.
  • a monosaccharide or oligosaccharide can be introduced into a synthetic polymer such as polyacrylamide, immobilized on a solid support, or the like.
  • Glycosylamine is characterized by its simple preparation method. Glycosylamine preparation does not require hydroxyl group protection / deprotection reactions frequently used in general carbohydrate synthesis reactions, and monosaccharides or oligosaccharides are dissolved in a saturated aqueous solution of ammonium bicarbonate for 3 days. It only needs to be left for 6 days [Journal of Carbohydrate Chemistry, Vol. 8, pp. 597-61 1 (19989)].
  • this method has a disadvantage that it is difficult to remove excessive ammonia contained after the reaction. If ammonia is present in the glycosylamine preparation, the ammonia will competitively inhibit the coupling reaction between glycosylamine and a compound having an active ester of carboxylic acid, a compound having an isothiocyanate group, or the like.
  • an active ester form of a carboxylic acid reacts with ammonia to form an amide, and an isothiocyanate group forms a thiourea, thereby inactivating a functional group that should react with glycosylamine.
  • An object of the present invention is to provide an array capable of easily detecting or quantifying an analyte (analyte :), in particular, a sugar, a sugar target, or a protein, and detecting the analyte using the array.
  • Another object of the present invention is to provide a quantification method, a kit containing the array, and a method for producing glycosylamine useful for producing the array.
  • the present inventors have developed a method for measuring the specificity of the interaction of a saccharide target with a saccharide receptor having a variety of sequences and binding modes. monosaccharides, oligosaccharides, glycolipids, glycopeptides, glycoproteins, and these complexes further extensive studies were added results for the adsorption process of the protein, at least 1 cm 2 per 2 to a particular area on Kataaimoto material We succeeded in adsorbing a plurality of the carbohydrate receptors at a density of more than one site. Furthermore, the binding between the saccharide receptor and the “substance having a binding property to the saccharide receptor” was successfully measured on the solid-phase substrate containing the saccharide receptor. The present invention has been completed.
  • Solid phase substrate 1 cm 2 per in an array of immobilized least two places targets quality in a predetermined region on the solid phase, two or more positions at different concentrations target substance that exceeds at least one stage Immobilized array,
  • the target substance is at least one substance selected from the group consisting of sugar, a sugar target, and a protein.
  • the saccharide is a monosaccharide, an oligosaccharide having two or more sugars, a polysaccharide, a glycosphingoglycolipid, a glycosphingolipid, a glycemic glycolipid, a glycoprotein, a glycopeptide, a glycosylamine, a glycosylasparagine, a glycosylserine,
  • the array according to (4), wherein the array is at least one compound selected from the group consisting of glycosylthreonine and proteoglycan or a complex containing the compound.
  • the sugar target is at least one kind of sugar target selected from the group consisting of cells, viruses, toxins, sugar-binding proteins, and antibodies;
  • the detection or quantification method according to (8) wherein when an interaction is detected, the test substance is used as an indicator that the test substance is an analyte having a binding property to the target substance.
  • step (B) a step of adding an alkali to the reaction product obtained in the step (A), thereby obtaining glycosylamine
  • alkali according to the above [12 :) to [15], wherein the alkali is at least one alkali selected from the group consisting of pyridine, triethylamine, trimethylamine, N-methylmorpholine, N-ethylmorpholine, piperidine and perforin.
  • alkali is at least one alkali selected from the group consisting of pyridine, triethylamine, trimethylamine, N-methylmorpholine, N-ethylmorpholine, piperidine and perforin.
  • FIG. 1 is a diagram showing the reactivity of an array on which lysoglycolipids are immobilized and cholera toxin B subunit.
  • FIG. 2 is a diagram showing the reactivity of an array having glycated polylysine immobilized thereon and FITC-labeled Con A.
  • FIG. 3 is a diagram showing the reactivity of an array having glycated polylysine immobilized thereon and a FITC-labeled DSA.
  • FIG. 3 is a diagram showing the reactivity with Jihachi 120.
  • FIG. 5 is a diagram showing the binding property between an array on which glycolipids are immobilized and lactic acid bacteria.
  • FIG. 6 is a diagram showing the format of an array on which glycolipids are immobilized.
  • FIG. 7 is a diagram showing the reactivity of an array (glass 1 (Glass # l)) on which glycolipids are immobilized and cholera toxin B subunit.
  • FIG. 8 is a diagram showing the reactivity of an array (glass 2 (Glass # 2)) on which glycolipids are immobilized and cholera toxin B subunit.
  • FIG. 9 is a view showing the reactivity of an array (glass 3 (Glass # 3)) on which glycolipids are immobilized and cholera toxin B subunit.
  • FIG. 10 is a diagram showing the reactivity of an array (glass 1 (Glass # l)) on which glycolipids are immobilized with lactic acid bacteria.
  • FIG. 11 is a diagram showing the reactivity of an array (glass 2 (Glass # 2)) on which glycolipids are immobilized with lactic acid bacteria.
  • FIG. 12 is a diagram showing the reactivity between an array (glass 3 (Glass # 3)) on which glycolipids are immobilized and lactic acid bacteria.
  • FIG. 13 is a diagram showing the reactivity of an array (glass 1 (G1ass # 1)) on which glycolipids are immobilized and an anti-GQ1b antibody.
  • FIG. 14 is a diagram showing the reactivity of an array (glass 2 (Glass # 2)) on which glycolipids are immobilized, with an anti-GQ1b antibody.
  • FIG. 15 is a diagram showing the reactivity of the glycolipid-immobilized array [Glass 3 (Glass # 3)] with an anti-GQ1b antibody. Detailed description of the invention 1. Definition
  • target substance refers to a substance immobilized on a predetermined region on an array, and particularly refers to a sugar, a sugar target, or a protein.
  • a substance having a binding property to a target substance immobilized on a predetermined area on the array is referred to as a “target substance” or “analyte (ana1yte)”.
  • the “analyte (target substance)” may be any substance that interacts with the target substance, in particular, a sugar, a sugar target, or a protein. Examples of such an analyte include a sugar target when the target substance is a sugar, and a sugar target as an analyte that interacts with the sugar.
  • the target substance is a sugar target
  • the analyte interacts with the sugar target.
  • a sugar can be cited as an analyte.
  • the target substance is a protein
  • examples include a protein, a compound, an antibody, a nucleic acid, a lipid, and a cell that interact with the protein.
  • the term “sugar” refers to a carbohydrate receptor or a complex carbohydrate receptor containing a carbohydrate receptor, which is immobilized on a predetermined region on an array, and Include monosaccharides, oligosaccharides of 2 or more sugars, polysaccharides, glycosphingolipids, lysosphingolipids, glycemic glycolipids, glycoproteins, glycopeptides, glycosylamines, glycosylasparagine, glycosylserine, and glycosylthreonine , Proteoglycans and complexes containing these, but are not limited thereto, and at least one type of saccharide can be used depending on the purpose of use. Alternatively, it may mean “sugar” as a substance having a binding property to a sugar target immobilized on a predetermined region on an array.
  • “sugar target” has a meaning as a substance having a binding property to a sugar immobilized on a predetermined region on an array, and includes a cell, a virus, a toxin, and a sugar bond. Sex protein, antibody.
  • the cells include, for example, gram-positive bacteria, gram-negative bacteria, molds, yeasts, protozoa, helminths, animal cultured cells, plant cultured cells, and the like.
  • Examples of toxins include: And the toxins described in BioBiochemistry, Vol. 58, pp. 309-350 (19989).
  • sugar-binding proteins include, for example, lectins. More specifically, lectins include lectins listed in plant lectins, animal lectins, insect lectins, and the Internet (eg, http: ⁇ lectin described in plab. ku. dk / tcbh / lectin-abbreviatins.htm).
  • lectins include lectins listed in plant lectins, animal lectins, insect lectins, and the Internet (eg, http: ⁇ lectin described in plab. ku. dk / tcbh / lectin-abbreviatins.htm).
  • the antibody include an anti-glycolipid antibody, an anti-glycoprotein antibody, an anti-proteoglycan antibody and the like.
  • the “sugar target” in the present specification is not limited to the above examples, and various substances may be used depending on the purpose of use if they have sugar-binding
  • the “sugar target” also has a significance as a substance immobilized in a predetermined region on the array in order to detect or quantify a sugar as an analyte (target substance) in a sample.
  • the “predetermined region” means that when the target substance is immobilized on the solid-phase substrate, the target substance is immobilized in a predetermined arrangement, and thereafter, The area where the target substance is immobilized at which concentration at which location is ascertained.
  • the array of the present invention is an array in which at least one selected from the group consisting of a target substance, particularly a sugar, a sugar target and a protein is immobilized on a solid-phase substrate.
  • the array of the present invention is an array in which two or more target substances are immobilized in a predetermined region on a solid phase at two or more locations per 1 cm 2 of a solid-phase substrate.
  • One feature is that it is immobilized at two or more locations at different concentrations that exceed the level. Therefore, according to the array of the present invention, it is possible to detect with high sensitivity the specificity of the interaction of the “substance having a binding property to the target substance” with respect to the target substance having various sequences and binding modes. Exhibits excellent properties.
  • the target substance may be continuously arranged at different concentrations exceeding at least one step, that is, at two or more locations so as to be vertically, horizontally, or diagonally adjacent to each other on the solid phase.
  • Arrays, etc. As described above, according to an array in which the target substance is continuously immobilized in a predetermined region on the solid phase at different concentrations exceeding at least one step, for example, the same target substance at different concentrations is continuously applied. Since they are arranged in a specific manner, they exhibit an excellent property that the tendency of the degree of binding of the target substance due to the difference in the concentration of the target substance can be determined more easily.
  • Such an array may be, for example, an array in which a target substance is immobilized in a predetermined area on a slide glass.
  • the solid-phase substrate used for preparing the array of the present invention is not particularly limited as long as it is a substrate that can be used for ordinary binding tests, and is not particularly limited as long as it is an insoluble substrate.
  • Plastic materials such as silicon chips, polypropylene, and polystyrene, or insoluble metal materials can be used.
  • the above-mentioned substrate may be used as it is, but it is preferable to use it after activation treatment.
  • the activation treatment method include a normal surface activation treatment method.
  • Activated substrates include, for example, substrates with exposed amino groups, epoxy A substrate having exposed groups, a substrate having halogen groups exposed, a substrate having thiol groups exposed, a substrate having acid anhydrides exposed, a substrate having hydrophobicity, and the like are included.
  • a method in which polylysine is adsorbed on the surface of the base material to expose the amino group on the surface of the base material can be mentioned.
  • a method of treating the glass substrate surface with a silylating agent to covalently bond an amino group to the substrate surface can also be mentioned.
  • an aminosilane compound can be used as the silylating agent.
  • aminosilane compound generally available compounds can be used. Such compounds include, for example, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N- (2-aminoethyl) 3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) Examples thereof include 3-aminopropylmethyltrimethoxysilane, p-aminophenyltrimethoxysilane, m-aminophenyltrimethoxysilane, and 3- (m-aminophenoxy) provirtrimethoxysilane.
  • the substrate having an exposed amino group a commercially available substrate can be used.
  • a slide glass having an amino group covalently bonded thereto such as Matsunami APS (manufactured by Matsunami Glass Co., Ltd.), silane prep slide ( Sigma) can also be used.
  • a silylating agent comprising a compound having an epoxy group
  • Such compounds include, for example, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 2- (3 , 4-epoxycyclohexyl) ethyltriethoxysilane, 5,6-epoxyhexyltriethoxysilane and the like can be used.
  • a silylating agent comprising a compound having a halogen group
  • Such compounds include, for example, 3-chloro Methyl propylmethyldimethoxysilane, 3-chloromethylpropylmethoxysilane, 2- (chloromethyl) propylmethyldichlorosilane, chloromethyltrichlorosilane, chloromethyltriethoxysilane, chloromethyltrimethoxysilane, ((chloromethyl) phenylethyl) tri Chlorosilane, ((chloromethyl) phenylethyl) trimethoxysilane, ( ⁇ -chloromethyl) phenyltrichlorosilane, ( ⁇ -chloromethyl) phenyltrimethoxysilane, (p-chloromethyl) phenyltri-n-propoxysilane, etc. Can be used.
  • a silylating agent composed of a compound having a thiol group may be used.
  • a compound having a thiol group for example, 3-mercaptopropyltrimethoxysilane can be used.
  • a silylating agent comprising a compound having an acid anhydride may be used.
  • a compound having an acid anhydride for example, 3- (triethoxysilyl) propylsuccinic anhydride can be used.
  • a silylating agent comprising a compound having a hydrocarbon chain
  • Such compounds include, for example, alkoxysilane compounds such as n-butyltrimethoxysilane, n-butyloctyltriethoxysilane, n-octyltrimethoxysilane, dodecyltriethoxysilane, dodecyltrimethoxysilane, eicosyltrichlorosilane, docosyltrichlorosilane Or a mixture thereof.
  • These silylating agents can be obtained, for example, from Chisso Corporation.
  • the silylation treatment can be performed by a method well known to those skilled in the art.
  • a solvent such as methanol, ethanol, propanol, isopropanol, acetonitrile, dimethylformamide, hexane, tetrahydrofuran, toluene, pyridine, and triethylamine is preferably used.
  • a solvent such as methanol, ethanol, propanol, isopropanol, acetonitrile, dimethylformamide, hexane, tetrahydrofuran, toluene, pyridine, and triethylamine is preferably used.
  • the processing time varies depending on the type of the silylating agent and the solid phase substrate. It can be processed in minutes to 60 minutes.
  • an alkoxysilane compound is used as a silylating agent
  • a silylating agent alkoxysilane compound
  • a 95% aqueous ethanol solution with stirring, and the mixture is stirred for an appropriate time, for example, 1 minute to 60 minutes.
  • the surface treatment is performed by adding a solid phase substrate.
  • the concentration of the silylating agent is not particularly limited, it can be used, for example, at a final concentration of about 1 to 10%.
  • the pH of the ethanol solution can be adjusted to a slightly acidic pH, preferably about 4 to 6, with acetic acid or the like, if necessary.
  • the silylating agent When a chlorosilane compound is used as the silylating agent, generally, the silylating agent is added to anhydrous ethanol, isopropanol, or the like with stirring.
  • the concentration of the silylating agent is not particularly limited, it can be used, for example, at a final concentration of about 1 to 10%.
  • the chlorosilane compound is added to anhydrous ethanol with stirring, and the mixture is stirred for an appropriate period of time, for example, 1 to 60 minutes to sufficiently generate alkoxysilane and silanol, and then the solid-phase substrate is added. Perform surface treatment.
  • the solution may be used, for example, by heating at a temperature of 30 to 50, if necessary.
  • the solid-phase substrate is washed once or twice with a solvent such as ethanol, and then left at 110 ° C for 5 to 30 minutes or at room temperature for about one day to obtain a substrate surface. Can be cured.
  • a solvent such as ethanol
  • the substrate can be used to create various functional groups on the substrate surface by introducing a suitable linker by a method well known to those skilled in the art.
  • a suitable linker for example, an aldehyde group, an isothiocyanate group, a carboxyl group, a succinimide carboxylate group, a thiol group, a maleimide group, a hydrocarbon chain, a hydrazide residue, or the like can be introduced.
  • streptavidin can be further introduced into the surface of the base material using these functional groups.
  • the surface of a substrate activated with succinimide ester groups may Streptavidin can be introduced by the method described in (Journal of Biochemistry :), Vol. 117, pp. 107-6-1082 (1995).
  • Immobilization of a saccharide as a target substance on an activated solid phase substrate can be carried out by a method suitable for each property of the saccharide to be used and the activated solid phase substrate.
  • a method suitable for each property of the saccharide to be used and the activated solid phase substrate for example, in the case of fat-soluble substances such as glycosphingolipids and glycolipid glycerol, they are obtained after dissolving and dispersing in a suitable solvent capable of dissolving and Z or uniformly dispersing or Z or dispersing them.
  • the solution and / or the dispersion can be directly adsorbed to the surface of the solid-phase base material provided with hydrophobicity by a hydrocarbon chain or the like by hydrophobic interaction.
  • the solvent examples include an aqueous solution, dimethyl sulfoxide, dimethylformamide, methanol, acetonitrile, chloroform, and a mixed solvent thereof.
  • the solution and / or dispersion may include a non-ionic or ionic surfactant.
  • a lipid such as phospholipid, cholesterol, or diacylglycerol, or a saccharide such as trehalose may be added to the solution and Z or the dispersion.
  • the aqueous solution may be a solution containing volatile salts. In order to adjust the shape of the spot, it is effective to add, for example, glycerol, polyethylene glycol, saccharides, and salts to the solution and / or dispersion.
  • sugar is glycoprotein, glycopeptide, glycosylamine, glycosylasparagine, glycosylserine or glycosylthreonine, the aldehyde group, isothiosinate group, carboxyl group, etc. Or a solid phase substrate activated with a carboxylic acid succinimide ester group or the like.
  • the aldehyde is converted to an aldehyde by utilizing the amino group created by converting each reducing terminal saccharide residue to glycosylamine.
  • Group, isothiosinate group, carboxyl group, carboxylate succinimide ester group and the like can be covalently bonded to a solid phase substrate activated. Conversion of the reducing terminal sugar residue to glycosylamine can be performed according to a known method.
  • reaction may be carried out for 1 hour to 1 day in a weakly acidic to weakly alkaline buffer solution, for example, a phosphate-buffered saline solution while heating to room temperature to 50 ° C.
  • a weakly acidic to weakly alkaline buffer solution for example, a phosphate-buffered saline solution while heating to room temperature to 50 ° C.
  • the reducing terminal saccharide residue is covalently bonded to a solid support activated with an amino group, a hydrazine residue, or the like.
  • This coupling reaction involves the reduction of the normal sugar and the amino
  • the reaction can be carried out according to the reaction with a group or a hydrazine-ligated product. For example, water, P
  • reaction In a solvent such as 5 to 7 buffer, dimethyl sulfoxide, methanol, ethanol, propanol, isopropanol, acetonitrile, dimethylformamide, tetrahydrofuran, etc.
  • a solvent such as 5 to 7 buffer, dimethyl sulfoxide, methanol, ethanol, propanol, isopropanol, acetonitrile, dimethylformamide, tetrahydrofuran, etc.
  • the reaction can be carried out for 1 hour to 1 day.
  • An organic acid such as acetic acid or an inorganic acid such as hydrochloric acid may be added to the solvent, if necessary.
  • the Schiff base may be reduced using a reducing agent such as sodium borohydride, sodium cyanoborohydride, dimethylamine porane, pyridine 'borane complex, etc. It is possible to stabilize the covalent bond between and the solid phase substrate.
  • a reducing agent such as sodium borohydride, sodium cyanoborohydride, dimethylamine porane, pyridine 'borane complex, etc. It is possible to stabilize the covalent bond between and the solid phase substrate.
  • the reducing terminal saccharide residue is, for example, a polymer such as polylysine, chitosan, polyamine, sugar-free protein, or DNA.
  • the resulting saccharide-polymer complex can be further covalently, hydrophobically or ionic-bonded to the activated solid-phase substrate.
  • a biotin molecule activated with a hydrazine residue can be covalently bound to the reducing terminal sugar residue.
  • the sugar-biotin compound can be immobilized on a streptavidin-activated solid-phase substrate via a streptavidin-biotin bond.
  • the saccharide is a monosaccharide
  • an oligosaccharide having two or more saccharides, or a polysaccharide, a thiol group, a maleimide group, and a phenylazide are used as the reducing terminal sugar residue by using an appropriate linker such as a hydrazide derivative.
  • Groups can be introduced.
  • a thiol group or maleimide group is introduced, a solid phase group activated with a thiol group or maleimide group It can be immobilized on the material via a disulfide bond or a thioether bond.
  • a phenylazide group When a phenylazide group is introduced, it can be nonspecifically bound to a solid phase substrate that has been activated by exposure to visible light or ultraviolet light.
  • the linker used to introduce a thiol group or a maleimide group is not particularly limited.
  • N— ( ⁇ -maleimid Docaproic acid) hydrazide ⁇ — (—maleimidoundecanoic acid) hydrazide
  • 4- ( ⁇ —maleimidomethyl) cyclohexane-1-carboxylhydrazide 4 -— (41-maleimidphenyl) butyric acid hydrazide
  • the linker used for introducing the phenylazide group is not particularly limited, but includes, for example, ⁇ -azidobenzoylhydrazide.
  • the amino group can be converted to a thiol group using an appropriate linker.
  • the linker in this case is not particularly limited.
  • ⁇ -succimidyl S-acetylthioacetate ⁇ -succimidyl S-acetylthiopropionate manufactured by Pierce
  • it can.
  • the amino group can be converted to a phenylazide group using an appropriate linker.
  • the linker in this case is not particularly limited.
  • the saccharide when it is immobilized on a predetermined region on the surface of the solid-phase substrate, it can be immobilized via double-stranded DNA. That is, one solid is applied to the surface of the solid-phase base material by the method described in U.S. Patent No. 5,445,934 or U.S. Patent No. 5,744,305.
  • the strand DNA is immobilized or solid-phase synthesized at a certain density, while a single-stranded DNA having a reactive group such as an amino group or a thiol group is synthesized using a commercially available DNA synthesizer.
  • a sugar-DNA complex To prepare a sugar-DNA complex. Then, if a sugar-DNA complex solution having a sequence complementary to the DNA sequence immobilized on the surface of the solid phase substrate is overlaid on the solid phase, the DNA hybridized Sugar can be immobilized in a predetermined area. Further, by adjusting the sequence and density of DNA on the solid phase, the type and density of the saccharide to be immobilized can be freely selected.
  • the density of the target substance immobilized on a predetermined region on the solid-phase substrate is not particularly limited, but the target substance and a substance having a binding property to the target substance. (Analyte) from the viewpoint of efficient binding and the possibility of preparing an array with a small amount of sample. For example, as many types of immobilized target substances as possible It is preferred to test the binding properties of the binding substance (analyte) and to minimize the amount of the substance to be tested.
  • density j refers to the number of individual areas (spots) where the target substance is immobilized per unit area on the solid-phase substrate. at least 1 cm 2 per two places or more density, preferably 2 to 1 0 0 0 0 places, More preferably, an array in which target substances are immobilized at a density of 200 to 1000 points, wherein at least one target substance is immobilized in a predetermined region at different concentrations exceeding at least one step. Arrays are preferably used.
  • ⁇ different concentrations exceeding one step '' means that at least one target substance is immobilized at different concentrations, and the concentration step is appropriately selected and set according to the purpose. Can be.
  • the “concentration J” used here is used as an index of the number of molecules of the immobilized target substance per unit area of the region on the solid-phase substrate occupied by the immobilized target substance spot.
  • An array with a step-wise, discontinuous density gradient on a solid-phase substrate is prepared by immobilization using a target substance solution that has been serially diluted two times, three times, etc.
  • the immobilization position is not particularly limited as long as at least one target substance is immobilized at different concentrations exceeding at least one step on the same array. That is, at least one target substance may be continuously immobilized at a predetermined concentration in at least one step at different concentrations in a predetermined region. May be immobilized in different regions on the same array for each concentration exceeding one step.
  • the present invention is achieved by spotting a target substance or a target substance into which an appropriate linker has been introduced as described above on an activated solid-phase base material suitable for each of the target substances using a micropipet cabillary. Can also be prepared.
  • an array preparation device for example, an arrayer manufactured by Affymetrix, so that the target substance was properly aligned and immobilized on the solid-phase substrate.
  • An array of the invention can be made.
  • the surface of the solid-phase substrate in which no spot of the target substance is present can be subjected to an inactivation treatment by a commonly used method.
  • the method of the inactivation treatment is not particularly limited.
  • bovine serum albumin and casein The treatment can be carried out by treating the cells with a phosphate buffer solution containing about 1 to 10% of a protein such as in 1 to 16 hours at 4 to 40 hours.
  • active residues on the surface of the activated solid phase substrate can be inactivated by treatment with a suitable small molecule that reacts with it.
  • suitable small molecule that reacts with it.
  • the low molecule used herein include, but are not limited to, amino acids such as glycine, triethanolamine, formaldehyde, and 2-mercaptoethanol.
  • the most efficient method may be selected depending on the sugar target, and the sugar target used and the activated solid phase may be selected. It can be carried out by a method suitable for each property of the substrate.
  • the solvent that can be used for dissolving or suspending the sugar target may be any solvent that can sufficiently exert the sugar binding activity of the sugar target, such as an aqueous solution, Dimethyl sulfoxide, dimethylformamide, or a mixed solvent thereof is preferred.
  • the sugar target solution or suspension may include a surfactant.
  • the sugar target is a hydrophobic substance, such as a protein
  • the harmful substance is obtained after dissolving and / or dispersing in a suitable solvent in which they can be dissolved and / or uniformly dispersed.
  • the resulting solution and / or dispersion can be directly adsorbed to the surface of the solid-phase substrate provided with hydrophobicity by a hydrocarbon chain or the like by hydrophobic interaction.
  • the sugar target has an amino group or a thiol group, for example, an amino group-reactive functional group such as an aldehyde group, an N-hydroxylsuccinimide ester group of a carboxylic acid, or an isothiocyanate group; a thiol group or a maleimide Thiol-reactive group, etc.
  • an amino group-reactive functional group such as an aldehyde group, an N-hydroxylsuccinimide ester group of a carboxylic acid, or an isothiocyanate group
  • a thiol group or a maleimide Thiol-reactive group etc.
  • a covalent bond between the sugar target and the solid-phase substrate can be formed by using a crosslinking agent and Z or a reaction accelerator.
  • cross-linking agents for example, ethylene glycol-bis- (succinimidyl succinate), N- ( ⁇ -maleimidocaproyloxy) succinimide ester and various reactive groups from Pierce.
  • Compounds having a molecular length are commercially available. In the present invention, it can be appropriately selected from such cross-linking agents depending on the combination of the solid phase substrate and the sugar target.
  • reaction accelerator examples include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dicyclohexyl carbodiimide and the like.
  • the timing of contact between the sugar target and the solid-phase substrate, the cross-linking agent and / or the reaction accelerator by the three or four members is not particularly limited. , For example,
  • the sugar target can be immobilized on the solid-phase substrate by utilizing specific binding between molecules.
  • Specific binding between molecules includes an antigen-antibody reaction, a biotin-avidin bond, a bond between glutathione and glutathione-S-transferase, and a bond between oligohistidine and a metal.
  • the sugar target when immobilized on the surface of the solid-phase substrate, it can be immobilized via double-stranded DNA. That is, U.S. Pat. Single-stranded DNA is immobilized or solid-phase synthesized at a free density by the method described in US Pat. No. 5,744,305 or US Pat. No. 5,744,305. A single-stranded DNA having a reactive group such as an amino group or a thiol group is synthesized using a synthesizer.
  • DNA with a reactive group synthesized using a commercially available synthesizer and a sugar target that has introduced a functional group such as a succinimide group, a thiol group, or a maleimide group that can react with the reactive group of the DNA To form a sugar target-DNA complex, and apply a sugar target-DNA complex solution having a sequence complementary to the DNA sequence immobilized on the solid-phase base material surface to the solid phase.
  • the sugar target can be immobilized to a predetermined region on the solid phase by DNA hybridization. Further, by adjusting the sequence and density of the DNA on the solid phase, the type and density of the sugar target to be immobilized can be selected.
  • the sugar target is a cell such as a gram-positive bacterium, a gram-negative bacterium, a mold, a yeast, a protozoan, a helminth, a cultured animal cell, a cultured plant cell, etc.
  • the cell is alive or dead.
  • the method described above can be used as a method for immobilization on a solid phase surface.For example, a method using cell hydrophobicity, a method using amino group / thiol group, and a specific binding between molecules can be used. The method can be based on the method used, the method using double-stranded DNA, or the like.
  • Cells can be immobilized by a commonly used microbiological or histochemical immobilization method.
  • immobilization means inactivating cells
  • immobilization on a solid-phase substrate is a concept distinct from solid-phase immobilization.
  • Microbiological and histochemical immobilization methods include, for example, immobilization by drying, immobilization with 70% to 90% ethanol, and immobilization with glutaraldehyde and formaldehyde.
  • Microbiological or histochemical immobilization may be performed before or after immobilization on a solid-phase substrate or solid-phase immobilization.
  • the immobilization can be performed by a method suitable for the properties of the protein to be used and the activated solid phase substrate. For example, after dissolving and Z or dispersing in a suitable solvent capable of dissolving or uniformly dispersing the protein, the resulting solution and / or dispersion is imparted with hydrophobicity by a hydrocarbon chain or the like. It can be directly adsorbed on the surface of the solid substrate by hydrophobic interaction.
  • an array library can be constructed.
  • array library refers to a library of target substances isolated from the natural world or synthesized artificially, and includes, for example, a glycosylamine library.
  • the glycosylamine library can be easily prepared by dissolving in an appropriate solvent and then spotting the obtained solution on a solid-phase substrate activated with, for example, succinimide carboxylate. be able to.
  • a secondary library having a carboxylate succinimide ester group can be prepared.
  • a coupling agent such as disuccinimidyl suberate (Pierce)
  • a secondary library having a carboxylate succinimide ester group can be prepared.
  • a chemically stable secondary library having a spacer can be constructed by coupling the glycosylamine library with succinic anhydride or succinimide ester of Fmoc-e-aminohexanoic acid. .
  • These glycosylamine libraries, secondary libraries derived therefrom, and higher-order libraries derived from the secondary library are used not only for array production but also for fluorescent labels and immobilized sugars. Raw materials for typical sugar derivatives This can be easily analogized by those skilled in the art.
  • Glycosylamine for preparing an array or constructing an array library is prepared by using a reducing sugar as a raw material, for example, Neoglycoconjugate, pp. 199-223, Y.C. Riichi, Reiko. C It can be synthesized according to the method described in Riichi, Academic Press, published in 1993.
  • a contact reaction between the reducing sugar and ammonia or ammonium ion is performed.
  • monosaccharides or oligosaccharides of two or more sugars are dissolved in a saturated aqueous solution of ammonium bicarbonate, and solid ammonium bicarbonate is added as appropriate while releasing at room temperature or at 50 ° C in a free system. If left for days, glycosylamines are formed.
  • an alkali base
  • a tally evaporator or a centrifugal concentrator it is preferable to add an alkali (base) to the reaction solution after the completion of the reaction, and to concentrate the mixture using a tally evaporator or a centrifugal concentrator.
  • step (B) a step of adding an alkali to the reaction product obtained in the step (A), thereby obtaining glycosylamine
  • glycosylamine can be produced.
  • Such a manufacturing method is also included in the scope of the present invention.
  • alkali refers to a so-called Brenstead base that accepts protons.
  • the protonated added alcohol that is, the conjugate acid of the added alcohol is accompanied by bicarbonate ions to form bicarbonate.
  • the alkali is preferably an alkali other than ammonia, primary and secondary amines, and furthermore, it is easy to evaporate.
  • an alkali having a boiling point of 200 C or less is preferable.
  • a substance which is a stronger base than ammonia is preferable.
  • the alkali used in the production method of the present invention preferably has a pKb value lower than the pKb value (4.76) of ammonia, in other words, the pKa value of the conjugate acid is an ammonium ion. It is desirable that the alkali is higher than the pKa value (9.24).
  • suitable alkalis include pyridine, N-methylmorpholine, N-ethylmorpholine, triethylamine, trimethylamine, piperidine, morpholine and the like. More preferably, it is desirable that the alkali bicarbonate can be distilled off.
  • the alkali to be added may be, for example, a fixed one such as an anion exchange resin.
  • the timing of addition of the alkali may be after the formation of glycosylamine, and the number of times and the amount of addition of the alkali are not particularly limited.
  • a solvent such as water, alcohol, ketone or dimethylsulfoxide may be added together with the alkali.
  • freeze-drying may be performed after concentration so that the sample is not lost during drying.
  • One feature of the method of the present invention for detecting or quantifying an analyte (target substance) is to use the array of the present invention.
  • the method for detecting or quantifying an analyte (target substance) of the present invention is characterized by using the array of the present invention. Specifically, the target substance immobilized on the array and the target substance are immobilized on an array in which the target substance is immobilized on a predetermined area at two or more places at different concentrations exceeding at least one step. To detect the interaction with the analyte to be bound, or to perform quantification based on the amount of binding. More specifically, the detection or quantification method of the present invention includes: (a) contacting the array of the present invention with a test substance;
  • test substance when an interaction is detected, the test substance is used as an indicator that the test substance is a substance (analyte) having a binding property to the target substance.
  • the interaction refers to a binding between a target substance and a substance having a binding property to the target substance.
  • the interaction can be detected or quantified by a visual method, a method using a fluorescent label or the like to read with a scanner, a method of applying a small amount of electricity to read the change, and a change in frequency transmitted by applying a laser.
  • these methods can be used according to the purpose of detection or quantification and the required sensitivity.
  • the sugar immobilized on the array and the sugar immobilized on the array are determined using two or more immobilized arrays at different concentrations exceeding at least one step in a predetermined area. Detecting an interaction with a substance having a binding property to a sugar [the analyte (target substance) in the present invention and corresponding to the sugar target], or performing a quantification based on the amount of the binding.
  • the sugar target is immobilized on the array using an array in which the sugar target is immobilized in a predetermined area at least at two or more different concentrations in at least one step.
  • the interaction between the sugar target and the substance having a binding property to the sugar target is detected, or the amount of the binding is determined. Perform quantification,
  • the sugar and sugar target are determined in advance.
  • a substance having a binding property to each of the sugar and the sugar target immobilized on the array (the present invention) Detects the interaction with the analyte (target substance), which corresponds to the sugar target and sugar, respectively.
  • the protein immobilized on the array and the protein are immobilized on an array in which the protein is immobilized in a predetermined area at two or more locations with different concentrations exceeding at least one step.
  • the interaction with a substance having a binding property is detected, or quantification is performed based on the amount of the binding.
  • the sugar is immobilized on the array and the sugar target is analyzed in the analyte (object).
  • analyte target substance
  • object The substance is described below as an example, but the reverse is true, and the same is true for proteins and protein receptors.
  • the interaction between the sugar and the sugar target is measured by bringing the array prepared in the above (iii) and (iv) into contact with the sugar target as a specimen by a generally known method. be able to.
  • the sugar target substance to be a sample is a cell
  • it can be prepared and used by culturing a standard cell, a mutant cell, or a cell isolated from a patient by a generally used method.
  • the sugar target is a virus, it can be prepared and used in the same manner as in the case of a cell.
  • the sugar target is a toxin, it can be used by purifying it from a culture supernatant obtained by culturing toxin-producing pathogenic cells or the like, or a cell lysate.
  • the sugar target is a sugar-binding protein
  • it can be extracted and purified from crushed animal or plant tissues by a generally used method.
  • the sugar target is an antibody, animal antiserum or monoclonal antibody prepared by a general method can be used.
  • the sugar and / or sugar target should be used.
  • a substance having a binding property can be labeled and used by an appropriate method.
  • the labeling method is not particularly limited, and examples thereof include a method using a substance such as a radioisotope, a fluorescent substance, a chemiluminescent substance, an antigen recognized by an appropriate antibody, or a substance having a luminophore.
  • a substance such as a radioisotope, a fluorescent substance, a chemiluminescent substance, an antigen recognized by an appropriate antibody, or a substance having a luminophore.
  • a substance that is taken into cells or viruses and binds to a gene a substance that is taken into a membrane, a substance that binds to a protein, or the like can be used.
  • the labeling can be performed while culturing the cells or viruses to be tested in an appropriate medium.
  • labeling toxins, sugar-binding proteins or antibodies commonly used protein labeling methods can be used.
  • the sample is brought into contact with the array without labeling beforehand, and then the cells or viruses are detected using fluorescent or chemiluminescent inducer substances
  • the number of cells or viruses serving as specimens can be adjusted to an appropriate number for each specimen and added to the array.
  • Sugar targets is if the cells of the specimen, the number of cells when added, is not particularly limited, for example, 1 0 7 ⁇ 1 0 9 cfu / ml , preferably about 1 0 8 cfu / ml A degree is desirable.
  • the sugar target substance to be a sample is a virus
  • the amount of the virus at the time of addition is not particularly limited, but is, for example, about 10 to 50 ag / ml, preferably about 30 zg / ml.
  • the amount of the toxin at the time of addition is not particularly limited, for example, about 0.1 to 10 tg / ml, preferably about 1 g / ml.
  • the solution used for preparing the specimen is not particularly limited.
  • physiological saline / phosphate buffered physiological saline can be used.
  • the liquid volume of the specimen may be a liquid volume enough to cover the entire array.
  • the signal intensity of radioactivity, fluorescence, chemical luminescence, etc., for an array that has been subjected to a binding test with a sugar target labeled by the above method is measured by a dedicated measuring instrument, such as a microscope equipped with a CCD camera, a chromatoscanner or By performing measurement using an image analyzer or the like, it is possible to detect a sugar target, which is a specimen, on an array, and to further identify sugars having affinity for the sugar target.
  • a quantification method a method generally can be used regardless of whether a saccharide target is quantified or a saccharide is quantified.
  • a simple method is a competition test method (competition attestee method).
  • an unlabeled sugar target with a known concentration is mixed in a system in which a labeled sugar target is brought into contact with a solid-phased array of sugars, the binding between the sugar and the labeled sugar target can be achieved.
  • the labeling signal from the labeled sugar target on the array is reduced.
  • Performing the above competition test on several different concentrations of the unlabeled sugar target provides a standard curve of labeled signal from the labeled sugar target on the array versus the concentration of unlabeled sugar target added.
  • a standard curve is created by mixing unlabeled sugars with known concentrations, and quantification of unknown concentration sugars is performed. can do.
  • the present invention further provides a kit for producing the array of the present invention as described above, or a kit for detecting or quantifying an analyte (a kit for detecting a bound substance), which contains the array of the present invention.
  • a kit for producing an array of the present invention immobilization
  • the target substance can be arbitrarily selected according to the substrate, buffer, and purpose. For example, as described in Example 8 below, a plurality of target substances can be contained.
  • the kit may contain a reagent for fluorescent labeling and a fluorescent labeling substance for control experiments in addition to the array of the present invention.
  • a kit described in Example 8 below examples include the kit described in Example 8 below.
  • the slide glass was immersed in a 1 Omg / ml EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, Nacalai Tesque, Inc.) PBS solution and allowed to stand at room temperature for 1 hour. Then, the slide glass was washed five times with purified water. After that, water droplets adhering to the glass surface were removed by centrifugation and dried overnight in a desiccant containing a desiccant, to produce a slide glass activated with glass.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, Nacalai Tesque, Inc.
  • a slide glass having amino groups covalently bonded specifically, Matsunami MAS (manufactured by Matsunami Glass Co., Ltd.) and silane prep slide (manufactured by Sigma) were immersed in a 0.1M anhydrous DMSO solution at room temperature for 6 hours. . Then, the slide glass was washed five times with purified water. Thereafter, the water droplets attached to the glass surface were removed by centrifugation, and the desiccant was dried overnight in a desiccant.
  • Matsunami MAS manufactured by Matsunami Glass Co., Ltd.
  • silane prep slide manufactured by Sigma
  • DMF Dimethylformamide
  • a glass slide with amino groups covalently bonded specifically, Matsunami APS (manufactured by Matsunami Glass Industry Co., Ltd.) and Silane Prep Slide (manufactured by Sigma) were loaded with 0.05 M polyoxyethylene diacetic acid (manufactured by Sigma), 20 mg / nil in an EDC (1-ethyl-3- (3-dimethylaminopropyl) carbopimid hydrochloride, Nacalai Tesque, Inc.) PBS solution and left at room temperature for 1 hour. Next, the slide glass was washed five times with purified water. Thereafter, water droplets attached to the surface of the glass were removed by centrifugation, and the whole was dried overnight in a desiccant containing a desiccant to prepare a carbodiimide-activated PEG slide glass.
  • Matsunami APS manufactured by Matsunami Glass Industry Co., Ltd.
  • Silane Prep Slide manufactured by Sigma
  • a glass slide with amino groups covalently bonded specifically, Matsunami APS (Matsunami Glass Industry Co., Ltd.) and Silane Prep Slide (Sigma) are immersed in a methylene chloride solution containing 5% steer chloride in room temperature. And left it alone. The slide glass was then washed three times with methylene chloride and three times with methanol. Thereafter, the solvent adhering to the glass surface was removed by centrifugation, and dried overnight in a desiccant containing a desiccant to prepare a hydrophobic slide glass.
  • Matsunami APS Matsunami Glass Industry Co., Ltd.
  • Silane Prep Slide Sigma
  • Example 2 the slide glass was washed five times with a 1% aqueous acetic acid solution. Thereafter, the solvent adhering to the glass surface was removed by centrifugation and dried overnight in a desiccator containing a desiccant to prepare a foil activated slide glass.
  • Example 2 the solvent adhering to the glass surface was removed by centrifugation and dried overnight in a desiccator containing a desiccant to prepare a foil activated slide glass.
  • Glycolipids [Glucosylceramide (Sigma), Galactosylceramide (Sigma), Lactosylceramide (Wako Pure Chemical Industries), Asiaguchi GM1 (Matrea) GM1 (Matrea), GM2 ( 1), GM3 (manufactured by Matreya), and GDIa (manufactured by Wako Pure Chemical) were each placed in a plastic tube with a screw cap, and dried under reduced pressure. To the dried product, 1001 of a 1M aqueous solution of triethylamine acetate (pH 7.3) was added, and the mixture was vigorously stirred while applying ultrasonic waves to prepare 100-glycolide solutions.
  • a dilution series spanning ⁇ 100 1 was made.
  • a slide glass (Matsunami Glass Industry Co., Ltd.) coated with a glycolipid diluent solution coated with n-butyl trimethoxysilane using an Affymetrix 417 Arrayer (Affymetrix) so as to have a diameter of 120 ⁇ m and a center interval of 300 m ).
  • the slide glass was placed in a desiccator containing silica gel and dried at 37 ° C for 1 hour.
  • the obtained slide glass was washed twice with PBS, and immersed in a PBS solution containing 1% psi serum albumin at room temperature for 2 hours. Thereafter, the plate was washed three times with PBS. Carefully take care not to dry the glass surface, and then cholera toxin B subunit fluorescently labeled with Fluorolink-1 AbCy 3 (manufactured by Amersham Pharmacia Biotech) (25 ⁇ g / ml containing 0.25% 25 serum albumin (PBS, manufactured by Risto Biological Laboratories) was layered on a slide glass. After covering the surface of the obtained slide glass with a paraffin film, the slide glass was allowed to stand at 37 under humidification for 1 hour under humidification.
  • the slide glass was washed three times with PBS and rinsed with a 10 mM aqueous solution of acetic acid-triethylamine (pH 7.0). Then, the solvent attached to the glass surface was removed by centrifugation, and the glass was dried at room temperature.
  • the slide glass was inspected using a fluorescence microscope BX60 (Olympus). BP 545-580 was used for the excitation filter, and B A610 IF was used for the absorption filter.
  • Lysoglycolipids [Glucopsychosin (manufactured by Matreya), Psychosin (manufactured by Matreya), lyso GM1 (manufactured by Takara Shuzo), lyso GM2 (manufactured by Takara Shuzo), lyso GM3 (manufactured by Takara Shuzo), lyso GD1a (Manufactured by Takara Shuzo Co., Ltd.)] was placed in a plastic tube with a screw cap at 10 nmo.
  • each lysoglycolipid solution with 5% OmM N-ethylmorpholine / formic acid aqueous solution (pH 7.2) containing 0.1% sodium cholate, and dilute over 32 nM to 100 OM A series was made.
  • the diluted solution of lysoglycolipid was purified using an Affymetrix 417 Arrayer (Affymetrix) to obtain a diameter of 120 ⁇ m.
  • a spot was placed on a glutaraldehyde-activated slide glass (manufactured by Matsunami Glass Industry Co., Ltd.) activated with glutaraldehyde at a center distance of 300 m and a center distance of 300 m, and the plate was left standing at 37 for 1 hour under humidification.
  • the slide glass was washed twice with pure water, the slide glass was immersed in a mixed solution of 0.1 g of sodium borohydride-containing water (3 Oml) and ethanol (10 ml), and allowed to stand at room temperature for 10 minutes. After washing the slide glass three times with pure water, the slide glass was immersed in a PBS solution containing 1% serum albumin for 2 hours at room temperature.
  • FIG. 1 is a diagram showing the reactivity of an array on which each lysoglycolipid is immobilized and cholera toxin B subunit, wherein row A shows psychosin, B shows glucosychosin, C shows lysoGM1, D indicates lyso GM2, E indicates lyso GM3, F indicates lyso GD Ia, columns indicate dilution series of each lysoglycolipid, column 1 is 100 M, 2 is 20 zM, 3 is 4 zM, 4 Indicates 800 nM, 5 indicates 160 nM, and 6 indicates 32 nM.
  • agarotetraose prepared according to the method described in International Patent Publication No. 99/24447 Pan Fret
  • galacto-N-neotetraose (Gal ⁇ l-4AhGaIl-3Gal ⁇ l-4AhGaK)
  • Oose (Gal 31-4G lcNAc iSl-SGal / Sl-4Glc, manufactured by Biocurve) was dried at 625 nmo 1 each, and the obtained dried product was dissolved in 2.5 zl of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • 501 DMSO containing 500 polylysine (average molecular weight: 5600, manufactured by Sigma) was added, and the mixture was stirred and heated at 37 ° C for 30 minutes.
  • a reducing reagent solution obtained by dissolving 2 Omg of the polan-dimethylamine complex with 1001 acetic acid was added to the mixture, and the mixture was further heated at 80 ° C. for 1 hour.
  • 1 ml of pure water was added to the obtained solution, and the obtained solution was sufficiently dialyzed against a 1 OmM acetic acid aqueous solution. Thereafter, the dialysate was concentrated to dryness to prepare PLL-agarotetraose and PLL-lacto-N-neotetraose.
  • a reducing reagent solution 251 in which 2 Omg of a borane-dimethylamine complex was dissolved in 100% of acetic acid was added, and the mixture was further heated at 80 for 1 hour. Thereafter, 1 ml of pure water was added to the obtained solution, and the resulting solution was sufficiently dialyzed against an aqueous solution of 10 mM acetic acid.
  • the obtained dialysate is concentrated to dryness, and PLL-i8-Man4, PLL- ⁇ -1 ⁇ & 113 ⁇ ? Each of 1- & 115 was prepared.
  • Glycopeptide 1 zmo1 obtained by pronase digestion of soybean aglutinin (Honen Corporation) was converted into 5 OmU endo-A [Applied and Environmental Microbiology, Vol. 55, (Prepared by the method described in pages 3107 to 3112 (1 989)] for 5 hours at 37 ° C.
  • the obtained solution was supplied to a cellulose cartridge (manufactured by Takara Shuzo Co., Ltd.) to purify sugar chains.
  • the resulting sugar chain 250 nmo 1 is dried in a plastic tube with a screw cap, and DMS 0 solution containing 1 mg of polylysine (average molecular weight: 56000, Sigma) and 10% acetic acid is added. did.
  • Glycated polylysine prepared in Example 4 (PLL-agarotetraose, PLL-lacto-N-neotetraose, PLL-Man4, PLL-a-Man 3 , PLL-1 Man5, PLL-1 Man 9) are diluted with 0.1% sodium cholate in 5 OmM N-ethylmorpholine'formic acid aqueous solution (pH 7.2) on a 96-well microplate. And dilution series ranging from 16 Ong / ral to 500 ⁇ g / ml.
  • the diluted solution of saccharified polylysine was prepared using Affymetrix 417 Arrayer (manufactured by Affymetrix) so as to have a diameter of 120 im and a center interval of 30 O ⁇ m.
  • the slide glass was inspected using a fluorescence microscope BX60 (Olympus). BP 460-490 was used for the excitation filter, and BA515 IF was used for the absorption filter.
  • FIGS. In other words, Figs. 2 to 4 Fig. 4 shows the reactivity of the gin-immobilized array with FITC-labeled ConA (Fig. 2), FITC-labeled DSA (Fig. 3), and FITC-labeled RCA120 (Fig. 4).
  • Row A is PLL—agarotetraose
  • B is PLL—Lacto N—neotetraose
  • C is PLL—y8_Man 4
  • D is PLL—One Man 3
  • E is PLL One and Man 5
  • F is PLL—
  • Column 9 indicates the dilution series of each glycated polylysine
  • column 1 indicates 500 / g / mU2, 100 ⁇ g / ml
  • 3 indicates 20 g / ml
  • 4 indicates 4 g / ml
  • 5 indicates 800 ng / ml and 6 indicate 160 ng / ml.
  • FITC-labeled C0nA preferentially shows spots on PLL-1 Man3, PLL-1 Man5 and PLLa-Man9, and these glycated polylysines appear. It can be seen that specific binding to ConA is suggested.
  • FITC-labeled DSA and FITC-labeled RCA120 preferentially form spots on PLL-agarotetraose and PLL-lacto-N-neotetraose. This indicates that these glycated polylysines are suggested to specifically bind to DSA and RCA120.
  • Example 6 Detection of Microbial Binding to an Array
  • Glycolipids [Glucosylceramide (Sigma), Galactosylceramide (Sigma), Lactosylceramide (Wako Pure Chemical Industries), Ashiaguchi GM1 (Matreya), GM1 (Matreane earth), GM2 ( (Wako Pure Chemical Co., Ltd.), GM3 (Matrea Co., Ltd.), and GD Ia (Wako Pure Chemical Co., Ltd.)] were each placed in a brass tube with a screw cap, and dried under reduced pressure. To the dried product was added a 1M aqueous solution of acetate-triethylamine (pH 7.3) 1001, and the mixture was vigorously stirred while sonicating to prepare ImM glycolipid solutions.
  • acetate-triethylamine pH 7.3
  • the slide glass was washed twice with PBS, and then immersed in a PBS solution containing 1% serum albumin for 2 hours at room temperature. Then, it was washed three times with PBS. Taking care to glass surface is not dry, 1% ⁇ shea serum albumin P BS solution to about 1 0 7 cells / ml lactic acid bacteria were suspended at a concentration of [Lactobacillus Ramunosa scan strain (I FO 3425)) The slide glass was overlaid. After leaving at room temperature for 1 hour, the slide glass was lightly washed twice with PBS.
  • a heron anti-lactic acid bacterium antiserum diluted 500-fold with a PBS solution containing 1% serum albumin [Biochemical and Biophysica 1 Research Communications. Vol. 228, pp. 148-152 (Biochemical and Biophysica 1 Research Communications). 1 996)] and left at room temperature for 1 hour.
  • the slide glass was washed twice with PBS for 2 minutes, and overlaid with Alexa 546-labeled goat anti-Peacock IgG (manufactured by Molecular Probes) diluted 500-fold with a PBS solution containing 1% Pseudoserum albumin. It was left at room temperature for 1 hour.
  • the slide glass was washed twice with PBS for 2 minutes, covered with a glass cover, and examined with a fluorescence microscope.
  • the fluorescence microscope used was BX60 (manufactured by Olympus), and the excitation filter was BP520-550, and the absorption filter was BA580IF.
  • FIG. 5 shows the results. That is, FIG. 5 is a diagram showing the binding properties between the array on which each glycolipid is immobilized and the lactic acid bacterium.
  • Row A shows galactosyl ceramide
  • B shows turcosyl ceramide
  • C shows lactosyl ceramide
  • D shows Asia mouth GM1
  • E is GM1
  • F is GM2
  • G is GM3
  • H GDIa
  • the column shows the dilution series of each glycolipid
  • the column 1 is lmM
  • 2 is 200 / zM
  • 3 is 100 ⁇
  • 4 indicates 50 ⁇ ⁇
  • 5 indicates 25 « ⁇
  • 6 indicates 12.5.
  • the flask was connected to a vacuum pump and aspirated at room temperature for 30 minutes.
  • the obtained residue was dissolved in 6 ml of pure water and freeze-dried overnight.
  • the dried product was dissolved again in 3 ml of pure water, freeze-dried, and weighed to find that it was 15 lmg.
  • a part of the reaction solution (6.25 ml, equivalent to lactose 10 Omg) was prepared according to the method of Karin et al. [Journal of Power Chemistry, Vol. 8, pp. 597-611] (1 989)], lyophilized twice, and weighed to obtain 159 mg of lactosylamine.
  • Dimethyl sulfoxide (101) was added to each of the solids obtained above to dissolve the solids. Transfer 11 to another plastic tube, add 0.1 M fluorescein isothiocynate (Sigma) in dimethyl sulfoxide, stir, and incubate at 37 ° C for 1 hour did. To each reaction solution, 81 of an 80% aqueous solution of acetonitrile was added, and 21 of them was spotted on silica gel 60 HPT LC (manufactured by Merck) and developed with 80% acetonitrile. The plate was scanned with blue light absorption using GS-700 Imaging Densitometer One-Year (Bio-Rad).
  • a kit for preparing a sugar array using Affymetrix 417 Arrayer (manufactured by Affimetrics) was constructed as follows.
  • the glycolipid contained in this kit contained a plurality of glycolipids shown in Table 1 so that the glycolipid could be arbitrarily selected according to the purpose.
  • Butylsilane-coated slide glass (Matsunami Glass Industry Co., Ltd.) 100 sheets 1M acetic acid-triethylamine solution (pH 7.3) containing 4% trehalic ⁇ -ose: 100 ml glycolipids listed in Table 1 (dry matter) 100 / g each
  • a slide glass (2.6 cm x 7.6 cm) spotted with sugar, a reagent for protein fluorescence labeling, and a carbohydrate bond consisting of fluorescently labeled protein for control experiments Construct protein search kit as follows did.
  • the sugar array slide glass was prepared using the sugar array preparation kit constructed in (8-1) of Example 8 and a 12-fold three-fold dilution series (lmM to 5.6 nM) for 25 kinds of glycolipids.
  • a total of 300) was prepared and spotted using Affymetrix 417 Arrayer (manufactured by Affymetrix) at a diameter of 120 m and a center interval of 300 m to form an array. Two replicas were also made on the same slide.
  • the glycolipid solution was diluted with a 1 M aqueous solution of acetic acid-triethylamine (pH 7.3) containing 4% trehalose on a 96-well microtiter plate to prepare a dilution series ranging from 3 to 100 M.
  • a series was prepared by dissolving and diluting glycolipids using a 1 M aqueous acetic acid-triethylamine solution (pH 7.3) containing no trehalose.
  • the glycolipid dilution solution was spotted on a slide glass (manufactured by Matsunami Glass Co., Ltd.) coated with n-butyltrimethoxysilane at a diameter of 120 m and a center distance of 300 m using an Affymetrix 417 Arrayer (manufactured by Affymetrix). .
  • the slide glass was placed in a desiccator containing silica gel and dried at 37 ° C for 1 hour.
  • the slide glass was washed twice with PBS, immersed in a PBS solution containing 1 ⁇ l serum albumin at room temperature for 2 hours, and then washed three times with PBS. Then, paying attention not to dry the glass surface.
  • cholera toxin B subunit 25 ⁇ g / ml 0.25% serum
  • Fluorolink AbCy 3 Fluorolink AbCy 3
  • Albumin-containing PBS manufactured by Risto Biological Laboratories
  • PBS was layered on a slide glass, covered with a paraffin film, covered with a paraffin film, protected from light, and allowed to stand at 37 ° C for 1 hour under humidification. Wash the slide glass 3 times with PBS, rinse with 1 OmM aqueous solution of triethylamine acetate (pH 7.0), remove the solvent adhering to the glass surface by centrifugation, and dry at room temperature.
  • the slide glass was inspected using a fluorescence microscope BX60 (Olympus). BP545-580 was used for the excitation filter, and BA610IF was used for the absorption filter.
  • Lactosylceramide (bovine butter mi lk)
  • each 6 ml of this glycolipid solution was placed in a tube, and a 5-fold dilution series was prepared in 5 steps. That is, the concentration of glycolipid in each dilution series was 1.5 mg / ml, 0.3 mg / ml, 0.06 rng / ml, 0.012 mg / ml, 0.0024 rag / ml. ml.
  • Cy3-polylysine [polylysine was fluorescently labeled with Fluorolink-AbCy3 (Amersham Pharmacia Biotech)] 25 O mg /
  • a solution having a concentration of 6001 was diluted by a factor of 100 to prepare a solution.
  • Cy 3-PL indicates the spot position of Cy 3-polylysine of the marker
  • rows B to M indicate the glycolipids to be spotted and their positions, respectively.
  • Columns 2 and 7 have a glycolipid concentration of 5 mg / ml
  • columns 3 and 8 have a glycolipid concentration of 0.3 mg / ml
  • columns 4 and 9 have a glycolipid concentration of 0.06 mg / ml
  • columns 5 and 10 have a glycolipid concentration of 0.06 mg / ml.
  • columns 6 and 11 show spot positions at glycolipid concentration 0.0024 mg / ml.
  • the slide glass was placed in a desiccated silica gel overnight and dried at 37 ° C for 1 hour, and this glycolipid array was used for the following various detections.
  • the glycolipid array prepared above was washed twice with PBS, and then blocked with a PBS solution containing 1% serum albumin for 2 hours at room temperature. After washing three times with PBS, cholera toxin B subunit fluorescently labeled with Fluorolink-Ab Cy 3 (manufactured by Amersham Pharmacia Biotech) (25 ⁇ g / ml 0.25% PBS containing serum albumin) (Rist Biological Laboratory Co., Ltd.) was overlaid on a slide glass. The surface of the slide glass was covered with parafilm so as not to dry, and the slide glass was allowed to stand at 37 ° C for 1 hour under humidification with light shielding.
  • the slide glass was washed three times with PBS for 3 minutes, rinsed lightly with 1 OmM acetic acid-triethylamine aqueous solution (pH 7.3), and then the solvent adhering to the glass surface was removed by centrifugation, and Affymetrix 418 was removed.
  • the fluorescence intensity was analyzed using Array Scanner (Affymetrix).
  • the same three slide glasses were used, and the results are shown in Figs. 7 to 9.
  • the horizontal axis represents the concentration of each glycolipid when the array was prepared, and the vertical axis represents the fluorescence intensity.
  • concentration-dependent specific binding of cholera toxin to GM1, GM2 and Fuc0sy1-GM1 is observed.
  • the glycolipid array prepared above was washed twice with PBS, and then blocked with a PBS solution containing 1% serum albumin at room temperature for 2 hours. After washing three times with PBS, a lactic acid bacterium (Lactobacillus rhamnosus strain (IFO 3425)) suspended at a concentration of about 10 7 cells / ml in a PBS solution containing 1% serum albumin was overlaid on the slide glass. The slide glass was covered with parafilm so as not to dry, and then left at room temperature for 1 hour. The slides were then lightly rinsed twice with PBS. Next, a heron anti-lactic acid bacteria antiserum [Biochemical Biophysical Research Communications, Vol. 228, pp.
  • the solvent on the glass surface was removed by centrifugation, and the fluorescence intensity was analyzed using an Affymetrix 418 Array Scanner (manufactured by Affymetrix).
  • the same three slide glasses were used, and the results are shown in Figs. 10 to 12.
  • the horizontal axis indicates the concentration of each glycolipid when the array was prepared, and the vertical axis indicates the fluorescence intensity.
  • the glycolipid array was blocked with a PBS solution containing 1% serum albumin at room temperature for 2 hours.
  • an anti-GQ1b antibody (clone number: GMR13, manufactured by Seikagaku Corporation) diluted 100-fold with a PBS solution containing 1 ⁇ C serum albumin was overlaid on a slide glass, and then placed on a slide glass. After the surface was covered with parafilm to prevent drying, it was left at 4 ° C for 16 hours.
  • the slide glass was washed three times with PBS, and a Cy3-labeled heron anti-mouse IgM antibody diluted 500-fold with a PBS solution containing 1% serum albumin (An antibody manufactured by Zymed, Fluorolink-Ab Cy3 ( Antibody labeled with Amersham Pharmacia Biotech) was overlaid on a slide glass, covered with parafilm so as not to dry the slide glass, and allowed to stand at 37 ° C for 1 hour. Then, the plate is washed 3 times with PBS for 3 minutes, rinsed lightly with 1 OmM aqueous solution of triethylamine acetate (pH 7.3), and then the solvent on the glass surface is removed by centrifugation.
  • PBS solution containing 1% serum albumin An antibody manufactured by Zymed, Fluorolink-Ab Cy3 ( Antibody labeled with Amersham Pharmacia Biotech) was overlaid on a slide glass, covered with parafilm so as not to dry the slide glass, and allowed to stand at 37
  • the analyte particularly sugar
  • the target substance or protein can be easily detected or quantified, and the difference between the recognition and binding of the substance (analyte) that recognizes and binds to the target substance in vitro can be determined at the same time as the concentration of the target substance is derived. Simple, fast and highly sensitive detection Can be issued.
  • glycosylamine of the present invention glycosylamine useful for preparing the array of the present invention can be efficiently obtained with high purity.

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Abstract

L'invention concerne un jeu ordonné d'échantillons présentant une substance cible immobilisée en deux ou plusieurs points dans une zone préétablie d'une phase solide par cm2 d'une base de phase solide. La substance cible est immobilée en deux ou plusieurs points, à des concentrations différentes dépassant au moins une étape. L'invention concerne un procédé de détection ou de quantification d'un sujet à analyser au moyen dudit jeu ordonné d'échantillons. Elle concerne un nécessaire pour détecter ou quantifier un sujet à analyser (c.-à-d. un nécessaire pour détecter une matière liante), et un procédé de production de glycosylamine. Ainsi, une substance sensible à la substance cible et se liant à cette dernière (c.-à-d. la substance à analyser) peut être détectée rapidement et sans inconvénient in vitro avec une sensibilité élevée, sur la base de la différence du niveau de reconnaissance et de liaison mesuré selon la concentration de la substance cible. Un glycosylamine très pur, utile pour la préparation du jeu ordonné d'échantillons, peut être obtenu avec un rendement supérieur.
PCT/JP2001/001247 2000-02-21 2001-02-21 Procede de detection d'une substance a analyser WO2001061353A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7053051B2 (en) 2003-10-28 2006-05-30 Medtronic, Inc. Methods of preparing crosslinked materials and bioprosthetic devices

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05222099A (ja) * 1991-10-15 1993-08-31 Monsanto Co 合成ｎ−結合グリコ複合体の製造法
JPH09325149A (ja) * 1996-02-06 1997-12-16 Boehringer Mannheim Gmbh 結合アッセイに関する材料および方法
JPH11508564A (ja) * 1995-06-30 1999-07-27 ナショナル リサーチ カウンシル オブ カナダ 合成ｎ−結合グリココンジュゲートを製造するための方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05222099A (ja) * 1991-10-15 1993-08-31 Monsanto Co 合成ｎ−結合グリコ複合体の製造法
JPH11508564A (ja) * 1995-06-30 1999-07-27 ナショナル リサーチ カウンシル オブ カナダ 合成ｎ−結合グリココンジュゲートを製造するための方法
JPH09325149A (ja) * 1996-02-06 1997-12-16 Boehringer Mannheim Gmbh 結合アッセイに関する材料および方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CARBOHYDRATE CHEMISTRY, vol. 8, no. 4, 1989, pages 597 - 611, XP002942006 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7053051B2 (en) 2003-10-28 2006-05-30 Medtronic, Inc. Methods of preparing crosslinked materials and bioprosthetic devices

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