WO2005010528A1 - Procede d'elimination d'une substance non specifique - Google Patents

Procede d'elimination d'une substance non specifique Download PDF

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Publication number
WO2005010528A1
WO2005010528A1 PCT/JP2004/011104 JP2004011104W WO2005010528A1 WO 2005010528 A1 WO2005010528 A1 WO 2005010528A1 JP 2004011104 W JP2004011104 W JP 2004011104W WO 2005010528 A1 WO2005010528 A1 WO 2005010528A1
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Prior art keywords
acid
immobilized
sample
solid support
ligand
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PCT/JP2004/011104
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English (en)
Japanese (ja)
Inventor
Akito Tanaka
Tomohiro Terada
Tsuruki Tamura
Takaaki Shiyama
Akira Yamazaki
Minoru Furuya
Masayuki Haramura
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Reverse Proteomics Research Institute Co., Ltd.
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Priority to JP2005512114A priority Critical patent/JP4496168B2/ja
Publication of WO2005010528A1 publication Critical patent/WO2005010528A1/fr

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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 a basic technology for intermolecular interaction using a solid support. More specifically, in the process of immobilizing a ligand on a solid-phase carrier, utilizing the intermolecular interaction on the solid phase, and measuring and analyzing the interaction, non-specific interaction that hinders analysis, etc.
  • the present invention relates to a technique for reducing the effect.
  • the specific protein is analyzed when the protein bound to the affinity resin is analyzed by SDS gel or the like.
  • Non-specific proteins that obscure the protein make it difficult to detect the specific proteins, or 2)
  • the presence of peaks caused by large non-specific protein adsorption The presence of non-specific proteins other than proteins that have specific molecular interactions with the immobilized ligand molecules (so-called target proteins) has become a problem, such as difficulty in distinguishing peaks due to specific protein binding. .
  • target proteins In general, it is rare that a target protein is present in a large amount in a living body.
  • proteins eg, albumin
  • these interfering proteins include structural proteins such as tubulin-actin in lysates prepared from rat brain as the former, and albumin in plasma as the latter.
  • IP imnmno-precipitation
  • a method of immobilizing antibodies on columns for example, F.F. Steel, et al.), “Molecular & Cellular Proteomics”, (USA), May 16, 2003, “Efficient and specific albumin from human serum samples. See Efficient and specific removal of albumin from human serum samples. "
  • many important compounds (proteins) are entrained in the precipitated mass, resulting in problems such as loss of the target product and its cost. A solution has been desired.
  • the present invention provides a method for searching for a marker or finding a marker in diagnosis or the like, in which an interfering non-specific protein is artificially removed or reduced from a sample before the start of a study.
  • the aim is to provide a method.
  • the present inventors have found in the course of intensive studies to solve the above-mentioned problems that the main factor of the adsorption of the non-specific protein is due to hydrophobic interactions not based on structural characteristics (WO 2004Z025297). ).
  • the sample biological component mixture
  • a solid support on which a substance such as stearic acid or the like, which has few structural features and high hydrophobicity, was immobilized.
  • the present invention is as follows.
  • Hydrophobic substances include pendecanoic acid, myristic acid, palmitic acid, linoleic acid, arachidonic acid, linolenic acid, oleic acid, stearic acid, 9- (naphthalene-11-yl) monononanoic acid, dodecanesulfonic acid,
  • the hydrophobic substance is at least one selected from the group consisting of myristic acid, palmitic acid, linoleic acid, arachidonic acid, linolenic acid, oleic acid, stearic acid, octadecanesulfonic acid and hexadecanesulfonic acid.
  • the solid support according to the above [6].
  • a method for removing a substance that non-specifically binds to a ligand and / or a ligand-immobilized solid phase carrier comprising treating the sample with a solid phase carrier on which a hydrophobic substance is immobilized.
  • the solid support obtained by immobilizing a hydrophobic substance is the solid support according to any one of the above [1] to [8], wherein the solid support is the solid support according to any one of the above [9] to [12].
  • the method described in the section. [14] Non-specific binding to ligand and / or ligand-immobilized solid support The method according to any one of the above [9] to [12], which is based on a hydrophobic interaction between the two.
  • a method for purifying albumin comprising: treating a sample with the solid phase carrier according to any one of the above [1] to [8], and adsorbing albumin in the sample to the solid phase carrier. .
  • FIG. 1 is a graph showing that FK506 non-specific substances can be adsorbed and removed by pretreatment with a stearic acid-immobilized resin.
  • Rat brain lysate was used as a sample. If pretreatment with stearic acid-immobilized resin was not performed, samples were pretreated with 100 ⁇ l of stearic acid-immobilized resin, and samples were treated with 200 ⁇ l of stearic acid-immobilized resin. The results of pre-processing are shown below.
  • FIG. 2 is an electrophoretic photograph showing the result of examining the degree of adsorption of a nonspecific substance to a resin on which various compounds are immobilized.
  • FIG. 3 is an electrophoretic photograph showing the results of examining the degree of adsorption of nonspecific substances between the stearic acid-immobilized resin and the CB—F3GA immobilized resin. Escherichia coli 1 ysate expressing DHFR was used as a sample.
  • FIG. 4 is an electrophoresis photograph showing the results of examining the removal of albumin from human plasma and the degree of albumin adsorbed to the stearic acid-immobilized resin on the stearic acid-immobilized resin.
  • the present invention has been problematic in analyzing the interaction between a ligand immobilized on a solid support and a molecule having a specific interaction with the ligand (ie, a target molecule).
  • a ligand immobilized on a solid support ie, a target molecule.
  • Technology to suppress or reduce non-specific binding to Z or ligand-immobilized solid phase carriers more specifically, ligand immobilization without loss or denaturation of the desired target molecule
  • ligand immobilization without loss or denaturation of the desired target molecule
  • non-specific substances to remove or reduce substances that non-specifically adsorb to immobilized solid phase carriers
  • the terms ligand and target molecule are intended to mean a combination having a specific intermolecular interaction. When one of the combinations is immobilized as a ligand on a solid phase, the other is used.
  • target molecules ie, their names can be changed depending on which one is immobilized on the solid phase. Not only one type of target molecule having a specific interaction with a ligand, and similarly, not only one type of ligand having an interaction specific to a target molecule.
  • “Specific interaction” can be compared to the “key-keyhole relationship” (Reference book: “Drug Receptors”, edited by Kazunari Takayanagi, Nanzando), and only specific ligands (specific target molecules) It acts to specifically recognize and bind to specific receptors for agonists or antagonists, enzymes for substrates, and FK506 binding proteins (eg, for FK506 (ligand))
  • the relationship between the target molecule), steroid hormone receptors for steroid hormones (eg, dexamethasone and glucocorticoid receptor), and HDAC for the anticancer drug trapoxin, etc. corresponds to “specific interaction”.
  • non-specific interaction refers to an action that causes a situation in which the target of binding thereby is not limited to a specific molecule and is variously changed depending on reaction conditions. It means the action between the ligand on the immobilized solid support and the unspecified molecule that binds and adsorbs to the surface of the solid support. “Non-specific interaction” hinders or confuses the binding of a ligand to a target molecule based on “specific interaction”, or the binding due to “specific interaction” There is a risk of overlooking.
  • Non-specific binding means binding / adsorption based on non-specific interaction.
  • “non-specific binding” is preferably based on non-specific hydrophobic interactions between molecules.
  • Non-specific substance that binds to the solid phase carrier on which the ligand is immobilized by non-specific interaction includes proteins, peptides, nucleic acids, fatty acids, carbohydrates and the like.
  • the present inventors have focused on non-specific binding of proteins and the like by hydrophobic interaction, and have immobilized a hydrophobic substance on a solid support (for convenience, also referred to as a hydrophobic substance-immobilized solid support, A method for removing the substance from the sample by establishing the binding of a non-specific substance to the solid phase carrier on which the ligand is immobilized is clearly established.
  • the “hydrophobic substance” is a substance that tends to be hydrophobic so as to bind to a non-specific substance when immobilized on a solid support.
  • the degree of hydrophobicity can be generally represented by a hydrophobic parameter
  • the hydrophobicity of the “hydrophobic substance” can be defined by a partition coefficient, specifically, LOGP. .
  • CLOGP Predicted value obtained by software for estimating the hydrophobic parameter of a compound by computer; for example, Corwin / Leo's
  • the hydrophobic parameter is not limited to CLOGP. Larger CLOGP means higher hydrophobicity.
  • the LOG P of the hydrophobic substance of the present invention is 4 or more, preferably 6 or more when calculated as CLOG P. If it is less than 4, a sufficient effect of removing nonspecific substances cannot be obtained. Also, the larger the LOGP, the higher the hydrophobicity, and a substance having such a high hydrophobicity can suitably achieve the object of the present invention, but when calculated as CLOGP, the effect significantly increases even if it exceeds about 20.
  • CLOGP is 20 or less from the viewpoint of not being seen and the ease of synthesis.
  • the degree of hydrophobicity of the “hydrophobic substance” is more strictly fixed to the solid support. It may be defined as the state in which the solid phase carrier is immobilized with the hydrophobic substance, that is, the hydrophobic property of the entire solid-phase carrier on which the hydrophobic substance is immobilized.
  • the hydrophobic substance that can be used in the present invention is not particularly limited as long as it has the above properties.
  • it has the above properties (that is, 4 or more, preferably 6 or more when LOGP is calculated as CLOGP).
  • R i and R 2 are the same or different and are a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted alkynyl group
  • the "substituted or unsubstituted alkyl group" for R 2 includes an aryl group which may have a substituent, an alkoxy group which may have a substituent, and an amide group which may have a substituent
  • decanyl pendecanyl, dodecanyl, tridecanyl, tetradecanyl, pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl, 2-phenylenyl, N-phenyl Ethylcarbamoylpropyl, 8-naphthyloctyl and the like. It preferably has 10 or more carbon atoms, more preferably 15 or more carbon atoms. Preferable examples include pentadecanyl and heptadecael.
  • an alkyl group having 10 to 99 carbon atoms (as defined above), an aryl group having 6 to 10 carbon atoms (eg, phenyl) , 1-naphthyl, 2-naphthyl, etc.), an aralkyl group having 7 to 30 carbon atoms (for example, benzyl, phenethyl, etc.), a halogen atom (for example, chlorine atom, iodine atom, bromine atom, fluorine atom), a hydroxyl group,
  • An amino group, an alkoxy group having 1 to 30 carbon atoms for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.
  • a carboxyl group and the like for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-but
  • the “aryl group” in the “aryl group optionally having substituent (s)” includes, for example, an aryl group having 6 to 10 carbon atoms such as phenyl, 1-naphthyl, and 2-naphthyl. .
  • substituted alkoxy group examples include an aryl group having 6 to 10 carbon atoms (eg, phenyl, 1_naphthyl, 2-naphthyl, etc.), a halogen atom (For example, chlorine atom, iodine atom, bromine atom, fluorine atom), hydroxyl group, amino group, carboxyl group and the like.
  • alkoxy group in the “alkoxy group optionally having substituent (s)” includes, for example, methoxy, ethoxy, n-propo Examples thereof include alkoxy groups having 1 to 30 carbon atoms, such as xy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.
  • the “substituent” in the “optionally substituted amide group” includes an alkyl group having 1 to 30 carbon atoms (for example, methyl, ethyl, propyl, etc.), a carbon atom having 7 to 3 carbon atoms. 0 aralkyl groups (eg, benzyl, phenethyl, etc.), halogen atoms
  • an alkyl group having 1 to 30 carbon atoms for example, methyl, ethyl, propyl, etc.
  • a carbon atom having 7 to 30 carbon atoms for example, methyl, ethyl, propyl, etc.
  • aralkyl groups eg, benzyl, phenethyl, etc.
  • halogen atoms eg, chlorine atom, iodine atom, bromine atom, fluorine atom
  • alkoxy groups having 1 to 30 carbon atoms eg, Ethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.
  • carboxyl group for example, methyl, ethyl, propyl, etc.
  • aralkyl groups eg, benzyl, phenethyl, etc.
  • halogen atoms
  • cycloalkyl group in the "optionally substituted cycloalkyl group", a cycloalkyl group having 3 to 30 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl can be mentioned. .
  • substituted in the “heteroaryl group optionally having substituent (s)” includes an alkyl group having 1 to 30 carbon atoms (for example, methyl, ethyl, propyl, etc.), a carbon atom having 7 to 30 carbon atoms.
  • aralkyl groups eg, benzyl, phenethyl, etc.
  • halogen atoms eg, chlorine atom, iodine atom, bromine atom, fluorine atom
  • hydroxyl groups amino groups
  • alkoxy groups having 1 to 30 carbon atoms (eg, Ethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.), and a carboxyl group.
  • heteroaryl group in the “optionally substituted heteroaryl group” include thiazolyl and ⁇
  • substituted carbonyl group examples include minothiazolyl, furanyl, thiophenyl, pyrrolyl, indolyl and the like.
  • alkyl groups for example, methyl, ethyl, propyl, etc.
  • aralkyl groups having 7 to 30 carbon atoms for example, benzyl, phenethyl, etc.
  • halogen atoms for example, chlorine atom, iodine atom, bromine
  • fluorine atom fluorine atom
  • hydroxyl group amino group
  • alkoxy group having 1 to 30 carbon atoms for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.
  • a lipoxyl group and the like for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.
  • the "substituted or unsubstituted alkenyl group" for R 2 includes an aryl group which may have a substituent, an alkoxy group which may have a substituent, and an amide group which may have a substituent A cycloalkyl group which may have a substituent, a heteroaryl group which may have a substituent, a carbonyl group which may have a substituent, a halogen atom (for example, a chlorine atom, an iodine atom, A alkenyl group having 2 to 30 carbon atoms or an unsubstituted carbon atom having 2 to 30 carbon atoms, which is substituted by one or more substituents selected from the group consisting of a bromine atom, a fluorine atom), a hydroxyl group and an amino group.
  • a halogen atom for example, a chlorine atom, an iodine atom, A alkenyl group having 2 to 30 carbon atoms or an un
  • alkenyl group is intended.
  • Examples of the “alkenyl group having 2 to 30 carbon atoms” include butyl, aryl, 1-propenyl, isopropenyl, 2-butenyl, 3-butenyl, 2-hexenyl, 8, 11-heptadecenyl, 4, 7, 10 and 13-nonadecatetraenyl, 8, 11 and 14-heptadecatrienyl, 8-heptadecenyl and the like.
  • the substituent of the “alkenyl group having 2 to 30 carbon atoms” has the same meaning as the substituent described above as the substituent of the “alkyl group having 1 to 99 carbon atoms”.
  • the “substituted or unsubstituted alkynyl group” for R 2 includes an aryl group which may have a substituent, an alkoxy group which may have a substituent, and an amide group which may have a substituent
  • a halogen atom for example, a chlorine atom, an iodine atom, 2 to 3 carbon atoms substituted by one or more substituents selected from the group consisting of a bromine atom, a fluorine atom
  • alkynyl group having 0 or an unsubstituted alkynyl group having 2 to 30 carbon atoms is intended.
  • alkynyl group having 2 to 30 carbon atoms include ethynyl, 1-propyl, 2-propynyl, 1-butyninole, 2-butyninole, 3-butyninole, 1-pentynyl, 2-pentynyl, and 3-pentyne Quinolone, 4-pentinole, 1-hexynole, 2-hexynole, 3-hexyl, 4-hexynyl, 5-hexynyl and the like.
  • they are ethynyl, 1-propynyl and 2-probyl.
  • the substituent of the “alkynyl group having 2 to 30 carbon atoms” has the same meaning as the substituents described above as the substituent of the “alkyl group having 1 to 99 carbon atoms”.
  • the hydrophobic substance is preferably a compound having a small structural feature, specifically, pendelic acid, myristic acid, palmitic acid, linoleic acid, arachidonic acid, linolenic acid, oleic acid, stearic acid, 9- (naphthaleneone). 11-yl) Monononanoic acid, octadecanesulfonic acid, hexadecanesulfonic acid and the like. Particularly preferred are stearic acid and palmitic acid.
  • the hydrophobic substance used for immobilization may be one kind or a mixture of two or more kinds.
  • hydrophobic substance may be a commercially available substance or a commercially available substance, or can be prepared according to various literatures.
  • new substances can also be appropriately prepared by utilizing various reactions in organic synthesis usually performed in this field. For example, reactions such as alkylation, arylation, alkoxylation, amidation, cycloalkylation, heteroarylization, and carbonitriol are used. Naturally, two or more reactions can be combined if necessary.
  • solid phase carrier for immobilizing the “hydrophobic substance” those usually used in the art can be suitably used. However, the purpose of the use, that is, the non-specific one prior to the analysis of the specific interaction between molecules, can be used.
  • a solid support suitable for removing the substance is selected.
  • resin polystyrene, methacrylate resin, polyacrylamide, etc.
  • glass metal (gold, silver, iron, silicon, etc.) are used.
  • metal gold, silver, iron, silicon, etc.
  • the beads are made of a resin, they can be packed into a column to simplify the subsequent operation, and a metal thin film or a glass plate is also suitable.
  • the solid phase carrier used for immobilizing the hydrophobic substance in the present invention is not particularly limited in its material and shape.
  • the hydrophobic substance is immobilized but the hydrophobic substance-immobilized solid-phase carrier has reduced hydrophobicity as a whole, it requires an extra step to use it, and the operation becomes complicated. It is not preferable for practicing the present invention because it may become unusable or unusable.
  • Immobilization of a hydrophobic substance on a solid support is usually carried out by a known method in the art and a method combining these methods, for example, amide bond, Schiff base formation, c_c bond, ester bond. Immobilization by covalent bond or non-covalent bond such as hydrogen bond, hydrophobic interaction and the like. All are carried out using materials and reactions known in the art. The individual conjugation is carried out utilizing reactions usually performed in the art. As a simple and reliable means, there is a method utilizing an amide bond formation reaction. This reaction can be carried out according to, for example, “Basics and Experiments of Peptide Synthesis” (ISBN 4-621-02962-2, Maruzen, First Edition in 1985).
  • reagents and solvents used in each reaction those commonly used in the art can be used, and are appropriately selected depending on the binding reaction to be employed. Whether or not the hydrophobic substance has been immobilized on the solid support can be confirmed from the reaction rate measured by quantification of amino groups on the surface of the solid support before and after the reaction (for example, ninhydrin test).
  • the “ligand-immobilized solid phase carrier” is one on which a specific interaction between a ligand and a target molecule occurs, and is suitably used for selection of a target molecule and the like.
  • the ligand or the ligand immobilized on the ligand-immobilized solid support is not particularly limited, and may be a known compound or a novel compound to be developed in the future. It may be a low molecular compound or a high molecular compound.
  • the low-molecular compound is a compound having a molecular weight of less than about 1000, and includes, for example, organic compounds and their derivatives and inorganic compounds that can be generally used as pharmaceuticals. Compounds and derivatives thereof, naturally occurring compounds and derivatives thereof, small nucleic acid molecules such as promoters, various metals, etc., and preferably organic compounds and their derivatives and nucleic acid molecules that can be used as pharmaceuticals.
  • the high molecular compound is a compound having a molecular weight of about 1000 or more, and includes proteins, polynucleic acids, polysaccharides, and a combination thereof, and is preferably a protein.
  • These low molecular weight compounds or high molecular weight compounds are commercially available as long as they are known, or can be obtained through steps such as collection, production, purification and the like according to each reported document. These may be of natural origin, may be prepared by genetic engineering, or may be obtained by semisynthesis or the like.
  • the present invention provides a method for removing a non-specific substance, which is non-specifically adsorbed to a ligand and Z or a ligand-immobilized solid phase carrier, from a sample, wherein the non-specific substance is, for example, a resin ( It can be non-specifically adsorbed on a solid support of any material such as polystyrene, methacrylate resin, polyacrylamide, etc., glass, metal (gold, silver, iron, silicon, etc.). Similarly, it can be non-specifically adsorbed to any shape such as a plate, a bead, a thin film, a thread, and a coil.
  • a resin It can be non-specifically adsorbed on a solid support of any material such as polystyrene, methacrylate resin, polyacrylamide, etc., glass, metal (gold, silver, iron, silicon, etc.).
  • it can be non-specifically adsorbed to any shape such as a plate, a bead,
  • the solid phase carrier for immobilizing the ligand may be the same material and the same shape as the solid phase carrier for immobilizing a hydrophobic substance, or may be a different material or a different shape.
  • the same material and different shapes may be used, or different materials and the same shape may be used.
  • the sample may contain a non-specific substance and a specific substance, and is preferably a liquid composition containing these substances. Samples composed entirely of specific substances are not preferred for use in view of the purpose of removing non-specific substances of the present invention. Also, a sample composed entirely of non-specific substances is not preferable for use in view of the purpose of removing non-specific substances of the present invention.
  • the sample may be composed of all known compounds but may contain some novel compounds. Or all may be composed of novel compounds. For example, it is a mixture of proteins prepared by genetic engineering using Escherichia coli or the like, or an extract (lysate) of cells or tissues. In addition, examples of compounds composed entirely of novel compounds include novel proteins whose functions and structures are not yet known, and mixtures of newly synthesized compounds. When the sample is a mixture, especially when the sample contains a known compound, the content of these compounds in the sample can be arbitrarily set to a desired value. In some cases, a surfactant is used for extraction from cells or tissues.
  • a sample derived from blood is particularly exemplified as a sample exhibiting the effect.
  • Samples derived from blood include, for example, whole blood, plasma, serum, and dilutions thereof.
  • Such a sample mainly contains albumin as a substance that non-specifically binds to the ligand and the Z or ligand-immobilized solid phase carrier.
  • the ability of the solid phase carrier of the present invention to adsorb albumin particularly makes it possible to use the solid phase carrier of the present invention for the purpose of concentrating or purifying albumin in a sample.
  • the method for removing a hydrophobic substance-immobilized solid support or a non-specific substance according to the present invention is a method for screening a target molecule having a specific interaction with the ligand using the ligand-immobilized solid support.
  • the method for removing a non-specific substance can be used for analyzing a sample from which a non-specific substance has been removed or reduced by the method for removing a hydrophobic substance-immobilized solid support or a non-specific substance of the present invention. It can also be used for a method of screening for a target molecule that can be included in the target.
  • the analysis method includes known methods such as electrophoresis, immunobutting using immunological reaction, immunoprecipitation, chromatography, mass spectrum, amino acid sequencing, and NMR (especially for small molecules). Depending on the method, there may be mentioned a combination of these methods. Removal of ligands and substances that bind non-specifically to Z or ligand-immobilized solid support Therefore, it is preferable that the type of the hydrophobic substance immobilized on the hydrophobic substance-immobilized solid phase carrier of the present invention is appropriately changed and set according to the characteristics of the ligand.
  • the screening method includes at least the following steps. In this screening method, the definitions of the sample, the ligand and the target molecule, the ligand-immobilized solid support, and the hydrophobic substance-immobilized solid support are as described above.
  • the shape of the sample can be changed as appropriate depending on what principle, means, and method are used for the next step or for the purpose of implementation.
  • a column filled with a bead resin is used as the solid support for hydrophobic substance immobilization, it is preferably liquid.
  • the method of bringing the sample into contact with the hydrophobic substance-immobilized solid phase carrier is not particularly limited as long as the non-specific substance in the sample is adsorbed and removed by the hydrophobic substance-immobilized solid phase carrier. It can be changed as appropriate depending on what principle, means, and method are used in the next step.
  • a column filled with a bead resin is used as a solid support for immobilizing a hydrophobic substance
  • the method is simply carried out by adding a liquefied sample to a column and passing it through the column (column method).
  • it can be conveniently carried out by mixing the bead resin and the sample for a certain period of time (batch method).
  • Optimal conditions for the adsorption and removal of non-specific substances are set based on the conditions usually used in ab-initial mouth chromatography, such as the amount of applied column, flow rate, elution treatment, and mixing time.
  • the following steps are included.
  • This step is a step of bringing the sample obtained in the previous step, that is, the sample from which non-specific substances have been removed, into contact with the ligand-immobilized solid phase carrier.
  • the method for bringing the sample into contact with the ligand-immobilized solid phase carrier is not particularly limited as long as the target molecule can be bound by specific interaction on the ligand-immobilized solid phase carrier when present in the sample. Appropriate depending on the solid phase carrier to be used and the principle, means and method used in the next step Can change. For example, ⁇ using a column filled with a bead resin as a ligand-immobilized solid phase carrier is simply carried out by adding a liquid sample to a force ram and passing it through the force ram (column method).
  • This step can be appropriately changed depending on the type of the solid phase carrier to be used, the type of the immobilized ligand, and the like, and is usually performed by various methods for identifying low molecular weight compounds or high molecular weight compounds, which are carried out in the art. Do. It could also be implemented by methods that will be developed in the future. For example, when a column packed with a bead resin on which the ligand is immobilized is used as the ligand-immobilized solid support, the step (1) is performed by adding a sample from which non-specific substances have been removed in advance. (2)], binding the target molecule to the ligand.
  • the bound target molecule is dissociated from the ligand by treatment such as changing the polarity of the buffer solution or adding excess ligand, and then identified or surface-active as it is when bound to the ligand on the solid phase. It can also be extracted and identified by an agent or the like. Specific examples of the identification method include electrophoresis, immunoblotting and immunoprecipitation using immunological reactions, chromatography, mass spectrum, amino acid sequencing, and NMR (especially for small molecules). The method is implemented by a known method or by combining these methods. The step of identifying a molecule that does not bind to the ligand can be performed according to the method for identifying a molecule that binds to the ligand.
  • the identification step is performed. It is preferable to carry out treatments such as concentration and rough purification in advance before entering the reaction. Based on the obtained data and existing reports, each molecule is identified, and whether or not the ligand is a target molecule is determined.
  • This step may be automated. For example, various types obtained by two-dimensional electrophoresis It is also possible to directly read the data of the molecule and identify the molecule based on the existing database.
  • the sample obtained after the contact with the hydrophobic substance-immobilized solid phase carrier obtained in the above step (1) can be analyzed as it is. For example, the following steps are included.
  • Carboxylic acid was immobilized on TO YO pearl resin (T SKgel AF-amino-650M (trade name), Tosoichi Co., Ltd. N-ring).
  • DMF acetic anhydride-dimethylformamide
  • the resin for immobilizing sulfonic acid-type saturated fatty acids was prepared by preparing acid chloride corresponding to 4 equivalents of sulfonic acid with respect to the amount of amino groups of 100 ⁇ l of the resin (amount of amino group: 100 ⁇ mo 1); Zero equivalent of triethylamine was dissolved in DMF 5 times the volume of the resin (vZv) and mixed with the resin. The reaction was carried out at room temperature for 12 hours. After completion of the reaction, the resin was washed with DMF, and the solvent was replaced with a 20% ethanol solution and stored.
  • the resin used was an affinity resin having an amino group introduced as a functional group (TOYOP EARL AF-amino-650 (trade name), manufactured by Tosoh Corporation). 4 equivalent force to amino group of 100 1 resin (amino group content: 10 mo 1) FK 506 with rubonic acid introduced, 4.8 equivalents of dimethylaminopropyl monoethyl-carbodiimide hydrochloride (EDC ⁇ HC 1) (WSC I), 4.8 equivalents of 1-hydroxybenzotriazole (HOBT) resin was dissolved in a DMF solution of 5 times the volume of the solution and mixed with the resin. The reaction was carried out by stirring at room temperature for 12 hours. After completion of the reaction, the resin was washed with DMF, replaced with a 20% ethanol solution and stored.
  • TOYOP EARL AF-amino-650 (trade name), manufactured by Tosoh Corporation). 4 equivalent force to amino group of 100 1 resin (amino group content: 10 mo 1) FK 506 with rubonic acid introduced,
  • Rat brain lysate was used as a sample (total protein) in order to remove hydrophobic non-specifically adsorbed proteins with stearic acid-immobilized resin.
  • the brain lysate was adjusted to 5 mg Zml with 0.25 M sucrose buffer (25 mM Tris-HCl, pH 7.4), and the stearic acid-immobilized resin was added to 1000 ⁇ l of the total protein solution. 1001 and 2001 were mixed and stirred at 4 ° C for 5 hours. Thereafter, the resin and the purified brain lysate were separated by centrifugation.
  • FKBP12 was selectively obtained from purified rat brain lysate using FK506-immobilized resin.
  • 10 ⁇ l of FK506-fixed resin was mixed and stirred at 4 ° C. for 12 hours.
  • 20 ⁇ l of sample buffer including 2-mercaptoethanol, SDS-PAGE (trade name), Nacalai
  • the mixture was added and stirred at 25 ° C for 10 minutes. Thereafter, the sample buffer was separated by centrifugation, and the protein obtained by SDS-PAGE was collected.
  • NBP Non-specific Binding Protein
  • sample buffer including 2-mercaptoethanol, SDS-PAGE (trade name), Nacaline clay
  • CB-F3GA dye chipaclonal blue F3GA
  • a dye chipaclonal blue F3GA which is commercially available as a ligand molecule of a pretreatment column for removing unnecessary proteins such as albumin
  • the column was immobilized on TO YO Pearl (TSKg el AF-am ino-650M (trade name), Tosoh Co., Ltd .: h3 ⁇ 4) and used as a control column.
  • the human DHFR expression vector was prepared using the GATEWAY system (Invitrogen). First, a plasmid was extracted from an MGC clone (MGC ID 857, Invitrogen) encoding the human DHFR gene, and a human DHFR entry 1 'vector was obtained by a two-step PCR reaction using this as a ⁇ M. Produced. A human DHFR expression vector having a His tag at the N-terminal was constructed by recombination (LR reaction) using c1onase from this entry 'vector, and the host E. coli strain BL2 1 star (DE 3) p LysS was transformed.
  • MGC clone MGC ID 857, Invitrogen
  • the host Escherichia coli was subjected to preculture and then main culture was started in SB medium. Three hours after the start of the cultivation, DHFR expression was induced by adding isopropyl isopropyl monolactogalactoylanoside (IPTG) to a final concentration of 0.1 mM. The cells were cultured at 20 ° C. and 130 rpm overnight. After completion of the culture, the cells were collected, and homogenated with ultrasonic waves to obtain DHFR-expressing Escherichia coli 1 ysate.
  • IPTG isopropyl isopropyl monolactogalactoylanoside
  • stearic acid-immobilized resin prepared in Example 1
  • CB-F3GA-immobilized resin prepared in Example (1)
  • Buffer A (20 mM phosphate buffer pH 7. 1, 0. 5 0 / oTw een 20)
  • stearate fixed that has been solvent substitution with buffer A to DH FR expression 1 Ysate diluted 5-fold And CB-F3GA immobilized resin were added in an amount of 250 ⁇ l each, followed by stirring at 4 ° C. for 3 hours.
  • each resin was dispensed in 10 ⁇ l portions and washed 5 times with 100 ⁇ l of buffer solution ⁇ . After washing, remove the protein adsorbed on the resin with buffer B (1.4M NaC1, 20m Elution was carried out in this order with three kinds of solutions: M phosphate buffer (pH 7.1), buffer C (8M rare aqueous solution), and buffer D (SD sample buffer).
  • M phosphate buffer pH 7.1
  • buffer C 8M rare aqueous solution
  • buffer D SD sample buffer
  • CB-F3GA immobilized resin which is commercially available as a ligand molecule of a pretreatment column for removing unnecessary proteins such as albumin, is mixed with DHFR-expressing Escherichia coli 1 ysate. As a result, it was revealed that DHFR protein binds to the resin in a large amount. On the other hand, for the stearic acid-fixed resin, no adsorption of the DHFR protein to the resin was observed under the present examination conditions. As is well known, CB-F3GA is inferred from its structural characteristics ((l) Bio-Rad catalog version 2023 p5 4-5 5, (2) N.
  • a sample that may contain the target molecule is immobilized with a hydrophobic substance.
  • pretreating with the solid phase carrier thus obtained, it is possible to remove or reduce substances non-specific to the ligand molecules present in the sample. Therefore, it is possible to more accurately analyze specific interactions between molecules and identify target molecules. As a result, the degree of purification of the target molecule can be increased.

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Abstract

L'invention a trait à un support phase solide, auquel est fixée une substance hydrophobe, telle que de l'acide stéarique, ainsi qu'à un procédé permettant d'éliminer d'un échantillon une substance pouvant être adsorbée de façon non spécifique sur un support phase solide auquel est fixé un ligand, ledit procédé consistant à traiter l'échantillon avec le support phase solide auquel est fixée une substance hydrophobe. Ledit procédé permet d'analyser l'interaction spécifique entre les molécules de ligand et les molécules cibles. Conformément au processus destiné à identifier/cribler des molécules cibles en fonction de ladite interaction spécifique, l'échantillon susceptible de contenir les molécules cibles est prétraité avec le support phase solide auquel est fixée une substance hydrophobe, ce qui permet à la substance qui est présente dans l'échantillon et agit de façon non spécifique sur les molécules de ligand d'être complètement ou partiellement éliminée.
PCT/JP2004/011104 2003-07-28 2004-07-28 Procede d'elimination d'une substance non specifique WO2005010528A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007121204A (ja) * 2005-10-31 2007-05-17 Denka Seiken Co Ltd 塩基性多糖類を含有する免疫測定法用検体処理液組成物及びキット、並びにこれらを用いる免疫測定法
JP2008533473A (ja) * 2005-03-11 2008-08-21 ワイス 弱分配クロマトグラフィー方法
US9017317B2 (en) 2012-12-06 2015-04-28 Medtronic Ardian Luxembourg S.A.R.L. Refrigerant supply system for cryotherapy including refrigerant recompression and associated devices, systems, and methods
JP2016516186A (ja) * 2013-03-13 2016-06-02 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 複合マトリックス中の微生物の分析を向上させるための方法
WO2016136863A1 (fr) * 2015-02-25 2016-09-01 積水メディカル株式会社 Procédé de dosage immunologique de l-fabp et réactif de dosage utilisé dans ledit procédé
US9645057B2 (en) 2012-04-05 2017-05-09 Becton, Dickiinson and Company Method for improving analysis of microorganisms in complex matrices

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JPS60256057A (ja) * 1984-06-01 1985-12-17 Dai Ichi Pure Chem Co Ltd 免疫学的測定法
JPH0245758A (ja) * 1988-08-08 1990-02-15 Kagakuhin Kensa Kyokai 水溶性高分子物質と低分子成分とが共存する試料の分析方法及び前処理方法並びにクロマトグラフイー用充填剤
JPH05203636A (ja) * 1991-09-13 1993-08-10 Merck Patent Gmbh 改質されたクロマトグラフィー用支持体材料
JPH07318551A (ja) * 1994-05-23 1995-12-08 Res Dev Corp Of Japan クロマトグラフィ−方法及び該方法に使用するクロマトグラフィ−用充填剤
JPH08506907A (ja) * 1993-12-21 1996-07-23 ベーリンガー マンハイム ゲーエムベーハー アシル化タンパク質凝集体および免疫検定における干渉の抑制についてのそれらの使用
JP2000039401A (ja) * 1998-03-24 2000-02-08 Dainippon Printing Co Ltd 表面プラズモン共鳴バイオセンサ―用測定セル及びその製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60256057A (ja) * 1984-06-01 1985-12-17 Dai Ichi Pure Chem Co Ltd 免疫学的測定法
JPH0245758A (ja) * 1988-08-08 1990-02-15 Kagakuhin Kensa Kyokai 水溶性高分子物質と低分子成分とが共存する試料の分析方法及び前処理方法並びにクロマトグラフイー用充填剤
JPH05203636A (ja) * 1991-09-13 1993-08-10 Merck Patent Gmbh 改質されたクロマトグラフィー用支持体材料
JPH08506907A (ja) * 1993-12-21 1996-07-23 ベーリンガー マンハイム ゲーエムベーハー アシル化タンパク質凝集体および免疫検定における干渉の抑制についてのそれらの使用
JPH07318551A (ja) * 1994-05-23 1995-12-08 Res Dev Corp Of Japan クロマトグラフィ−方法及び該方法に使用するクロマトグラフィ−用充填剤
JP2000039401A (ja) * 1998-03-24 2000-02-08 Dainippon Printing Co Ltd 表面プラズモン共鳴バイオセンサ―用測定セル及びその製造方法

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008533473A (ja) * 2005-03-11 2008-08-21 ワイス 弱分配クロマトグラフィー方法
JP2007121204A (ja) * 2005-10-31 2007-05-17 Denka Seiken Co Ltd 塩基性多糖類を含有する免疫測定法用検体処理液組成物及びキット、並びにこれらを用いる免疫測定法
JP4718301B2 (ja) * 2005-10-31 2011-07-06 デンカ生研株式会社 塩基性多糖類を含有する免疫測定法用検体処理液組成物及びキット、並びにこれらを用いる免疫測定法
US9645057B2 (en) 2012-04-05 2017-05-09 Becton, Dickiinson and Company Method for improving analysis of microorganisms in complex matrices
US9017317B2 (en) 2012-12-06 2015-04-28 Medtronic Ardian Luxembourg S.A.R.L. Refrigerant supply system for cryotherapy including refrigerant recompression and associated devices, systems, and methods
JP2016516186A (ja) * 2013-03-13 2016-06-02 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 複合マトリックス中の微生物の分析を向上させるための方法
WO2016136863A1 (fr) * 2015-02-25 2016-09-01 積水メディカル株式会社 Procédé de dosage immunologique de l-fabp et réactif de dosage utilisé dans ledit procédé
JPWO2016136863A1 (ja) * 2015-02-25 2017-11-30 積水メディカル株式会社 Ｌ−ｆａｂｐの免疫学的測定方法及び該方法に用いられる測定試薬

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