Glycolipid capture agent and uses thereof

Abstract

To provide a glycolipid capture agent, a glycolipid recovery method, a method for diagnosing glycolipid metabolic disorders, and kits therefor.SOLUTION: A glycolipid capture agent containing graphite carbon is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a glycolipid capture agent, a glycolipid recovery method, a method for diagnosing glycolipid metabolic disorders, and a kit therefor. [Background technology]

[0002] Recently, attempts to detect biomarkers from liquid biological samples such as blood, known as liquid biopsy, have attracted attention. Some measurement parameters require sample pretreatment, and extraction and purification of target substances has wide applications, making it a valuable research tool. Glycolipids are one such target substance, but the difficulty of basic procedures such as extraction and purification limits their use and research scope.

[0003] On the other hand, many diseases caused by impaired glycolipid metabolism have been reported, and glycolipid metabolism is dysfunctional in several lysosomal storage diseases, including Gaucher disease and Fabry disease. It is known that in these diseases, glycolipids themselves accumulate without being metabolized, or intermediate metabolic products produced by unusual pathways, mainly lysoglycosylglycosides (glycosphingosines), are present in the blood, and analyses related to such blood biomarkers are being attempted.

[0004] For example, methods for detecting mucopolysaccharidoses are known, using human urine or serum samples as samples and glycosaminoglycan (GAG) chains, which are biomarkers for mucopolysaccharidoses, as a sandwich assay or an inhibition assay using antibodies against each GAG chain (Patent Document 1, Published Japanese Translation of PCT International Publication No. 2005-524074). However, it is considered difficult to identify multiple lysosomal diseases by measuring a single type of glycosaminoglycan. Furthermore, antibodies (unlabeled and biotin-labeled) against each GAG chain are required.

[0005] There are also methods for diagnosing lysosomal diseases using sandwich immunoassays in which human cell extracts, blood, plasma, or urine are used as samples and the causative enzymes of each lysosomal disease are used as antigens (Patent Document 2, Published Japanese Translation of PCT International Publication No. 2006-523300). Because this method involves measuring each target compound using a capture antibody and a detection antibody specific to that compound, it is necessary to develop a method for each lysosomal disease.

[0006] Because glycolipids are metabolic substances that accumulate in the blood, it is expected that they can be detected as biomarkers using liquid biopsy, but a simple collection method has not yet become widespread. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication 2005-524074 [Patent Document 2] Special Publication 2006-523300 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a capturing agent, kit, and method for capturing or recovering glycolipids, particularly glycolipids selected from glycosphingolipids, glyceroglycolipids, and glycosphingosines. Another object of the present invention is to provide a capturing agent, kit, and method for capturing or recovering glycolipids simply and inexpensively. Furthermore, another object of the present invention is to provide a kit and method for qualitatively or quantitatively determining the recovered glycolipids and, based on the results, identifying glycolipid metabolic disorders or determining the degree of progression thereof. [Means for solving the problem]

[0009] As a result of extensive research to solve the above problems, the present inventors have found that glycolipids can be captured or recovered by using graphite carbon, and have completed the present invention. That is, the present invention includes the following aspects. [1] A glycolipid sequestering agent, comprising graphitic carbon. [2] The capture agent according to [1], wherein the glycolipid is selected from glycosphingolipids, glyceroglycolipids, and glycosphingosines. [3] The capture agent according to [1] or [2], which is porous. [4] A kit for recovering glycolipids, comprising the capture agent according to any one of [1] to [3]. [5] The kit according to [4], wherein the kit is for diagnosing a glycolipid metabolism disorder. [6] The kit according to [4] or [5], further comprising a reagent capable of qualitatively or quantitatively determining glycolipids. [7] A method for recovering glycolipids, comprising: a) contacting a sample containing glycolipids with the capturing agent according to any one of [1] to [3] to capture the glycolipids; and b) recovering the glycolipid from the capturing agent that has captured the glycolipid A method comprising: [8] The method according to [7], further comprising the step of c) qualitatively or quantitatively determining the recovered glycolipids. [9] A method for assisting in the diagnosis of a glycolipid metabolism disorder, comprising: a) contacting a sample containing glycolipids with the capturing agent according to any one of claims [1] to [3] to capture the glycolipids; b) recovering the glycolipid from the capturing agent that has captured the glycolipid; c) qualitatively or quantitatively determining the recovered glycolipids; and d) A step of identifying a glycolipid metabolic disorder or assisting in determining the degree of progression thereof based on the obtained qualitative or quantitative results of glycolipids. A method comprising:

[10] The method according to [9], wherein the glycolipid metabolism disorder is a lysosomal disease. [Effects of the Invention]

[0010] According to the present invention, glycolipids, particularly glycolipids selected from glycosphingolipids, glyceroglycolipids, and glycosphingosines, can be captured or recovered. Furthermore, according to the present invention, glycolipids can be detected, captured, recovered, or concentrated simply and inexpensively. Furthermore, according to the present invention, glycolipids can be captured or recovered, and by qualitatively or quantitatively analyzing the recovered glycolipids, it is possible to identify glycolipid metabolic disorders or determine the degree of progression of such disorders based on the results of the qualitative or quantitative analysis. [Brief explanation of the drawings]

[0011] [Figure 1] In Example 1, the recovery of sphingosine sugars was evaluated using any of the following beads: (1) Glycan Capturing Beads, (2) iSPE, (3) CHROMABOND, and (4) HyperCarb. The results are shown below. [Figure 2] In Example 2, the results of evaluating the recovery of glycolipids using graphite carbon beads are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, "glycolipid" refers to a compound in which one or more sugars are bound to a fatty acid. Examples of glycolipids include sphingoglycolipids, glyceroglycolipids, and glycosphingosines. Glycolipids can also be classified according to the fatty acid they contain; for example, a compound containing a ceramide is called a sphingoglycolipid, and a compound containing an acylglycerol or alkylglycerol is called a glyceroglycolipid.

[0013] As used herein, "glycosphingolipid" refers to a compound in which one or more sugars are bound to a ceramide, and here "ceramide" refers to a compound in which sphingosine and a fatty acid are amide-bonded. Therefore, as used herein, "glycosphingolipid" refers to a glycolipid in which a sugar is bound to a ceramide having a structure in which a fatty acid is amide-bonded to a sphingoid. Examples of sphingoglycolipids include glucosylceramide (GlcCer), globotriaosylceramide (Gb3), galactosylceramide (GalCer), gangliosides (e.g., GM1 ganglioside, GM2 ganglioside, GM3 ganglioside, etc.), cerebrosides, sulfogalactosylceramide, ceramide oligohexosides, globosides, etc.

[0014] As used herein, "glyceroglycolipid" refers to a compound in which one or more sugars are bound to an acylglycerol or alkylglycerol, and preferably refers to a compound in which one or more sugars are bound to a diacylglycerol. Examples of glyceroglycolipids include monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG).

[0015] "Glycosphingosine" as used herein refers to the lyso form of glycosphingolipid, where the lyso form refers to a structure containing sphingosine that has become an amino group as a result of the release of a fatty acid from the ceramide moiety of glycosphingolipid. Therefore, "glycosphingosine" as used herein refers to a glycolipid in which a sugar is bound to sphingosine. Examples of glycosphingosines include globotriaosylsphingosine (Lyso-Gb3), glucosylsphingosine (Lyso-Gb1), lyso-GM2 ganglioside (Lyso-GM2), glucosylsphingosine (Lyso-GlcCer), lactosylsphingosine (Lyso-LacCer), and the like.

[0016] As used herein, "graphitic carbon" refers to carbon having a planar structure in which carbon atoms are arranged hexagonally, preferably forming a layered structure. Graphitic carbon can be used as an adsorbent or capture agent in, for example, chromatography or solid-phase extraction. In chromatography or solid-phase extraction, graphitic carbon provides retention of highly polar substances and separation of structurally related substances.

[0017] As used herein, "glycolipid metabolism disorder" refers to a condition in which some abnormality occurs in the glycolipid metabolic pathway, causing the blood concentration of glycolipids or their metabolites to be unable to be maintained within an appropriate range (e.g., a condition in which the blood concentration is higher or lower than the appropriate range). Glycolipid metabolism disorders include, for example, lysosomal diseases. As used herein, "lysosomal storage disease" refers to a group of diseases caused by the accumulation of substances in the body due to genetic deficiencies or mutations in enzymes present in lysosomes. Examples of "lysosomal storage disease" as used herein include mucopolysaccharidoses (types I, II, III, IV, VI, and VII), Fabry disease, Gaucher disease, Pompe disease, Niemann-Pick disease (types A and B), GM1 gangliosidosis, GM2 gangliosidosis, α-mannosidosis, β-mannosidosis, fucosidosis, mucolipidosis, galactosialidosis, sialidosis, Krabbe disease, heterogeneous leukodystrophy, and other lysosomal storage diseases in which the biomarker is a carbohydrate-related substance, preferably Fabry disease or Gaucher disease. For example, Fabry disease is a metabolic disorder of glycosphingolipids caused by decreased activity of α-galactosidase, and Gaucher disease is a metabolic disorder of glycosphingolipids caused by decreased activity of glucocerebrosidase.

[0018] As used herein, a "glycolipid-containing sample" may be derived from a biological sample. Examples of biological samples include body fluids, extracts obtained from cells or tissues, cell or tissue secretions, and feces. Examples of body fluids include blood, urine, serum, serous fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretions, vaginal secretions, ascites, pleural fluid, pericardial fluid, peritoneal fluid, and sweat. As used herein, a "blood sample" refers to a sample containing at least a portion of a blood component, and may be, for example, whole blood, serum, or plasma, or any of these diluted forms. The blood sample is preferably serum or plasma, and more preferably serum. The blood sample can be prepared by known methods.

[0019] As used herein, the term "reagent capable of qualitatively or quantitatively determining glycolipids" refers to a reagent capable of qualitatively or quantitatively determining glycolipids, or any of the sugar chains, ceramides, sphingosines, or fatty acids that constitute glycolipids, by a known qualitative or quantitative method. Reagents capable of qualitatively or quantitatively determining glycolipids include, for example, glycolipid labeling reagents, labeling substance detection reagents, and solvents for these.

[0020] [Glycolipid sequestrant] In one embodiment, the present invention includes a sequestrant for glycolipids, the sequestrant comprising graphitic carbon. The composition, shape, state, etc. of the scavenger are not particularly limited. The scavenger may contain one or more types of graphite carbon. When two or more types of graphite carbon are used, they may be different types of graphite carbon having different particle diameters, pore sizes, etc., or may be the same type of graphite carbon.

[0021] In the capture agent, the graphite carbon may be immobilized on a support or packed in a column. Examples of the support include well plates (e.g., 96-well microplates), membranes (e.g., nitrocellulose membranes, polyvinylidene fluoride membranes), slide glasses, magnetic beads, and latex particles. The graphite carbon can be immobilized on the support by a known method selected depending on the material of the support. The column may be, for example, a syringe type, a lure type, a well plate type, a spin column type, or the like.

[0022] In the scavenger, the graphite carbon may be in any shape, for example, in the form of particles or beads. When the graphite carbon is in the form of particles or beads, the average particle size may be, for example, at least 1 μm, at least 10 μm, at least 20 μm, at least 25 μm, or at least 30 μm, and at most 200 μm, at most 120 μm, at most 40 μm, at most 35 μm, or at most 30 μm, preferably about 1 to 200 μm or about 1 to 120 μm, more preferably about 20 to 40 μm. The average particle size can be measured, for example, by a particle size distribution analyzer using laser diffraction / scattering, centrifugal sedimentation, particle trajectory analysis, or dynamic light scattering.

[0023] In the scavenger, the graphite carbon may be porous or non-porous, and is preferably porous. When the graphite carbon has pores, its porosity can be expressed using, for example, the average pore size (or medium pore size), pore volume, specific surface area, etc. When the graphite carbon has pores, the average pore diameter (or mesopore diameter) may be, for example, at least 100 Å, at least 150 Å, at least 200 Å, or at least 250 Å, and at most 400 Å, at most 350 Å, at most 300 Å, or at most 250 Å, preferably about 200 to 300 Å, and more preferably about 250 Å. The average pore diameter can be measured, for example, by pore distribution measurement using a gas adsorption method, a gas permeation method, a mercury intrusion method, or a bubble point method. When the graphite carbon has pores, the pore volume is, for example, 0.1 m as a lower limit. 3 / g or more, 0.3m 3 / g or more, 0.5m 3 / g or more, 0.6m 3 / g or more or 0.7m 3 / g or more, with an upper limit of 1.5m 3 / g or less, 1.0m 3 / g or less, 0.9m 3 / g or less, 0.8m 3 / g or less or 0.7m 3 / g or less, preferably about 0.1 to 1.5m 3 / g, about 0.3~1.1m 3 / g or approximately 0.5 to 0.9 m 3 / g, more preferably about 0.6 to 0.8 m 3 The pore volume can be measured, for example, by pore distribution measurement using a gas adsorption method.

[0024] Furthermore, in the scavenger, the specific surface area of ​​the graphite carbon is, for example, 1 m 2 / g or more, 5m 2 / g or more, 10m 2 / g, 100m 2 / g or more or 110m 2 / g or more, with an upper limit of 200m 2 / g or less, 150m 2 / g or less, 140m 2 / g or less, 130m 2 / g or less, 120m 2 / g or less, 20m 2 / g or less or 15m2 / g or less, preferably about 1 to 200m 2 / g, about 1~150m 2 / g or approximately 5 to 140 m 2 / g, and more preferably about 5 to 15 m 2 / g, about 90~110m 2 / g or approximately 110-130m 2 / g, e.g., about 10m 2 / g, about 100m 2 / g or approximately 120m 2 The specific surface area can be measured, for example, by the Brunauer Emmett Teller (BET) method using a gas adsorption method.

[0025] In one embodiment, when graphite carbon is packed in a column, the packed weight of the graphite carbon is, for example, about 1 to 10,000 mg, and the column volume is, for example, about 0.1 to 60 mL. The graphite carbon can be produced by a known method, but commercially available graphite carbon may also be used. Examples of commercially available columns packed with graphite carbon include HYPERCARB (registered trademark) and Supelclean (registered trademark) ENVI-Carb.

[0026] In one embodiment of the present invention, capture of glycolipids may be carried out by solid-phase extraction using graphite carbon. The conditions for solid-phase extraction are not particularly limited as long as they allow adsorption or capture of glycolipids by graphite carbon.

[0027] In one embodiment, the present invention includes a kit containing the above-mentioned capture agent. The kit may be used for recovering glycolipids.

[0028] Here, the type or amount of glycolipids present in a biological sample can be used to identify or determine the degree of progression of glycolipid metabolism disorders, which are disorders of glycolipid breakdown or metabolism, resulting in the accumulation of glycolipids in the body or the presence of glycolipids as intermediate metabolites produced via an abnormal pathway in the body.

[0029] The types of glycolipids accumulated or present may differ depending on the type of glycolipid metabolism disorder. Therefore, by qualitatively determining the types of glycolipids present in a biological sample, glycolipid metabolism disorders can be identified. The results of the glycolipid qualitative analysis can then be used as an indicator to assist in identifying glycolipid metabolism disorders. Therefore, the capture agent and the kit can be used for diagnosing abnormalities in glycolipid metabolism, assisting in the diagnosis, collecting data for the diagnosis, and the like.

[0030] Furthermore, the amount of glycolipids accumulated or present may vary depending on the degree of progression of the glycolipid metabolism disorder. Therefore, the degree of progression of the glycolipid metabolism disorder can be determined by quantifying the amount of glycolipids present in a biological sample. For example, a reference value or cutoff value (pathological condition identification value) (hereinafter collectively referred to as "reference value / cutoff value") can be determined by statistically processing data obtained from multiple healthy individuals or patients, and the degree of progression of the glycolipid metabolism disorder can be evaluated based on this value. For example, a value below the first reference value / cutoff value can be evaluated as mild progression, a value between the first reference value / cutoff value or more and less than the second reference value / cutoff value can be evaluated as moderate progression, and a value above the second reference value / cutoff value can be evaluated as severe progression. The first reference value / cutoff value can be an upper reference limit determined by multiple healthy individuals, and the second reference value / cutoff value can be the upper reference limit multiplied by a specific number. Therefore, the quantitative results of glycolipids can be used as an index to assist in determining the degree of progression of glycolipid metabolic disorders. Therefore, the above-mentioned capture agent and the above-mentioned kit can be used for diagnosing the degree of progression of abnormalities in glycolipid metabolism, assisting in such diagnosis, collecting data for such diagnosis, and the like.

[0031] The sample to be measured using the above capture agent or kit may be a biological sample collected and prepared from a human subject (subject), and the subject may or may not be confirmed to have a glycolipid metabolism disorder. Here, the presence of a disease can be confirmed by known methods, such as antibody tests, PCR tests, biomarkers, or a doctor's diagnosis.

[0032] The kit may contain, in addition to the capture agent, a reagent capable of qualitatively or quantitatively determining glycolipids. As described above, the reagent capable of qualitatively or quantitatively determining glycolipids includes, for example, a glycolipid labeling reagent, a labeling substance detection reagent, and a solvent for these. The kit may further include other elements, such as a glycolipid standard sample, a reagent for preparing a glycolipid-containing sample, a diluent, a buffer solution, a support, and instructions for use.

[0033] [Method for capturing glycolipids] In one embodiment, the present invention provides a method for recovering glycolipids, comprising the steps of: a) contacting a sample containing glycolipids with the capture agent to capture the glycolipids; and b) recovering the glycolipid from the capturing agent that has captured the glycolipid The present invention includes a method comprising the steps of:

[0034] The sample used in the above method includes a sample that is the target of the capture agent. In addition, the sample used in the above method may contain other components, such as contaminants such as free fatty acids, lipids, and salts, as long as it contains glycolipids. The scavenger used in the above method may contain one type of graphite carbon or two or more types. When two or more types of graphite carbon are used, they may be graphite carbons of different types having different particle sizes, pore sizes, etc., or may be the same type of graphite carbon. The above method can also be carried out using one or more of the above-mentioned scavengers.

[0035] The step a) is a step of capturing glycolipids contained in the sample by contacting the sample with the capturing agent. Therefore, the step is not particularly limited as long as it is performed under conditions that allow the capturing agent to capture or adsorb the glycolipids. For example, the step a) can be performed according to a known solid-phase extraction method using graphite carbon.

[0036] The step b) is a step of recovering the glycolipid captured in the step a) from the capturing agent. The glycolipid can be recovered by a known solid-phase extraction method.

[0037] The method for recovering the glycolipids is as follows: c) A step of qualitatively or quantitatively determining the recovered glycolipids It may further include:

[0038] The step c) is a step of qualitatively or quantitatively analyzing the glycolipids recovered in the step b). The glycolipids can be qualitatively or quantitatively analyzed by, for example, mass spectrometry, liquid chromatography, capillary electrophoresis, NMR, or a method using a conjugate that specifically binds to the sugar chains in the glycolipids, according to known methods and procedures.

[0039] Examples of the liquid chromatography method or capillary electrophoresis method include a method in which a functional group is introduced into a glycolipid, the glycolipid is labeled with a labeling substance such as a fluorescent substance, and the labeled glycolipid is analyzed by liquid chromatography or capillary electrophoresis to estimate, identify, detect, qualify, or quantify the glycolipid.

[0040] Examples of the mass spectrometry or NMR method include methods in which glycolipids are analyzed by mass spectrometry such as MALDI-TOF MS or ESI-MS or NMR to estimate, identify, detect or qualify the glycolipid structure.

[0041] Furthermore, examples of the method using a binder that specifically binds to the sugar chain in the glycolipid include a method in which a partial structure of the sugar chain is estimated, identified, detected, or qualified by a sandwich method or the like using a lectin or antibody that recognizes the sugar chain as the binder. The above-mentioned "binding substance that specifically binds to a sugar chain" is not particularly limited as long as it specifically binds to the sugar chain, and examples thereof include lectins, antibodies, and aptamers. Here, "specifically binding to a sugar chain" means essentially binding only to a specific sugar chain structure or partial structure.

[0042] In one embodiment, the present invention provides a method for diagnosing a glycolipid metabolism disorder, comprising: a) contacting a sample containing glycolipids with the capturing agent to capture the glycolipids; b) recovering the glycolipid from the capturing agent that has captured the glycolipid; c) qualitatively or quantitatively determining the recovered glycolipids; and d) identifying a glycolipid metabolic disorder or determining the degree of progression thereof based on the obtained qualitative or quantitative results of glycolipids; The present invention includes a method comprising the steps of:

[0043] Steps a), b) and c) in the above diagnostic method can be carried out in the same manner as in the above method for recovering glycolipids.

[0044] Step d) in the above diagnostic method includes, for example, diagnosing using the qualitative or quantitative results (identification, concentration, amount) of glycolipids as an indicator, for example, identifying glycolipid metabolic disorders based on the qualitative analysis of glycolipid types, or determining a cutoff value, and evaluating the progression of glycolipid metabolic disorders based on the cutoff value.

[0045] In one embodiment, the present invention provides a method for assisting in the diagnosis of a glycolipid metabolism disorder, comprising: a) contacting a sample containing glycolipids with the capturing agent to capture the glycolipids; b) recovering the glycolipid from the capturing agent that has captured the glycolipid; c) qualitatively or quantitatively determining the recovered glycolipids; and d) A step of identifying a glycolipid metabolic disorder or assisting in determining the degree of progression thereof based on the obtained qualitative or quantitative results of glycolipids. The present invention includes a method comprising the steps of:

[0046] Steps a), b) and c) in the above-mentioned diagnostic aiding method can be carried out in the same manner as in the above-mentioned method for recovering glycolipids.

[0047] Step d) in the above-mentioned method for assisting diagnosis includes assisting a physician in making a diagnosis (e.g., collecting, organizing, and providing to a physician, detection results or measurement values) in steps that require a physician's judgment, such as making a diagnosis using the qualitative or quantitative results (identification, concentration, and amount) of glycolipids as an indicator, for example, identifying a glycolipid metabolic disorder based on the qualitative analysis of the type of glycolipid, or determining a cutoff value and evaluating the progression of the glycolipid metabolic disorder based on the cutoff value.

[0048] In one embodiment, the present invention provides a method for collecting data for diagnosing a glycolipid metabolism disorder, comprising: a) contacting a sample containing glycolipids with the capturing agent to capture the glycolipids; b) recovering the glycolipid from the capturing agent that has captured the glycolipid; c) qualitatively or quantitatively determining the recovered glycolipids; and d) collecting data for identifying glycolipid metabolic disorders or determining the degree of progression thereof based on the obtained qualitative or quantitative results of glycolipids; The present invention includes a method comprising the steps of:

[0049] Steps a), b) and c) in the data collection method can be carried out in the same manner as in the glycolipid recovery method described above.

[0050] Step d) in the data collection method includes collecting data (e.g., detection results or measurement values) necessary for a physician's judgment, such as making a diagnosis using the qualitative or quantitative results (identification, concentration, amount) of glycolipids as an index, for example, identifying a glycolipid metabolic disorder based on the qualitative analysis of glycolipid types, or determining a cutoff value and evaluating the progression of a glycolipid metabolic disorder based on the cutoff value. In addition to collecting data, this step also includes organizing the data and providing the data to a physician.

[0051] Furthermore, one embodiment of the present invention includes a sequestrant for glycolipids, the sequestrant comprising graphite carbon, and the use of graphite carbon for producing the sequestrant.

[0052] Furthermore, one embodiment of the present invention includes use of graphite carbon for producing a glycolipid recovery kit including a glycolipid capture agent containing graphite carbon.

[0053] The glycolipid capture agent, lipid recovery kit, and graphite carbon used in the above-mentioned use are the same as those explained for the capture agent and kit. [Example]

[0054] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0055] Example 1 Recovery of the sugar sphingosine by solid phase extraction 1. Any one of the following beads (1) to (4) (2.5 mg) was placed in a spin tube with a filter column (Mobicol, MoBiTec) and activated according to the manufacturer's operating instructions for each bead. 2. Lyso-LacCer (lactosylsphingosine (d18:1), Cayman Chemical) (400 pmol) of glycosphingosine was added to 0.1% TFA / MilliQ (475 μL) and dissolved. The resulting solution was added to the column prepared in 1 above in several portions, and centrifuged each time to adsorb Lyso-LacCer to the beads activated in 1 above. The filtrate was discarded each time. 3. The column was washed with Milli-Q (400 μL), followed by elution twice with 50% acetonitrile (25 μL) to recover glycosphingosine. 4. The recovered glycosphingosine was measured by mass spectrometry (MALDI-TOF MS). <Analysis conditions> Measurement equipment: Ultraflex III mass spectrometer (Bruker Daltonics) Measurement mode: Positive mode, Reflectron mode Matrix:DHB

[0056] The beads used in Example 1 and their configuration are shown below. (1) Glycan Capturing Beads (EZGlyco O-Glycan Prep Kit, part number BS-41601) Constituents: [ka] (2) iSPE (product number 200.006.1000, HILICON AB) Constituents: [ka] (3) CHROMABOND (product number 730594, Macherey-Nagel) Constituents: [ka] (4) HyperCarb (product number 60106-304, Thermo Fisher Scientific) Composition: Graphite carbon Particle shape: spherical Specific surface area: 120m 2 / g Medium pore diameter: 250Å Pore ​​volume: 0.7m 3 / g Average particle size: 30μm

[0057] The results are shown in Figure 1. The beads (1) to (3) did not allow the recovery of glycosphingosine. On the other hand, the graphite carbon beads (4) HyperCarb allowed the recovery of glycosphingosine.

[0058] Example 2 Recovery of glycolipids 1. Graphite carbon (2.5 mg) was placed in a spin tube with a filter column (Mobicol, MoBiTec) and activated according to the manufacturer's instructions. The graphite carbon beads used were HyperCarb (Thermo Fisher Scientific, 60106-304), the same as those used in Example 1 above. 2. Glycolipids (GM1 ganglioside, MERCK) (4 nmol) were dissolved in methanol, and 70 μL of 0.1% TFA / MilliQ was added and mixed. The resulting solution was added to the column prepared in 1 above and centrifuged to adsorb the glycolipids to the beads activated in 1 above. The filtrate was discarded each time. 3. The column was washed with 0.1% formic acid (200 μL), and then eluted twice with 0.1% formic acid and 50% acetonitrile (25 μL) to recover glycolipids. 4. The collected glycolipids were measured by mass spectrometry (MALDI-TOF MS). <Analysis conditions> Measurement equipment: Ultraflex III mass spectrometer (Bruker Daltonics) Measurement mode: Positive mode, Reflectron mode Matrix:DHB

[0059] The results are shown in Figure 2. A mass derived from the glycolipid GM1 was detected, confirming that GM1 was recovered by graphite carbon. [Industrial Applicability]

[0060] The present invention relates to a glycolipid capture agent, a glycolipid recovery method, a method for diagnosing glycolipid metabolic disorders, and a kit therefor. Accordingly, the present invention can be used in fields requiring simple and specific capture or recovery of glycolipids, such as liquid biopsy for diseases associated with glycolipids or their analogs, and development of diagnoses and treatments for lipid metabolic disorders.

Claims

1. A sequestrant for glycolipids, comprising graphitic carbon.

2. The capture agent of claim 1, wherein the glycolipid is selected from glycosphingolipids, glyceroglycolipids, and glycosphingosines.

3. The capture agent of claim 1, which is porous.

4. A kit for recovering glycolipids, comprising the capture agent according to any one of claims 1 to 3.

5. The kit according to claim 4, wherein the kit is for diagnosing a glycolipid metabolism disorder.

6. The kit of claim 4, further comprising a reagent capable of qualitatively or quantitatively determining glycolipids.

7. A method for recovering glycolipids, comprising: a) contacting a sample containing glycolipids with the capture agent according to any one of claims 1 to 3 to capture the glycolipids; and b) recovering the glycolipid from the capturing agent that has captured the glycolipid A method comprising:

8. The method according to claim 7, further comprising the step of c) qualitatively or quantitatively determining the recovered glycolipids.

9. A method for assisting in the diagnosis of a glycolipid metabolism disorder, comprising: a) contacting a sample containing glycolipids with the capture agent according to any one of claims 1 to 3 to capture the glycolipids; b) recovering the glycolipid from the capturing agent that has captured the glycolipid; c) qualitatively or quantitatively determining the recovered glycolipids; and d) A step of identifying a glycolipid metabolic disorder or assisting in determining the degree of progression thereof based on the obtained qualitative or quantitative results of glycolipids. A method comprising:

10. The method according to claim 9, wherein the glycolipid metabolism disorder is a lysosomal disease.

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