JP4251485B2 - Glycolipid measurement method, disease detection method and kit - Google Patents

Description

  The present invention relates to a method for measuring glycolipid and a measurement kit using the method. The present invention further relates to a disease detection method and a detection kit.

  Non-Patent Document 1 discloses that a sample is brought into contact with a solid phase to which a sugar chain-recognizing antibody is fixed, a sugar chain-containing substance in the sample is bound to the sugar chain-recognizing antibody, and then labeled sugar chain recognition Describes a method for measuring a sugar chain antigen in a specimen, which comprises at least a step of detecting a recognizing antibody bound to the sugar chain-containing substance by contacting a specific antibody. However, the above measurement principle has a problem that a sugar chain-containing substance such as a glycolipid containing only one antibody binding structure per molecule cannot theoretically be measured.

  Non-Patent Document 2 describes a technique for covalently binding a sugar chain to a protein using a chemical method. However, there is no description or suggestion regarding measurement of glycolipids or detection of diseases.

  Non-Patent Document 3 describes that it is known that a specific glycolipid concentration increases in blood and urine due to an inborn error of metabolism, and those glycolipids in blood and urine. Is described as being useful in screening for these inborn errors of metabolic disorders. Furthermore, in Non-Patent Document 4 and Non-Patent Document 5, sugar chain-binding proteins that extract these glycolipids from blood and urine using an organic solvent, adhere this to a solid phase, and bind to this are described. There is described a method for measuring these glycolipids in a specimen, which comprises at least a step of detecting the sugar chain-binding protein bound to the glycolipid in advance.

On the other hand, Patent Document 1 discloses a method for chemically binding a protein to a monosaccharide.
However, there is no description or suggestion of measuring a glycolipid in a body fluid using a solid phase to which a sugar chain to which a protein is covalently bonded is fixed, and relating this to a disease.

International Publication WO87 / 02777 Pamphlet Cancer Res. 46, 2619-2626, (1986) Methods On Glycoconjugates, pp 171-176 (1995) Glycopathology, pp 217 (1991) Analytical Biochemistry 267, 104-113 (1999) Japanese Journal of Inborn Errors of Metabolism 17 (2), 189 (2001)

  If a method capable of measuring glycolipids with higher sensitivity, specificity and accuracy, good reproducibility and quantification and a simple method can be provided, it is extremely practical as a method for measuring glycolipids. A kit was desired. If such a measurement method and measurement kit can be provided, it becomes extremely easy to detect a disease in which the amount of glycolipid in the body fluid changes.

  That is, an object of the present invention is to provide a very practical glycolipid measurement method and measurement kit, as well as a disease detection method and detection kit.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that glycolipids are more sensitive, specific and accurate than conventional methods by using a solid phase to which a sugar chain to which protein is bound is fixed. In addition, the inventors have found that the measurement can be easily performed with good reproducibility and quantitativeness, and the present invention has been completed.

That is, the present invention is as follows.
(1) “A conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is a functional group formed at the reducing end of the sugar chain and the function of the protein. A method for measuring a glycolipid in a specimen, comprising using a solid phase to which the conjugate, which is due to a covalent bond with a group, is fixed.
(2) A method for measuring a glycolipid in a sample, comprising at least the following steps (A) and (B).
Step (A): “a conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is formed at the reducing end of the sugar chain and the protein A sample and a sugar chain-binding protein are brought into contact with a solid phase to which the conjugate, which is due to a covalent bond with a functional group of the sample, is fixed, and the glycolipid and the sugar in the sample are contacted with the sugar chain-binding protein. A step of competitive reaction with a chain.
Step (B): a step of detecting the sugar chain-binding protein bound to the sugar chain fixed to a solid phase.
(3) The measurement method according to (1) or (2), wherein the covalent bond is a bond via a spacer compound capable of binding to both a sugar chain and a protein contained in the glycolipid.
(4) The measuring method according to (2) or (3), wherein the sugar chain binding protein is labeled with a labeling substance or can be labeled.
(5) The measuring method according to any one of (2) to (4), wherein the sugar chain binding protein is an anti-glycolipid antibody.
(6) The measuring method according to any one of (1) to ( 5) , wherein the glycolipid is a glycosphingolipid.
(7) The measuring method according to any one of (1) to (6), wherein the sugar chain is a sugar chain contained in Gb3 having a structure represented by the following formula 1.
Galα1-4Galβ1-3Glc (1)
Here, Gal represents D-galactose, Glc represents D-glucose, in the formula, − represents a glycoside bond, the number represents the carbon number in which the glycoside bond is present, and α and β are in position 1 An anomer of the glycosidic bond is shown.
(8) A glycolipid contained in a body fluid as a specimen is measured by the measurement method according to any one of (1) to (7), and the measurement result of the glycolipid in the body fluid and the glycolipid in vivo A method for detecting a disease by associating a disease whose amount varies.
(9) The detection method according to (8), wherein the disease is a disease of abnormal glycolipid metabolism.
(10) The detection method according to (8), wherein the disease is Fabry disease.
(11) A glycolipid measurement kit comprising at least the following component (A).
(A) A conjugate of a sugar chain and a protein contained in a glycolipid, wherein the bond between the sugar chain and the protein in the conjugate is a functional group formed at the reducing end of the sugar chain and a functional group of the protein. solid phase conjugates conjugate is secured is by covalent bonds.
(12) The kit for measuring glycolipid according to claim 11, wherein the covalent bond is a bond via a spacer compound capable of binding to both a sugar chain and a protein contained in the glycolipid.
(13) The glycolipid measurement kit according to (11) or (12) , further comprising the following component (B).
(B) Sugar chain binding protein.
(14) The measurement kit according to ( 13 ), wherein the sugar chain binding protein is labeled.
(15) The measurement kit according to ( 13) or (14), wherein the sugar chain binding protein is an anti-glycolipid antibody.
(16) Any one of ( 13) to (15) , wherein the sugar chain is a sugar chain contained in Gb3 having a structure represented by the following formula 1, and the sugar chain-binding protein is an anti-Gb3 antibody : The measurement kit described.
Galα1-4Galβ1-3Glc (1)
Here, Gal represents D-galactose, Glc represents D-glucose, in the formula, − represents a glycoside bond, the number represents the carbon number in which the glycoside bond is present, and α and β are in position 1 An anomer of the glycosidic bond is shown.
(17) A detection kit for a disease in which the amount of glycolipid in a living body varies, comprising at least the components of the measurement kit according to any one of (11) to ( 16) .
(18) The detection kit according to ( 17), wherein the disease is a disease of abnormal glycolipid metabolism.
(19) The detection kit according to ( 17) , wherein the disease is Fabry disease.

  In the present invention, the sugar chain binding protein is preferably labeled. Further, the sugar chain binding protein is preferably an anti-glycolipid antibody, more preferably the glycolipid is Gb3, and the anti-glycolipid antibody is preferably an anti-Gb3 antibody.

The measurement method of the present invention is a method that can measure glycolipids with high sensitivity, specificity and accuracy, good reproducibility and quantification, and simple, and is very practical. In addition, the detection method of the present invention is an application of the measurement method of the present invention, and can detect various diseases very easily, and is therefore a highly practical method. In addition, since the measurement kit and the detection kit of the present invention are kits used for carrying out the measurement method and the detection method of the present invention, glycolipids can be detected with high sensitivity, specificity and accuracy using this. -It can be easily measured with good quantitativeness, and various diseases in which the amount of glycolipids in a living body can be detected extremely easily.
The measurement kit and the detection kit of the present invention have a simple configuration and can be provided at low cost.
The present invention can be applied not only to the detection of various diseases described in the specification, but also to the determination of disease state, progress, determination of treatment policy, confirmation of treatment effect, evaluation of drug development, etc. Is extremely useful.

<1> Measurement method of the present invention The measurement method of the present invention is a “conjugate of a sugar chain and a protein contained in a glycolipid” , and the bond between the sugar chain and the protein in the conjugate is at the reducing end of the sugar chain. A method for measuring glycolipids in a specimen, comprising using a solid phase to which the conjugate, which is formed by a covalent bond between a formed functional group and a functional group of a protein, is used. More specifically, a method for measuring glycolipid in a sample, comprising at least the following steps (A) and (B).

Step (A): “a conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is formed at the reducing end of the sugar chain and the protein A sample and a sugar chain-binding protein are brought into contact with a solid phase to which the conjugate, which is due to a covalent bond with a functional group of the sample, is fixed, and the glycolipid and the sugar in the sample are contacted with the sugar chain-binding protein. A step of competitive reaction with a chain.
Step (B): a step of detecting the sugar chain-binding protein bound to the sugar chain fixed to a solid phase.

  The “conjugate” that can be used in the measurement method of the present invention is not limited as long as the protein and the sugar chain are bound directly or indirectly. Such a bond may be a chemical bond (covalent bond, hydrogen bond, ionic bond, etc.) or a bond by physical adsorption, but is preferably a chemical bond, and more preferably a covalent bond.

  In a conjugate in which both are directly covalently bonded, a substance in which a sugar chain and a protein are covalently bonded via a spacer substance capable of binding to both substances (compounds) (sugar chain-spacer substance- Protein). As a conjugate in which both are indirectly covalently bonded, for example, a non-protein substance (for example, biotin) having a binding property to a protein is covalently bonded to a sugar chain, and this is combined with a protein such as avidin or streptavidin. And the like (eg, sugar chain biotin-avidin) and the like. The bond between biotin and avidin is not a covalent bond, but the complex (biotin-avidin) formed by combining these two is extremely stable (the specific substance that forms such a complex is defined as “specific”. It is also referred to as a “direct binding pair”). In addition, what is directly covalently bonded here is preferable.

The position where the protein is covalently bonded is preferably the reducing end of the sugar chain. Such a conjugate can be prepared, for example, by the method described in the Examples.
The protein is not particularly limited as long as the sugar chain to which the protein is covalently bonded can be fixed to the solid phase described later and is not released even under the conditions of the competitive reaction or the washing described later. A protein containing a lot of amino groups or a protein having a molecular weight of 5,000 or more is preferred. A protein having both of these properties, that is, a protein containing many amino groups and having a molecular weight of 5,000 or more is particularly preferred. Specific examples of such proteins include serum albumin (bovine serum albumin (hereinafter also referred to as BSA), etc.), human serum albumin, ovalbumin, casein, avidin, polyamino acid (polylysine, etc.), etc. Albumin is preferred and BSA is more preferred.

  As the sugar chain to which the protein is bound, the same sugar chain as the sugar chain structure of the glycolipid to be measured or a part thereof is used. That is, the sugar chain in the present invention is a sugar chain contained in a glycolipid. The glycolipid to be measured is not particularly limited, but a glycosphingolipid is preferable, and Gb3 is particularly preferable. Therefore, a sugar chain for covalently binding a protein is also a “glycan included in Gb3”. Further, the chain length of the sugar chain for covalently binding the protein is not particularly limited. As such a sugar chain, a sugar chain contained in Gb3 represented by the following formula 1 is specifically mentioned.

          Galα1-4Galβ1-3Glc (1)

  Here, Gal represents D-galactose, Glc represents D-glucose, in the formula, − represents a glycoside bond, the number represents the carbon number in which the glycoside bond is present, and α and β are in position 1 An anomer of the glycosidic bond is shown.

  The method for covalently binding a protein and a sugar chain (a method for producing a sugar chain in which a protein is covalently bound) is not limited as long as it is a method capable of allowing a target covalent bond to be used, and a known method is appropriately adopted. For example, the method described in Methods on Glycoconjugates pp 171-176 (1995) can also be employed.

  Examples of methods for covalently binding a protein and a sugar chain are shown below, but are not limited thereto.

  Example (1) After introducing an amino group into the formyl group at the reducing end of the sugar chain via a suitable spacer compound, reacting with SPDP (N-Succinimidyl-3- (2-pyridyldithio) propionate), and converting to PDP Separately, PDP is introduced into the amino group of the protein, reacted with the reduced activated protein, and covalently bound. The resulting covalent bond is a disulfide bond (—S—S—).

  Example (2) An amino group is introduced into a formyl group at the reducing end of a sugar chain via an appropriate spacer, and SMCC (Succinimidyl-trans-4 (N-maleimidylmethyl) cyclohexane-1-carboxylate) or GMBS (N- (4 -Maleimidobutylryloxy) succinimide), then PDP is separately introduced into the amino group of the protein, reacted with the reduced activated protein, and covalently bonded.

  Example (3) A method in which a carboxyl group is introduced into a formyl group at the reducing end of a sugar chain via an appropriate spacer and covalently bound using a protein amino group and carbodiimide. The resulting covalent bond is an amide bond (—CO—NH—).

  Example (4) Using biotin hydrazide in which a hydrazide group is introduced into the carboxyl group of biotin, this amino group and a carboxyl group introduced into the formyl group at the reducing end of the sugar chain via an appropriate spacer are converted into EDC (1- It is covalently bonded using a condensing agent such as Ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride) (the resulting covalent bond is an amide bond (-CO-NH-)), and this biotin moiety is bonded to avidin. Method.

  Example (5) A method in which a p-aminophenyl group is introduced into the formyl group at the reducing end of a sugar chain, and the p-aminophenyl group is converted to phenylisothiocyanate by thiocarbonyl dichloride (thiophosgene) to be covalently bonded to the amino group of the protein.

In addition to the methods described above, the methods described in Japanese Patent No. 2728757 and Japanese Patent No. 2792332 can be employed.
The “sugar chain to which a protein is covalently bonded” produced by such a method is used while being fixed to a solid phase.

The shape, material, etc. of the solid phase are not limited as long as it is a water-insoluble solid phase to which a sugar chain to which a protein is covalently bonded can be fixed.
Examples of the shape of the solid phase include plates (for example, microplate wells), tubes, beads, membranes, gels, latexes, and the like. Examples of the solid phase material include polystyrene, polypropylene, nylon, and polyacrylamide. Among these, a plate made of polystyrene is preferable.

  As a method for fixing a sugar chain in which a protein is covalently bonded to these solid phases, a general method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bond method, a comprehensive method (an immobilized enzyme, 1975, Kodansha publication, see pages 9-75) can be applied. Among these, the physical adsorption method is preferable because the operation is simple and frequently used.

Specific examples of the physical adsorption method include and are preferably the following methods.
The sugar chain to which the protein is covalently bonded is dissolved in a buffer solution having a pH of about 7 to 9 (for example, phosphate buffer, phosphate buffered saline (PBS), carbonate buffer, etc.), added to the solid phase, and then dissolved at 4 ° C. A method of allowing to stand still and fix.

In addition, it is preferable to add a blocking substance to the solid phase after this to coat a portion where the sugar chain to which the protein is covalently bonded is not fixed. Examples of such blocking substances include serum albumin, casein, skim milk, gelatin, and the like, and commercially available blocking substances can also be used.
By the above method, a solid phase to which a sugar chain to which a protein is covalently bonded can be produced.

  In the step (A), the solid phase produced in this manner is brought into contact with the sample and the sugar chain-binding protein, and the glycolipid and the sugar chain in the sample compete with each other for the sugar chain-binding protein. Process.

  The specimen is not limited as long as the glycolipid to be measured is contained or possibly contained. In addition, the specimen need not be purified in advance. That is, even if the sample contains a substance having a sugar chain structure other than the glycolipid to be measured, and other various proteins, the measurement method of the present invention specifically measures the glycolipid to be measured. be able to.

  Specific examples of the specimen include glycolipid-containing solutions (pharmaceutical production process samples and the like), cell culture supernatants, body fluids, and the like. Of these, body fluids are preferred. Examples of the body fluid include blood, serum, plasma, urine, saliva, joint fluid, pleural effusion, ascites, bone marrow fluid, spinal fluid, and the like, and urine, serum, and plasma are particularly preferable.

  In the present specification, the “glycan binding protein” means a protein having a property of specifically binding to a sugar chain part of a glycolipid. In the measurement method of the present invention, it is particularly preferable to use glycolipid Gb3 as a measurement target, and therefore, a protein that specifically binds to Gb3 is preferable as the sugar chain binding protein.

  Specific examples of the sugar chain-binding protein include the following, but are not limited to these as long as the protein has the property of binding to a sugar chain.

(1) Sugar chain-recognizing antibodyAnti-Gb3 antibody, an antibody that recognizes a neutral glycolipid sugar chain such as an anti-Lactosyl ceramide antibody, an anti-GM1 antibody, an anti-GM2 antibody, an anti-GD1 antibody, an antibody that recognizes an acid glycolipid, An antibody that recognizes a sugar chain of a glycoprotein. These antibodies may be monoclonal antibodies or polyclonal antibodies, and the antibody molecules may be complete molecules, or fragments such as Fab, Fab2, F (ab ′) 2, and the like.

(2) Plant and bacterial sugar chain binding proteins
Bacterial glycan binding proteins such as plant lectins such as AALABAConA, LCA, PHA, PNA, RCA, WGAANA, and verotoxin.

(3) Animal-derived sugar chain binding proteins selectin, galectin, collectin, MBP, cell receptor having a sugar chain recognition site, proteoglycan having a sugar chain recognition site, and the like. These proteins may be complete molecules or molecular fragments containing a sugar chain recognition site.

  The origin of these sugar chain binding proteins is not particularly limited. For example, it may be a protein separated from a natural material or a recombinant protein produced by genetic engineering techniques. Among these, since it is most preferable that the measurement object of the present invention is the glycolipid Gb3, the anti-Gb3 antibody is most preferable as the sugar chain binding protein. The anti-Gb3 antibody is sold as “anti-Gb3” by Seikagaku Corporation.

  In the measurement method of the present invention, it is preferable that the sugar chain-binding protein is labeled with a labeling substance because detection is easy in step (B).

  Labeling substances used for labeling sugar-binding proteins include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), fluorescent dyes (luminol, fluorescein isothiocyanate (FITC), etc.), chemiluminescence Substances, biotin, avidin, radioisotopes and the like can be mentioned, but there are no particular limitations as long as they are usually capable of labeling proteins. The labeling method is a known method suitable for the labeling substance, for example, glutaraldehyde method, periodate crosslinking method, maleimide crosslinking method, carbodiimide method, activated ester method, etc. ("Protein Chemistry (below)", Tokyo Chemical Dojin , Published in 1987). For example, when using biotin as a labeling substance, a method using a hydrazide derivative of biotin (see Avidin-Biotin Chemistry: A Handbook, p57-63, PIERCE CHEMICAL COMPANY, 1994), or when using fluorescein isothiocyanate The method can be appropriately selected from the methods described in JP-B 63-17843.

  In addition, when the glycan-binding protein is not labeled in advance and is detected in the step (B), for example, a labeled antibody that recognizes the glycan-binding protein and the glycan-binding protein are recognized. The mode which may be finally labeled by making it react with sex protein may be sufficient.

  When a sample and a sugar chain-binding protein are brought into contact with the solid phase (solid phase to which a sugar chain covalently bonded to a protein is fixed), the glycolipid in the sample and the solid phase are fixed to the sugar chain-binding protein. The resulting sugar chain (covalently bound to the protein) is bound by a competitive reaction.

  There are three methods for contacting a sample and a sugar chain-binding protein to a solid phase to which a sugar chain to which protein is bound is fixed: a sugar chain fixed to the solid phase, a glycolipid in the sample, and a sugar chain-binding protein. Is not particularly limited as long as it contacts and reacts, but the method of the present invention uses a competitive reaction of a sugar chain with a sugar chain-binding protein and measures glycolipid in a sample. It is preferable to contact the solid phase last, or at least simultaneously with or after contacting the specimen and the sugar chain binding protein. Specifically, the following embodiments are exemplified. In addition, the following aspect is an illustration to the last, and this invention measuring method is not limited to these. The order and method of bringing the three members into contact can be appropriately determined by those skilled in the art with reference to, for example, the following embodiments.

Aspect (1): “A conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is formed at the reducing end of the sugar chain and the protein A sample is added to and brought into contact with the solid phase to which the conjugate is fixed, which is due to a covalent bond with the functional group, and solid-liquid separation (including washing) is performed as necessary. Add sex protein and contact.

Aspect (2): A specimen is a “conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is formed at the reducing end of the sugar chain. A solid phase to which the conjugate, which is due to a covalent bond between a group and a protein functional group, is added and brought into contact, and solid-liquid separation (including washing) is performed as necessary. Add binding protein and contact.

Aspect (3): “Conjugate of sugar chain and protein contained in glycolipid” , wherein the bond between the sugar chain and protein in the conjugate is formed at the reducing end of the sugar chain and the protein A sample and a sugar chain-binding protein are simultaneously added and brought into contact with a solid phase to which the conjugate, which is due to a covalent bond with the functional group, is fixed.

Aspect (4): A sugar-binding protein is added to and contacted with a specimen and incubated, and then this is a “conjugate of a sugar chain and a protein contained in a glycolipid” , and the sugar in the conjugate The bond between the chain and the protein is due to the covalent bond between the functional group formed at the reducing end of the sugar chain and the functional group of the protein, and is added to and brought into contact with the solid phase to which the conjugate has been fixed.

  In addition, it is preferable to incubate at 0 to 45 ° C., preferably 32 to 40 ° C., in order to sufficiently perform a competitive reaction after contacting these three members. The incubation time is not particularly limited as long as the above three can sufficiently react, and can be exemplified by about 30 minutes to 1 hour, and is preferable. In order to cause an inhibition reaction in which the three are reacted separately, about 10 to 1 hour is exemplified as the incubation time under the same temperature condition.

After the three reactions, it is preferable to perform solid-liquid separation, in particular, to wash the surface of the solid phase with a washing liquid. This washing is a “conjugate of a sugar chain and a protein contained in a glycolipid” fixed to a solid phase, and the sugar chain and the protein in the conjugate are formed at the reducing end of the sugar chain. It is carried out under the condition that the conjugate and the sugar chain-binding protein bound thereto are free from covalent bonds between the functional group and the protein functional group . As the washing solution, for example, a buffer solution added with a nonionic surfactant such as a Tween surfactant (for example, phosphate buffer solution, phosphate buffered saline (PBS), Tris hydrochloride buffer solution, etc.) ) Is preferably used.

After passing through the above-mentioned process (A), the process proceeds to process (B).
Step (B) is a “conjugate of a sugar chain and a protein contained in a glycolipid” fixed to a solid phase, and the bond between the sugar chain and the protein in the conjugate is at the reducing end of the sugar chain. This is a step of detecting a sugar chain binding protein bound to the conjugate, which is due to a covalent bond between the formed functional group and the functional group of the protein.

As described above, the measurement method of the present invention is a conjugate of a sugar chain and a protein contained in a glycolipid to a sugar chain-binding protein, and the binding between the sugar chain and the protein in the conjugate is a reduction of the sugar chain. Since it utilizes the competitive reaction between the conjugate (adhered to the solid phase) and the glycolipid in the sample, which is a covalent bond between the functional group formed at the end and the functional group of the protein , The glycolipid in the sample can be measured by detecting the sugar chain-binding protein bonded to the sugar chain fixed to the phase.

  That is, it is determined that the amount of glycolipid in the sample is small if the detected amount of the sugar chain binding protein bound to the sugar chain fixed to the solid phase is large. Conversely, if the amount of sugar chain binding protein bound to the sugar chain fixed to the solid phase is small, it is determined that the amount of glycolipid in the sample is large.

A “conjugate of a sugar chain and a protein contained in a glycolipid” fixed to a solid phase, wherein a bond between the sugar chain and the protein in the conjugate is a functional group formed at the reducing end of the sugar chain The detection of the glycan-binding protein bound to the conjugate, which is due to a covalent bond with the functional group of the protein, may be performed using an antibody that specifically reacts with the glycan-binding protein, and the step ( In the case of using a protein obtained by labeling a sugar chain binding protein with a labeling substance in A), a person skilled in the art can appropriately select a detection method according to the labeling substance used.

  For example, when biotin is used as a labeling substance, an enzyme (for example, peroxidase) to which avidin, streptavidin or the like is bound is added to bind biotin to avidin or streptavidin, and then to avidin, streptavidin or the like. Examples include a method in which a bound enzyme substrate or a chromogenic substrate is added, and the degree of color development of the product by the enzyme reaction is measured by a change in absorbance. Moreover, when using a fluorescent substance and a chemiluminescent substance, the method etc. which measure the fluorescence and light emission of the solution after reaction are mentioned.

  In the measurement method of the present invention, the concentration of glycolipid in the sample is prepared in advance by using a standard solution of glycolipid of known concentration and preparing a calibration curve for the relationship between the concentration of glycolipid and the detection result (for example, absorbance) of the labeling substance. In addition, it can be obtained by using the detection result of the sample having an unknown concentration and the calibration curve.

<2> Detection method of the present invention The detection method of the present invention measures glycolipids in body fluids by the measurement method of the present invention, and relates the amount of glycolipids in body fluids to diseases of animals (humans, pets, livestock, etc.). It is the detection method of the disease by.

The disease to be detected by the detection method of the present invention is not particularly limited as long as the disease causes a change in the amount of glycolipid in the body fluid.
Depending on the disease to be detected, the type of body fluid used as the specimen and the type of glycolipid to be measured can be appropriately selected.
In addition, the correlation between the amount of glycolipid in the body fluid and the disease, and the detection of the disease can be appropriately performed according to the disease to be detected.

  For example, when the disease to be detected is a disease in which the amount of glycolipid in the body fluid increases, the amount of glycolipid in the body fluid of a healthy animal (animal free of the disease) When the amount of the glycolipid measured by the measurement method of the present invention is larger than the amount in the body fluid of a healthy animal, the “is suffering from the disease” or “ It is related to “the possibility of suffering from the disease is high”, whereby the disease can be detected.

  For example, when the disease to be detected is a disease in which the amount of glycolipid in the body fluid decreases, the glycolipid in the body fluid of an animal with a healthy amount of glycolipid in the body fluid (an animal without the disease) Therefore, if the amount of glycolipid measured by the measurement method of the present invention is small compared to the amount in the body fluid of a healthy animal, “is suffering from the disease”, or It is associated with “high possibility of suffering from the disease”, whereby the disease can be detected.

  Further, in the case where a disease in which the amount of glycolipid in the body fluid is increased or decreased is detected, the amount of glycolipid measured by the measurement method of the present invention is the amount of glycolipid in the body fluid of a healthy animal. If it is equivalent to the amount, it can be related to “not suffering from the disease” or “not likely to suffer from the disease”.

  The detection method of the present invention includes not only the presence or absence of the disease but also the detection of the degree of the disease. For example, the amount of glycolipid in the body fluid of each individual is periodically measured by the measurement method of the present invention, and when the amount of glycolipid tends to deviate from the level of healthy animals, “the disease is progressing” Alternatively, it can be associated with “the possibility that the disease is progressing”. In addition, when the amount of glycolipid measured by the measurement method of the present invention tends to approach the level of a healthy animal, “the above disease is in an improvement direction” or “the above disease is in an improvement direction” Can be associated. Further, when the amount of glycolipid measured by the measurement method of the present invention does not change within the range of the level of healthy animals, “there is no change in health” or “there is a high possibility that there is no change in health” If the amount of glycolipid does not change outside the level of healthy animals, there is a high possibility that “the degree of the disease does not change” or “the degree of the disease does not change” Can be associated.

  The amount of glycolipid that serves as a detection criterion for the disease may be the concentration of glycolipid obtained using a calibration curve prepared for the relationship between the concentration of the standard glycolipid product and the detection result of the labeling substance. It may be a ratio to the amount of glycolipid in the body fluid of a healthy animal (an animal having no disease) without using the calibration curve.

Table 1 summarizes examples of body fluids that can be used in the detection method of the present invention, sugar chains to be measured, diseases that can be detected thereby, and changes in the amount of glycolipids in body fluids associated with the diseases.
Table 1 is merely an example, and the detection method of the present invention is not limited to these.

  For example, when detecting Fabry disease, Gb3 in urine is measured by the measurement method of the present invention, and this Gb3 is increased compared to the amount of Gb3 in urine of a healthy animal (animal not Fabry disease) Can be associated with “I have Fabry disease” or “highly likely to have Fabry disease”.

  The detection method of the present invention is preferably a method of detecting Fabry disease by measuring Gb3 in urine by the measurement method of the present invention and associating the amount of Gb3 in urine with Fabry disease.

<3> Measurement kit of the present invention The measurement kit of the present invention is a glycolipid measurement kit containing at least the following components (A) and (B).

(A) “A conjugate of a sugar chain contained in a glycolipid and a protein” , wherein the bond between the sugar chain and the protein in the conjugate is a functional group formed at the reducing end of the sugar chain and the function of the protein. A solid phase to which the conjugate, which is due to a covalent bond with a group, is fixed.
(B) Sugar chain binding protein.
The description of the solid phase (A) and the protein (B) is the same as in <1> the measurement method of the present invention.

  The measurement kit of the present invention is not particularly limited as long as it contains at least the above (A) and (B), and further, a glycolipid standard product having a known concentration as a standard for preparing a calibration curve, a detection reagent for a labeling substance, a sugar chain A reagent for labeling a binding protein or a reagent for detecting a sugar chain binding protein (labeled antibody against the protein, etc.) can be added as a constituent component. In addition to these components, the blocking substance, the washing solution, the sample diluent, the enzyme reaction stop solution, and the like may be included.

These components can be stored in separate containers and stored as a kit that can be used according to the measurement method of the present invention at the time of use.
The measurement of glycolipid using the measurement kit of the present invention can be performed according to the measurement method of the present invention <1>.

<4> Detection kit of the present invention The detection kit of the present invention is a disease detection kit containing at least the components of the measurement kit of the present invention. The description of the components and the like of the detection kit of the present invention is the same as that of the measurement kit of the present invention <3>.
Disease detection using the detection kit of the present invention can be performed according to the detection method of the present invention <2>.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
First, the materials used in this example and their availability will be described together.

(material)
Immunoplate (Narugenunk)
Sugar chain contained in glycolipid Gb3 (formula (1))-ethanolamine-BSA (Gb3 sugar chain-BSA conjugate)
BSA (Seikagaku Corporation sales)
Anti-Gb3 antibody (mouse IgG antibody: manufactured by Seikagaku Corporation)
Gb3 (Seikagaku Corporation)
Horseradish peroxidase-labeled anti-mouse IgG + IgM antibody (hereinafter referred to as HRP-labeled secondary antibody; manufactured by Seikagaku Corporation)
Tetramethylbenzidine solution (hereinafter referred to as TMB solution; manufactured by Moss)

The Gb3 sugar chain-BSA conjugate was produced by the following method.
After adding SPDP 9.5mg / 1mL ethanol solution dropwise to BSA 350mg / 3mL 0.1mol / L PB solution and stirring at room temperature for 30 minutes, gel filtration with Sephadex G-50 (trade name: Pharmacia) A fraction was obtained. Five times the amount of dithiothreitol (DTT) was added to the active fraction, and the mixture was stirred at room temperature for 60 minutes, followed by gel filtration with Sephadex G-50 (trade name: Pharmacia). The obtained active fraction was designated as active fraction 1.

  Next, Gb3 sugar chain-ethanolamine was prepared. That is, Gb3 sugar chain (212.3 mg, 0.421 mmol) was dissolved in pyridine (5 ml), acetic anhydride (5 ml) was added, and the mixture was stirred at room temperature for 2 hours. The solution was distilled off while azeotroping with toluene, and the residue was purified by silica gel column chromatography (Toluene: AcOEt = 2: 2.5) to obtain Compound 1 (407 mg, qu.).

Rf = 0.39, 0.31 (Toluene: AcOEt = 1: 1)
¹ H-NMR δ (CDCl ₃ )
6.251 (d, 0.5H, J = 3.9Hz, H-1aα), 5.690 (d, 0.5H, J = 8.3Hz, H-1aβ)
2.175, 2.135, 2.130, 2.124, 2.115, 2.098, 2.096, 2.090, 2.086, 2.072, 2.069, 2,065, 2.063, 2.059, 2.051, 2.042, 2.032, 2.012, 1.991, 1.985 (20s, 33H, 11Ac)

  Compound 1 (407 mg, 0.421 mmol) was dissolved in N, N-dimethylformamide (2 ml). Hydrazine / acetic acid (66.5 mg, 1.7 eq.) Was added, and the mixture was stirred at −2 ° C. to 5 ° C. for 1 hour. Furthermore, hydrazine / acetic acid (3.4 eq.) Was directly purified by silica gel column chromatography (hexane: AcOEt = 1: 3) to obtain Compound 2 (389 mg, qu).

Rf = 0.32 (hexane: AcOEt = 1: 3)

  Compound 2 (389 mg, 0.421 mmol) was dissolved in dichloromethane (3.0 ml) and trichloroacetonitrile (652 μl, 15.0 eq.), 1,8-diazabicyclo [5.4.0] undec-7-ene (1,8-Diazabicyclo [ 5.4.0] undec-7-ene: 45 μl, 0.7 eq.) Was added, and the mixture was stirred at 0 ° C. for 3 hours. The reaction solution was directly purified by silica gel column chromatography (Toluene: AcOEt = 2: 3) to obtain Compound 3 (417.5, mg, 92.8%).

Rf = 0.37 (Toluene: AcOEt = 2: 3)
¹ H-NMR δ (CDCl3)
8.650 (s, 1H, NH), 6.483 (d, 1H, J = 3.9Hz, H-1a), 5.589 (bd, 1H, H-1c or H-4c), 5.566 (bt, 1H), 5.000 (d , 1H, J = 3.4Hz, H-1c or H4c), 4.540 (d, 1H, J = 7.8Hz, H-1b), 4.025 (d, 1H, J = 2.4Hz, H-4b), 2.131, 2.108 , 2.096, 2.092, 2.074, 2.067, 2.062, 2.041, 2.012, 1.986 (10s, 30H, 10Ac)

  Compound 3 (417 mg, 0.390 mmol) and compound 7 (151.8 mg, 0.778 mmol) were dissolved in dichloromethane (5 ml), and the mixture was stirred at room temperature for 5 minutes with argon gas in the presence of 4 parts of molecular sieve. The reaction solution was cooled to −10 ° C., trimethylsilyl trifluoromethanesulfonate (TMSOTf: 21.7 μl, 0.3 eq.) Was added, and the mixture was stirred as it was for 1 hour. Further, TMSOTf (43.4 μl, 0.6 eq.) Was added and stirred as it was for 1 hour. The reaction solution was filtered through celite, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The obtained organic layer was dried over magnesium sulfate and filtered, and then the solvent was distilled off. The residue was purified by silica gel column chromatography (Toluene: AcOEt = 1: 3) to obtain Compound 4 (203,7 mg, 47.4%).

Rf = 0.38 (Toluene: AcOEt = 1: 3)
¹ H-NMR δ (CDCl3)
7.376-7.287 (m, 5H, ph), 5.583 (d, 1H, 3.4Hz, H-4c), 5.392 (dd, 1H, J = 3.4, 10.7Hz), 4.986 (d, 1H, J = 3.4Hz, H-1c), 4.874 (dd, 1H, J = 8.3, 9.3Hz), 4.735 (dd, 1H, J = 3.0, 10.7Hz), 4.010 (d, 1H, J = 2.0Hz, H4b), 2.126-1.981 (30H, 10Ac)

Compound 4 (197 mg, 0.178 mmol) was dissolved in a methanol: tetrahydrofuran (1: 1) (5 ml) solution, 1N-NaOH (1 ml) was added, and the mixture was stirred at room temperature for 1 day. The solution was neutralized with Amberlyst 15E (trade name: manufactured by Organo Corporation), and then the solvent was distilled off. The residue was purified by Sephadex LH-20 (trade name: Pharmacia, CHCl ₃ : MeOH: H ₂ O = 1: 4: 2), and further silica gel column chromatography (CHCl ₃ : MeOH: H ₂ O = Purification by 5: 5: 1) gave compound 5 (114.6 mg, 94%).

Rf = 0.63 (CHCl ₃ : MeOH: H ₂ O = 5: 5: 1)
¹ H-NMR δ (CD3OD)
7.436-7.409 (m, 5H), 5.115 (bs, 2H, 2CH ₂ ), 4.936 (d, 1H, J = 3.9Hz, H-1c), 4.480 (d, 1H, J = 7.8Hz, H-1a or h-1b), 4.446 (bd, 1H, J = 7.3Hz), 4.340 (t, 1H, J = 6.4Hz)

Compound 5 (114.6 mg, 0.168 mmol) is dissolved in a methanol: water (2: 1) (10 ml) solution, palladium on carbon (Pd-C: 10%) (120 mg) is added, and the mixture is stirred at room temperature under hydrogen for 5.5 hours. did. After filtration through Celite, purification was performed with Sphadex LH-20 (trade name: Pharmacia, MeOH: H ₂ O = 3: 2) to obtain Compound 6 (92 mg, qu.).

Rf = 0.38 (MeOH: H2O: NH ₃ (28%) = 1: 3: 1)
¹ H-NMR δ (CD3OD)
4.934 (d, 1H, J = 3.9Hz, H-1c), 4.534 (d, 1H, J = 8.3Hz, H-1a or H-1b), 4.497 (d, 1H, J = 7.8Hz, H-1a or H-1b)

  After adding 3 equivalents of SPDP to Gb3 sugar chain-ethanolamine prepared in this way and stirring at room temperature for 30 minutes, gel filtration was performed with Sephadex G-10 (trade name: Pharmacia) to activate. Fraction 2 was designated. The obtained active fraction 2 was added dropwise in an equal amount to the active fraction 2 and stirred at 4 ° C. for 16 hours to obtain a sugar chain-BSA conjugate.

<Example 1> Quantification of Gb3 and preparation of calibration curve 100 μL of Gb3 sugar chain-BSA conjugate solution (0.5 μg / mL) was added to the well of an immunoplate and allowed to stand at 4 ° C. overnight to be solid-phased. Thereafter, the plate was blocked with a 1% BSA solution, and the plate was washed with a washing solution (PBS containing 0.05% Tween 20 (trade name)).
50 μL of a Gb3 aqueous solution having various concentrations (0.125-8 μg / mL) was added to this plate, and 50 μL of an anti-Gb3 antibody solution (1600 ng / mL) was further added, followed by reaction at room temperature for 1 hour.

Thereafter, the plate was washed again, 100 μL of HRP-labeled secondary antibody solution (1 μg / mL) was added, and reacted at 37 ° C. for 1 hour. Thereafter, the plate was washed again, and 100 μL of TMB solution was added to cause color development. The reaction was stopped by adding 100 μL of 1 mol / LH ₂ SO ₄ , the absorbance at 450 nm was measured, and a calibration curve was prepared. The results are shown in FIG.

<Example 2> Addition / recovery test of Gb3 100 μL of a Gb3 sugar chain-BSA conjugate solution (0.5 μg / mL) was added to the well of an immunoplate and allowed to stand at 4 ° C. overnight to be solid-phased. After blocking with BSA, add 50 μl of 2-fold diluted urine (hereinafter also referred to as Gb3-added urine) to which Gb3 (final concentration 4 μg / mL) has been added, and then add 50 μl of anti-Gb3 antibody solution (1000 ng / ml). After reacting at room temperature for 1 hour, the reaction was carried out in the same manner as in Example 1, and the amount of Gb3 in the Gb3-added urine was measured to determine the addition recovery rate. Table 2 shows the results when Gb3-added urine was used.

As shown in Table 2, the measured value of normal urine without Gb3 is similar to the measured value of normal urine 0.9 ± 0.4 μg / ml reported in Analytical Biochemistry 267, 104-113 (1999). Met. In addition, a recovery rate was obtained in the range of 95 to 100% even in an addition recovery test at a concentration close to the lower limit of 6 μg / ml of Gb3 concentration in sick urine reported in the above-mentioned literature. Was shown to be high.
From this result, it was shown that a glycolipid can be measured specifically and accurately by using a sugar chain to which a protein is covalently bonded.

<Example 3> Preparation of the measurement kit of the present invention and the detection kit of the present invention It is conceivable to prepare the measurement kit of the present invention and the detection kit of the present invention having the following configurations.

1. 1. One immunoplate (96 wells) with Gb3 sugar chain-BSA conjugate attached to the wells 2. One anti-Gb3 antibody (mouse IgG antibody) One HRP-labeled secondary antibody (rabbit anti-mouse IgG antibody) 4. One TMB (tetramethylbenzidine) solution5. One stop solution (1mol / L hydrochloric acid) 1 set of Gb3 standard solution
1 Gb3 standard solution 1 (0.125μg / mL)
1 Gb3 standard solution 2 (0.25μg / mL)
1 Gb3 standard solution 3 (0.5μg / mL)
1 Gb3 standard solution 4 (1μg / mL)
1 Gb3 standard solution 5 (2μg / mL)
1 Gb3 standard solution 6 (4μg / mL)
1 Gb3 standard solution 7 (8μg / mL)

It is a figure which shows the preparation scheme of Gb3 sugar chain- ethanolamine. It is a figure which shows the analytical curve of Gb3 at the time of using the plate which solidified the Gb3 sugar chain-BSA conjugate | bonded_body.

Claims (19)

“A conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the binding between the sugar chain and the protein in the conjugate is between the functional group formed at the reducing end of the sugar chain and the functional group of the protein. A method for measuring a glycolipid in a specimen, which comprises using a solid phase to which the conjugate is covalently bonded . A method for measuring glycolipid in a sample, comprising at least the following steps (A) and (B).
Step (A): “a conjugate of a sugar chain and a protein contained in a glycolipid” , wherein the bond between the sugar chain and the protein in the conjugate is formed at the reducing end of the sugar chain and the protein A sample and a sugar chain-binding protein are brought into contact with a solid phase to which the conjugate, which is due to a covalent bond with a functional group of the sample, is fixed, and the glycolipid and the sugar in the sample are contacted with the sugar chain-binding protein. A step of competitive reaction with a chain.
Step (B): a step of detecting the sugar chain-binding protein bound to the sugar chain fixed to a solid phase. The measurement method according to claim 1 or 2, wherein the covalent bond is a bond via a spacer compound capable of binding to both a sugar chain and a protein contained in the glycolipid. 4. The measuring method according to claim 2, wherein the sugar chain binding protein is labeled with a labeling substance or can be labeled. The measuring method according to any one of claims 2 to 4, wherein the sugar chain binding protein is an anti-glycolipid antibody. The measurement method according to any one of claims 1 to 5 , wherein the glycolipid is a sphingoglycolipid. The measuring method according to any one of claims 1 to 6, wherein the sugar chain is a sugar chain contained in Gb3 having a structure represented by the following formula 1.
Galα1-4Galβ1-3Glc (1)
Here, Gal represents D-galactose, Glc represents D-glucose, in the formula, − represents a glycoside bond, the number represents the carbon number in which the glycoside bond is present, and α and β are in position 1 An anomer of the glycosidic bond is shown. A disease in which a glycolipid contained in a body fluid as a specimen is measured by the measurement method according to any one of claims 1 to 7, and the measurement result of the glycolipid in the body fluid and the amount of glycolipid in the living body vary. A disease detection method by associating The detection method according to claim 8, wherein the disease is a glycolipid metabolic disorder. The detection method according to claim 8, wherein the disease is Fabry disease. A kit for measuring glycolipid, comprising at least the following component (A).
(A) “A conjugate of a sugar chain contained in a glycolipid and a protein” , wherein the bond between the sugar chain and the protein in the conjugate is a functional group formed at the reducing end of the sugar chain and the function of the protein. A solid phase to which the conjugate, which is due to a covalent bond with a group, is fixed. 12. The glycolipid measurement kit according to claim 11, wherein the covalent bond is a bond via a spacer compound capable of binding to both a sugar chain and a protein contained in the glycolipid. The kit for measuring glycolipid according to claim 11 or 12 , further comprising the following component (B).
(B) Sugar chain binding protein. The measurement kit according to claim 13, wherein the sugar chain binding protein is labeled. The measurement kit according to claim 13 or 14, wherein the sugar chain binding protein is an anti-glycolipid antibody. The measurement kit according to any one of claims 13 to 15 , wherein the sugar chain is a sugar chain contained in Gb3 having a structure represented by the following formula 1, and the sugar chain-binding protein is an anti-Gb3 antibody.
Galα1-4Galβ1-3Glc (1)
Here, Gal represents D-galactose, Glc represents D-glucose, in the formula, − represents a glycoside bond, the number represents the carbon number in which the glycoside bond is present, and α and β are in position 1 An anomer of the glycosidic bond is shown. A kit for detecting a disease in which the amount of glycolipid in a living body varies, comprising at least the components of the measurement kit according to any one of claims 11 to 16 . 18. The detection kit according to claim 17 , wherein the disease is a glycolipid metabolic disorder. The detection kit according to claim 17 , wherein the disease is Fabry disease.

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