JP2000065837A - Measurement of glycosaminoglycan or glycosaminoglycan bonding molecule and measurement kit thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably measure a glycosaminoglycan bonding molecule and the like in a specimen by an ease operation by using a carrier fixedly carrying lipid bonding glycosaminoglycan on its surface. SOLUTION: Using a plate, in which lipid bonding glycosaminoglycan (L-GAG) is bonded to a carrier such as an immunoassay plate by using a lipid residue as an anchor molecule, glycosaminoglycan (GAG) or a GAG bonding molecule in a specimen is measured. As GAGs constituting L-GAG, hyaluronic acid, chondroitin, chondroitin sulfate, and a GAG fragment prepared by decomposing these GAGs by a GAG decomposing enzyme are available. As a lipid, a lipid, which has a functional group bonding GAG directly or via a spacer and can be fixed to the carrier, is used, and a primary amino group containing lipid or a phospholipid is suitable. Preferably, L-GAG is fixed to the carrier through a hydrophobic bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule contained in a subject and a kit for measuring the same.

[0002]

BACKGROUND ART Glycosaminoglycans (hereinafter also referred to as GAGs) are a general term for polysaccharides having a basic skeleton of a repeating disaccharide structure containing hexosamine. Many of them are composed of uronic acid as a constituent sugar, and a sulfate group is added to a hydroxyl group of the sugar. Are often substituted to form an acidic polysaccharide. Naturally, GAGs other than hyaluronic acid have a structure in which one or several hundreds of GAGs are bound to various kinds of proteins, and their sugar-protein conjugates are collectively referred to as proteoglycans. The protein part is called a core protein, and a tetrasaccharide called a binding region is bonded to the serine residue in the order of xylose, galactose, galactose, and glucuronic acid, from which the disaccharide repeating structure described above is linked to several to several thousand sugars. GAG chains are bound. However, in reality, it does not have a single repeating disaccharide structure, but rather the position of the sulfate group, the number of substitutions of the sulfate group, the degree of epimerization of uronic acid (the ratio of glucuronic acid to iduronic acid), molecular weight, etc. , Animal species, tissue, age, disease and the like. Even in one proteoglycan molecule, the GAG chain has various structures.

[0003] Such various GAG structures are generated, aged,
It is known that it changes due to lesions such as canceration and Alzheimer's disease, and is considered to be closely related to life phenomena. In particular, it has recently become known that a specific GAG ultrastructure specifically binds to a specific protein.
Attention has been paid. In other words, it is thought that the GAG chain has a specific fine structure, and important bioactivity is cultivated by binding of a bioactive protein that specifically acts on the fine structure. Therefore, it is very important clinically to measure the GAG chain while distinguishing various structures of the GAG chain. In fact, fluctuations of various GAG chains in blood and tissues can cause diseases (arthritis, hepatitis, nephritis, Alzheimer, cancer, dermatitis,
(Inflammation, parturition, etc.). For example, in arthritis and cirrhosis, it is said that the concentration of hyaluronic acid in the blood increases, and that certain heparan sulfate changes due to nephritis, diabetes, Alzheimer's disease, and canceration.

On the other hand, phosphatidylethanolamine (P) is a phospholipid at the reducing end of glycosaminoglycan.
E) was developed by covalent bonding
80201, J. Biol. Chem. (1993) 268, 15779-15787), and the phospholipid-bound glycosaminoglycan derivative (hereinafter, also referred to as GAG-PE) is solid-phased as a culture substrate in a plastic culture dish. Culturing and culturing hepatocytes and using them to make spherical agglomerates (spheroidization) to create artificial livers etc. (Japanese Patent Laid-Open No. 5-236951) or to produce antibodies against hyaluronic acid (HA) Immunizing mice with HA-phosphatidylethanolamine in which HA and phosphatidylethanolamine are covalently bound as antigens,
An example in which a product obtained by dissolving HA-phosphatidylethanolamine in distilled water was used as an antigen solution in order to measure the titer of an antibody present in the serum of an immunized mouse (Japanese Patent Application Laid-Open No.
12600). It is also known that this phospholipid-linked glycosaminoglycan derivative is hydrophobically bound to tissues such as blood vessel walls and articular cartilage, cell surfaces, etc., and exhibits various biological and pharmacological activities (Japanese Patent Application Laid-Open No. Hei 4 -80201, JP-A-4-80202,
JP-A-4-82836, JP-A-6-72893). Furthermore, a method for directly immobilizing hyaluronic acid, which is a kind of glycosaminoglycan, and measuring hyaluronic acid in a sample (JP-A-63-15066)
9, Cancer Research 57,773-777, February 15, 1997)
Although all of the above are known, any of the above-mentioned methods is used for immobilizing glycosaminoglycan by binding to a solid surface at a lipid residue site of lipid-bound glycosaminoglycan and measuring GAG or a GAG-binding molecule. There is no mention that it was used.

[0005] Various attempts have been made to measure the GAG concentration in body fluids such as blood to assist in diagnosis, and some hyaluronic acids are actually marketed as diagnostic agents. In many of them, a GAG-binding protein or an anti-GAG antibody is applied to an immunoassay plate such as an ELISA, and a sample containing GAG is reacted therewith, and GAG bound to the immobilized GAG-binding molecule is further labeled. A method of measuring the concentration of GAG in a sample by binding to a GAG-binding molecule is generally used (see Japanese Patent Publication No. 6-41952). Attempts have also been made to apply hyaluronic acid directly to the plate.However, high-molecular-weight hyaluronic acid is treated with a high-concentration solution to be adsorbed on the plate, and the hyaluronic acid adsorbed on this plate and the hyaluronic acid in the sample are removed. Hyaluronic acid binding protein (HA
BP) with a hyaluronic acid binding region,
Hyaluronic acid concentration is measured (AJ Fosang et al.,
Matrix, (1990) 10, 306-313).

Although the former method has relatively good sensitivity, the measurement result largely depends on the molecular weight of the GAG chain, and the relatively low-molecular GAG has a disadvantage that the reactivity is extremely poor. Is applied to the plate by using the non-specific adsorption phenomenon (actually, it is considered to be the adsorption of the protein mixed with the hyaluronic acid sample and the plate). There is a problem that a high concentration (100 μg / ml) of hyaluronic acid is required for the treatment, and the amount of adsorption is not stable and the results are often varied. In addition, even with other GAGs of relatively low molecular weight, even when the same type of treatment is performed, no adsorption phenomenon to the plate is observed at all, and a measurement method cannot be established by this method.

[0007] In addition, since specifying or estimating the structure of GAG present in a specimen can provide useful information for diagnosing a disease or the like, a simple analysis method is desired.

[0008]

SUMMARY OF THE INVENTION The present invention provides a method for stably measuring glycosaminoglycan or a glycosaminoglycan-binding molecule in a sample by a simple operation, and a kit used therefor. Aim.

[0009]

SUMMARY OF THE INVENTION The present invention comprises the following constitutions, which can solve the above-mentioned problems. 1. A method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule, comprising using a carrier having lipid-bound glycosaminoglycan immobilized on the surface of the carrier.

[0010] 2. 2. The method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule according to 1 above, wherein the lipid-bound glycosaminoglycan is covalently bonded to a lipid at a reducing end of the glycosaminoglycan. 3. 3. The method for measuring a glycosaminoglycan or a glycosaminoglycan-binding molecule according to the above 1 or 2, wherein the glycosaminoglycan constituting the lipid-bound glycosaminoglycan is hyaluronic acid or chondroitin sulfate.

4. The glycosaminoglycan constituting the lipid-bound glycosaminoglycan has a molecular weight of 3 kDa to 200 kDa.
The above-mentioned 1 characterized by being a hyaluronic acid of KDa
4. The method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule according to any one of the above items 3. 5. 5. The glycosaminoglycan or glycosamily according to any one of the above items 1 to 4, which comprises a step of reacting the lipid-bound glycosaminoglycan fixed to the carrier with a glycosaminoglycan-binding molecule. A method for measuring a noglycan binding molecule.

6. Glycosaminoglycan binding molecule,
The method for measuring a glycosaminoglycan or a glycosaminoglycan-binding molecule according to any one of the above items 1 to 4, which is an anti-glycosaminoglycan antibody or a glycosaminoglycan-binding protein. 7. The glycosaminoglycan according to any one of the above items 1 to 4, wherein the glycosaminoglycan-binding molecule is one that is directly labeled with a labeling substance or that can be indirectly labeled with a labeling substance. Or a method for measuring a glycosaminoglycan binding molecule.

8. A kit for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule, comprising a carrier having a lipid-bound glycosaminoglycan fixed to the surface of the carrier. 9. A glycosaminoglycan measurement kit comprising at least a reagent for binding a lipid to a glycosaminoglycan to be measured and a carrier for fixing the lipid-bound glycosaminoglycan.

10. 10. The glycosaminoglycan measurement kit according to the above 9, further comprising an antibody against chondroitin sulfate, heparan sulfate antibody, keratan sulfate, or a degradation product thereof. 11. A probe for analyzing or retrieving glycosaminoglycan-binding molecules, comprising a carrier having lipid-linked glycosaminoglycans fixed to the surface of the carrier.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, the use of lipid-linked glycosaminoglycans allows GAG to be efficiently fixed to a carrier such as an ELISA plate, and the GAG binding property is improved. By combining with a substance (such as a GAG-binding protein or an anti-GAG antibody), it has been found that GAG or a GAG-binding molecule can be measured stably and with high sensitivity, and that GAG can be analyzed. In particular, it has been found that by attaching a lipid to low-molecular-weight GAG, it becomes possible to efficiently adhere to a carrier such as a plate.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. In the present invention, `` measurement of glycosaminoglycan or glycosaminoglycan binding molecule '' means, not only quantitatively or qualitatively measuring a known glycosaminoglycan or glycosaminoglycan binding molecule, The type, binding property, etc. of unknown glycosaminoglycan or glycosaminoglycan-binding molecule, using a plurality of known glycosaminoglycan-binding molecules or glycosaminoglycans that may bind thereto, It is used as a concept that includes analysis and search.

In the present invention, "glycosaminoglycan-binding molecule" and "glycosaminoglycan-binding molecule" are used as terms representing the same concept. Specifically, an anti-heparan sulfate antibody An anti-GAG antibody, which is an immunoglobulin obtained by immunizing an animal with glycosaminoglycan or a fragment thereof, such as an anti-chondroitin sulfate antibody, an anti-keratan sulfate antibody or the like, and having a specific binding affinity to these; Hyaluronic acid-binding protein (HABP; hyaluronic acid-binding domain) having an acid-binding property, fibroblast growth factor (FGF) having a binding property to CD44 and heparan sulfate / heparin, hepatocyte growth factor (HGF), etc. , Which exhibit binding affinity for GAGs but are not antibodies but include GAG binding proteins. The present invention provides a lipid-linked glycosaminoglycan (hereinafter, referred to as
"L-GAG"), a plate for measuring the duty,
Method for measuring GAG or GAG-binding molecule in a specimen using a plate in which lipid residues are bound to a carrier such as beads, gels, tubes, strips, and membranes by acting as anchor molecules, and a kit for the measurement Is provided. Further, the carrier to which the L-GAG is fixed is also used as a probe effective for analysis and search of a novel GAG-binding molecule.

GAGs constituting L-GAG include hyaluronic acid (HA), chondroitin, chondroitin sulfate (CS) (eg, chondroitin sulfate A, dermatan sulfate (chondroitin sulfate B), chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate). Sulfate E, chondroitin sulfate K, chondroitin polysulfate, etc.),
Heparin (Hep), heparan sulfate (HS), keratan sulfate (KS), keratan polysulfate, or a GAG fragment obtained by decomposing the GAG with a GAG-degrading enzyme (eg, a non-reducing terminal unsaturated oligosaccharide); The lipid is not particularly limited as long as it has a functional group capable of binding GAG directly or via a spacer and can be fixed to a carrier, but a lipid or a phospholipid having a primary amino group is preferable. -(Α-phosphatidyl) ethanolamine, D, L-phosphatidyl-L-serine,
Ethanolamine plasmalogen, serine plasmalogen, stearylamine, palmitylamine, sphingosine and the like can be used. In particular, phosphatidylethanolamine is preferable as the lipid. In the examples herein, dipalmitoyl phosphatidylethanolamine (PE) was used.

The GAG constituting the L-GAG used in the present invention is appropriately selected according to the purpose of measurement. For example,
When used for quantification of a specific known GAG, the GA
The same GAG as G or a structurally similar GAG should be used, especially when measuring GAGs in a specific molecular weight range, GAGs in that molecular weight range should be selected.

When used for quantification of a specific known GAG-binding molecule, GAG having a type and / or molecular weight that specifically binds to the molecule should be selected. For example, when quantifying an antibody against an unsaturated disaccharide of a certain GAG, a GAG whose non-reducing end is an unsaturated analogous disaccharide having a specific binding affinity for the antibody is used. Furthermore, when analyzing or searching for an unknown GAG-binding molecule, one or more GAGs whose molecules are expected to have a binding affinity are bound to a carrier according to the present invention. It is necessary to prepare.

Furthermore, in order to analyze the disaccharide repeating structure of GAG, for example, to analyze the position of a sulfate group in a disaccharide unit, the GAG to be analyzed is bound to a carrier according to the present invention. In this case, the GAG fixed to the carrier is analyzed using an anti-GAG antibody or the like whose binding specificity is clear. In the present invention, L-GAG may have a structure in which GAG and a lipid are covalently bonded. Preferred structures include a carboxyl group (including lactone), a formyl group, and a carboxyl group of GAG.
A hydroxyl group or a primary amino group, or the group separately introduced into GAG by a chemical treatment, and a carboxyl group of a lipid;
An acid amide bond (—CONH) formed between a formyl group or a primary amino group, or the group separately introduced into a lipid.
-), An ester bond or an aminoalkyl bond (—CH ₂ N
It is a structure covalently bonded by H-). Above all, G
AGs having a structure in which a pre-group formed or introduced into the reducing terminal sugar of the AG and a primary amino group of the lipid are preferable.

The method for producing the above-mentioned phospholipid or L-GAG is not particularly limited, and a known synthesis method (for example, JP-A-4-8020)
1, JP-A-4-80202) can be employed. A typical synthesis method will be described below.

<Reducing Terminal Limited Oxidation Method>
The xylose residue, galactose residue, uronic acid residue or hexosamine residue, which is the sugar residue at the reducing end of G, is reduced and subjected to limited oxidation (partial oxidation) to specifically open the pyranose ring at the reducing end. And the GAG
A formyl group is formed at the reducing end of the compound to form an aldehyde compound, the formyl group of the aldehyde compound is reacted with a primary amino group of lipid to form a Schiff base, and then the Schiff base is reduced to form an aminoalkyl bond. Then, the GAG and the lipid are covalently bonded.

<Reducing terminal lactonization method>
By oxidizing a xylose residue, a galactose residue, a uronic acid residue or a hexosamine residue that is a sugar residue at the reducing end of AG, the pyranose ring at the reducing end is specifically opened to reduce the reducing end of the GAG. By subjecting the reducing end of the GAG to a lactone structure and reacting the lactone with a primary amino group of a lipid to form an acid amide bond, This is a method of covalently bonding GAG and lipid.

In the examples of the present specification, L-GAG was prepared by the reducing terminal lactone method. The carrier to which L-GAG is fixed is not particularly limited as long as it has a hydrophobic surface that can be fixed to lipid by hydrophobic interaction. As the carrier, for example, a plate (E
LISA and other immune measurement plates, etc.), beads, gels, tubes, strips, membranes and the like. Examples of the material include polystyrene, polypropylene, nylon, polyacrylamide, cellulose, nitrocellulose, and the like. In particular, a material that can form a hydrophobic bond with lipid and has a hydrophobic surface is preferable. In particular, it is preferable to use a plate as a carrier because the measurement procedure is simpler.

As a method for fixing L-GAG to these carriers, a general method used for immobilizing lipids can be applied. In particular, hydrophobic bonding is preferred, and in this embodiment, a hydrophobic bonding method was used. Further, the portion where L-GAG is not fixed may be, and preferably is, blocked by serum albumin, gelatin, casein, skim milk or the like.

The method of measuring GAG using the method of the present invention is as follows.
The L-GAG fixed to the carrier and the GAG in the sample are GA
GAG reacts competitively with G-binding substances and
This is a method of measuring GAG in a sample by detecting a GAG-binding substance bound to L-GAG in inverse proportion to the concentration. For example, it can be performed by the following steps, but is not limited thereto. In particular, using a plate as a solid surface, an ELISA (Enzyme-
Linked ImmunosorbentAssa
An example of applying the method y) will be described. 1) L-GAG is fixed to an ELISA plate. 2) A mixture of a sample for which GAG is to be measured and a fixed amount of an anti-GAG antibody is applied to the plate, and L-GAG on the plate and GAG in the sample are allowed to react competitively with the anti-GAG antibody. 3) After the reaction, the plate is separated from the sample and washed. 4) A certain amount of a secondary antibody directly labeled with a labeling substance is applied to the plate and reacted with the anti-GAG antibody. 5) The amount of GAG in the sample is calculated from the quantification of the labeled secondary antibody bound to L-GAG fixed to the plate.

In the above ELISA method, the anti-G
Anti-GAG directly labeled with a label instead of AG antibody
Using an antibody, quantify the labeled anti-GAG antibody bound to L-GAG on the plate. Using a GAG binding protein directly labeled with a labeling substance instead of the anti-GAG antibody, bind to L-GAG on the plate For example, a method of quantifying the labeled GAG-binding protein may be employed. further,
In each of the above methods, instead of direct labeling, for example, a specific antibody: a ligand for a receptor binding pair, biotin, is used as a secondary antibody, an anti-GAG antibody,
Use the protein bound to the binding protein, etc.
-An indirect labeling method may be employed in which the biotin bound to GAG is detected by binding a labeling substance to a binding substance such as apidine, streptapidine or the like.

In the above method, the type of the labeling substance and the detection method thereof are not particularly limited, but the following methods are exemplified. The labeling substance is not usually limited as long as it can be used for labeling proteins, but enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), isotopes ( ¹²⁵ I,
¹³¹ I, ³ H, etc.), fluorescent substances (fluorescein isothiocyanate, umbelliferone, etc.), chemiluminescent substances (luminol, etc.), avidin, biotin and the like.

Known methods can be used for the labeling method. For example, when labeling with an enzyme, for example, glutaraldehyde method, periodic acid crosslinking method, maleimide crosslinking method, carbodiimide method, activated ester method, etc. When labeling with an isotope, the chloramine T method, lactoperoxidase method, etc. (Sequence Chemistry Experiment Course 2 “Protein Chemistry (bottom)”,
Tokyo Kagaku Dojin, published in 1987).

The method for detecting the labeling substance differs depending on the labeling substance used. For example, a fluorescent substance as a labeling substance,
When a chemiluminescent substance is used, a method of measuring fluorescence or luminescence of a solution after the reaction may be used. The case where an enzyme is used as a labeling substance will be described. When peroxidase is used as the enzyme, after the reaction of the sample, the activity of the peroxidase bound to the separated plate is added with a coloring agent O-phenylenediamine or tetramethylbenzidine and an aqueous hydrogen peroxide solution, and the amount of coloring is measured by an absorptiometer. And the like.

The concentration of GAG in the sample is determined by preparing a calibration curve in advance using the GAG standard solution whose concentration is known and measuring the relationship between the GAG concentration of the standard solution and the detection intensity of the GAG-binding substance. In advance, it can be measured by a method using the detection intensity of the labeling substance corresponding to the GAG-binding substance in the case of using the sample and the above-mentioned calibration curve. The ELISA method is an example of the measurement method of the present invention, and the GAG-binding substance used in the method of the present invention is not limited to an anti-GAG antibody or a GAG-binding protein.

Examples of the anti-GAG antibody include antibodies against various GAGs, and polyclonal antibodies, monoclonal antibodies, and fragments of these antibodies can be used. Monoclonal antibodies are preferred in terms of specificity and stable supply.
In addition, by using GAGs constituting L-GAGs isolated from tissues or cells of a disease such as a tumor or Alzheimer's disease, diagnosis or contribution of various diseases can be achieved. Similarly, G is strongly correlated with a certain disease.
By selecting AG, the diagnosis of the disease or its contribution becomes possible.

In the present invention, the GA for which the structure is to be analyzed
G is attached to a lipid, fixed to a carrier, and the above-described measurement method is repeated using a plurality of different monoclonal antibodies, whereby GAG and L-GAG, which are known antigenic determinants of monoclonal antibodies, are obtained. The analysis of GAGs constituting L-GAG can be performed based on the relationship such as the difference in binding force to GAG. For example, the composition, sequence, structure, etc. of the constituent sugars of GAG can be estimated or contributed to.

In the above method, when an enzymatic degradation product of a certain GAG, for example, an antibody against an unsaturated disaccharide is used, L
After the GAG is fixed to the carrier, the LAG is treated with an enzyme that decomposes the GAG constituting the L-GAG into an unsaturated disaccharide, for example, chondroitinase ABC and AC, and the non-reduced GAG is fixed. Anti-GA as terminal unsaturated disaccharide
A method for analyzing the reactivity with the G antibody is exemplified.

When HABP is used as a measuring substance, L-GA
HA constituting G preferably has a molecular weight of 3 to 20.
Using 0 kDa HA, more preferably 10 to 30 kD
Use a. The sample to be subjected to the measurement method of the present invention is not particularly limited as long as it is a liquid that can contain GAG or a GAG-binding molecule. In particular, a body fluid sample (urine, blood, plasma, serum, etc.) taken out of a living body , An organ extract, a cell extract or a culture supernatant.

When a novel GAG-binding molecule is searched for using a carrier to which L-GAG having a known structure is immobilized as a probe, any unknown sample solution can be subjected to various conditions (pH,
Ionic strength, etc.) to contact the L-GAG
After binding the unknown substance to AG, the unknown substance is eluted under various conditions, and if necessary, isolation is performed by using another separation means in combination. The isolated unknown substance is specified by a known analysis means.

[0038]

The present invention will be described specifically with reference to the following examples. Embodiment 1 FIG. Reactivity between various GAG-PE and monoclonal antibody MO225 Various GAG-, ie, chondroitin sulfate derived from shark cartilage [CS (S) or simply CS (composition ratio of disaccharide unit: -HexA-
GalNAc (6-SO ₃ ⁻ ) -residue (hereinafter, 6S); 72,9%, -HexA-GalNAc
(4-SO ₃ ^{-) -} residue (hereinafter, 4S); 15.4%, -HexA (2-SO 3 -) -GalN
Ac (6-SO ₃ ⁻ ) -residue (hereinafter diSD); 9.3% (where HexA:
Hexuronic acid, GalNAc: refers to N-acetylgalactosamine. (6-SO ₃ ⁻ ) is a sulfate group at the 6-position, and (4-SO ₃ ⁻ ) is 4
Sulphate group, (2-SO ₃ ⁻ ) indicates the sulphate group at position 2), weight average molecular weight 20,000)), chondroitin sulfate derived from bovine tracheal cartilage [CS (T) (6S; 39.3%, 4S; 54, 9%, diSD; 0.4%,
Weight average molecular weight 12,000), whale cartilage-derived chondroitin sulfate [CS (W) (6S; 19.3%, 4S; 76.2%, diSD; 2.7
%, Weight average molecular weight 13,000)], pig kidney-derived dermatan sulfate [DS (4S; 91.3%, weight average molecular weight 16,000)],
Hyaluronic acid [HA (weight average molecular weight 2
3000, and limited hydrolysis with bovine testicular hyaluronidase, preparations were purified by molecular sieve)], porcine intestinal heparin _{[Hep (-HexA (2-SO 3} -) -GlcN-SO 3 - (6-SO 3 -) - residues (hereinafter, Tris); 68.8% (here, GlcN-SO ₃ ^- shows the N- glucosamine sulfate), manufactured by a weight average molecular weight 10,000)] (all Seikagaku Corporation) - as a raw material A conjugate (GAG-PE) of each phospholipid (dipalmitoyl phosphatidylethanolamine; PE, manufactured by NOF Corporation) was synthesized according to the method of Example 1 of Japanese Patent Application Laid-Open No. 4-8021 (CS (S) -PE or simply CS-PE, CS (T) -PE, CS (W) -PE, D
S-PE, HA-PE, Hep-PE).

The GAG-PE was dissolved in phosphate buffered saline (PBS, pH 7.2) at different concentrations, and the solution was mixed with a 96-well polystyrene EL.
Each ISA plate (Sumitomo Bakelite MS-8496F)
100 μl was added. After allowing to stand at 4 ° C. overnight, the plate is washed three times with PBS to thereby fix the plate on which the GAG-PE is fixed (hereinafter, also referred to as “GAG-PE-fixed plate”. The same applies to a specific case of GAG. ) Was made. 100 µl of a PBS solution (10 mg / ml) of bovine serum albumin (BSA) was added to the plate, and a blocking treatment was performed at 37 ° C for 1 hour. After washing with PBS, 0.05% of chondroitin sulfate-specific mouse monoclonal antibody MO225 (Seikagaku Corporation) was used as the primary antibody.
(w / v) Tween 20-containing PBS (PBS-Tween) diluent (1/500) 10
Add 0 μl, treat for 1 hour at room temperature, PBS / Tween 20 (150 μl
l) was washed three times. Subsequently, 100 μl of a PBS-Tween diluent (1/500) of a peroxidase-conjugated goat anti-mouse IgM antibody (manufactured by Organon Technica), which is a secondary antibody, was added, and the mixture was treated at room temperature for 1 hour, and then PBS-Tween (150 μl) For 4 times. 0.1 mg / ml o-phenylenediamine-0.003% (V /
V) Add 150 μl of aqueous hydrogen peroxide (OPA-H ₂ O ₂ ) and
After reacting for 3 minutes, 50 μl of 3M aqueous sulfuric acid was added. 4 of reaction solution
The absorbance at 92 nm was measured with a microplate reader (IMMUNO-MINI NJ-2300, manufactured by Nippon Intermed Co., Ltd.) (see FIG. 1). Monoclonal antibody MO225 has the 6S structure of CS, ie, -HexA-Ga
Recognizes lNAc (6-SO ₃ ⁻ ) -residues, especially the diSD structure, ie, -H
^{_{exA (2-SO 3 -)}} -GalNAc (6-SO 3 -) - strongly react with residues. Therefore, it reacted very strongly with shark cartilage-derived CS. Then, the MO derived from bovine tracheal cartilage-derived CS and whale cartilage-derived CS in this order.
The reactivity with 225 decreased. In addition, dermatan sulfate (DS) derived from porcine liver showed a much lower reactivity than CS derived from whale cartilage because most of it had a 4S structure. In addition, the PE conjugate of HA, HS, and Hep without the CS sugar chain skeleton did not show any reactivity with MO225. High concentration of free CS not bound to PE (1 mg / ml; 50 nmol / ml)
Even after treatment, no reactivity with MO225 was shown, indicating that it was not fixed to the plate.

Embodiment 2 FIG. MO225 antibody reactivity due to CS molecular weight difference Chondroitin sulfate derived from shark cartilage with different molecular weight (CS;
Weight average molecular weight 46,000, 20,000, 11,000, 8,000, 4,60
0, 2,800; a sample having a molecular weight of 10,000 or more was purified from a natural product, and a sample having a molecular weight of less than 10,000 was subjected to limited digestion of bovine testis-derived hyaluronidase (manufactured by Seikagaku Corporation) and purified by molecular sieve. )
The PE conjugate was synthesized in the same manner as in Example 1, fixed to an ELISA plate at a different concentration as in Example 1, and reacted with MO225. (See FIG. 2) The higher the molecular weight, the higher the reactivity, and a significant change was observed in the reactivity at around 10,000. In addition, molecular weight 3000
The following CS-PE showed little reactivity.

Example 3 Antibody Reactivity After Treatment of CS-PE-Fixed Plate with Chondroitinase (Analysis of CS Constituting CS-PE) CS-PE (shark cartilage-derived) fixed to the plate in the same manner as in Example 2 , Weight average molecular weight 20,000, 10 μg / ml; 100 μl / wel
l) was treated at 37 ° C. for 1 hour while changing the enzyme concentration of chondroitinase ABC (C-ABC), and then, as anti-CS specimens having different specificities, anti-CS antibody MO225 and anti-unsaturated 6S disaccharide antibody 3B3, anti-unsaturated 4S disaccharide antibody 2B6, anti-unsaturated 0S disaccharide antibody 1B5 (diluted 1/500 each; Seikagaku Corporation)
Were reacted in the same manner as in Example 1 (FIG. 3).

The reaction with MO225 sharply decreased by treatment with C-ABC of 0.5 mU / well or more, supporting the result of Example 2 that a certain molecular weight or more as an antigen was required for the antigen-antibody reaction. The reactivity of the 3B3 antibody was immediately increased by the C-ABC treatment, whereas the response of the 2B6 antibody had a lower delayed peak. This is 6S from shark cartilage derived CS from 4S
Not only is the content high, but also suggests more 6S disaccharide at the non-reducing end. The one that did not react very much with the 1B5 antibody was the OS disaccharide (-HexA-G) in this CS-PE.
This may be due to the low alNAc-) content.

As described above, by converting GAG into PE, the sequence structure of GAG can be estimated. Embodiment 4 FIG. CS Concentration Measurement Using a CS-PE-fixed plate (1, 10, 100 μg / ml), the CS concentration in the sample was measured by the inhibition method using the MO225 antibody. That is, 100 μl of CS-PE PBS solution (1, 10, 100 μg / ml)
At room temperature overnight, and add 10 mg / ml BSA in PBS (100 μl
After blocking in l), the plate was washed with PBS-Tween to prepare a CS-PE fixing plate. 50 μl of PBS solution containing various concentrations of free chondroitin sulfate (CS; shark cartilage-derived weight average molecular weight 20,000) and monoclonal antibody MO225
Of PBS-Tween diluted solution (1/500) was added and mixed, and shaken at room temperature for 2 hours. After washing 4 times with PBS-Tween (150μl), peroxidase-conjugated goat anti-mouse as secondary antibody
After adding 100 μl of a diluted solution of IgM antibody in PBS-Tween (1/1000) and treating at room temperature for 1 hour, color the OPA-H ₂ O _{2 in} the same manner as in Example 1 and measure the absorbance at 492 nm using a microplate reader. (See Fig. 4) From the dose response curve of the amount of CS added and the antibody response, CS-PE 10μ
Free CS could be measured in the concentration range of 10 to 1000 µg / ml with an amount of fixation of g / ml or more. That is, it was shown that the CS concentration in the sample in this concentration range can be quantified.

The concentration of chondroitin sulfate having a different molecular weight in the specimen was also measured according to the molecular weight. Example 5. Reactivity of immobilized HA-PE with hyaluronic acid-binding protein, comparison with free hyaluronic acid Hyaluronic acid with weight average molecular weight of 104,000 and 20,000 (HA; HB in the same manner as in Example 1 using a sample that has been limitedly degraded with bovine testicular hyaluronidase and purified through a molecular sieve.
A-PE conjugate was synthesized.

In the same manner as in Example 1, these PBS solutions of HA-PE were fixed at different concentrations to a 96-well ELISA plate, and then
Blocking was performed with a mg / ml BSA solution to prepare an HA-PE-fixed plate. A biotin-labeled hyaluronic acid binding protein (bHA) from bovine cartilage proteoglycan on the plate
BP) in PBS-Tween was added at 0.4 μg / ml (100 μl), and the mixture was treated at room temperature for 2 hours. After washing three times with PBS-Tween, peroxidase-conjugated avidin (Amersham: 1 /
PBS-Tween solution (100 μl) was added, and the mixture was treated at room temperature for 1 hour, and then washed four times with PBS-Tween (150 μl). OPA-
H ₂ O ₂ reagent (150 μl) was added and reacted at room temperature for 30 minutes.
After the reaction, 3 M sulfuric acid (50 μl) was added to stop the reaction, and the absorbance at 492 nm was measured using a microplate reader (see FIG. 5).

The reaction between HA and bHABP on the HA-PE-fixed plate increases in a dose-dependent manner in proportion to the concentration of HA-PE in the PBS solution of HA-PE used for fixation, and is less affected by the HA molecular weight. , And both showed highly sensitive reactivity. It should be noted that instead of HA-PE, hyaluronic acid (HA, weight-average molecular weight 27,000) derived from low-molecular weight chicken cockscomb and hyaluronic acid (uHA, weight-average molecular weight 1,000,000) derived from human umbilical cord were used.
000) PBS solution was prepared and coated in the same manner as above. HA with low molecular weight (27,000) showed no reactivity with bHABP even at high concentration (100 μg / ml). It was shown that it was not stuck to. In the treatment of high molecular weight (1,000,000) uHA (containing 1% protein as an impurity), a reaction with HABP was observed in a high concentration region.

Embodiment 6 FIG. Measurement of HA Concentration by Fixation of HA-PE Using hyaluronic acid (HA) having a weight average molecular weight of 20,000 as a raw material, a conjugate with a phospholipid (dipalmitoyl phosphatidylethanolamine: PE, manufactured by NOF Corporation) was prepared. It was prepared by the method described in 1. The HA-PE was dissolved at different concentrations in phosphate buffered saline (PBS), and dissolved in 0.03.
μg / ml, 0.1 μg / ml, 0.3 μg / ml, 1 μg / ml, 3 μg / ml
HA-PE solutions were prepared at ml, 9 μg / ml and 27 μg / ml.

100 μl of each was added to a 96-well plate, allowed to stand at 4 ° C. overnight, and then 200 μl of PBS was used.
Wash three times and add bovine serum albumin (BS
100 μl of a PBS solution (1%) of A) was added thereto, and a blocking treatment was performed at room temperature for 2 hours.
-A PE fixing plate was prepared. HA samples of various concentrations (HA: weight average molecular weight 10,000, PBS-
50 μl of a solution serially diluted with Tween) is added, and then the biotinylated hyaluronic acid binding protein (PBS-Tw
solution diluted with een; 0.4 μg / ml), stirred, treated at 37 ° C. for 1 hour, and then PBS-Tween 200 μl
And washed three times. 50 μl of peroxidase-conjugated avidin (solution diluted with PBS-Tween) was added, and 37
The mixture was reacted at 30 ° C. for 30 minutes, and then washed three times using 200 μl of PBS-Tween. Then, OPA-H ₂ O ₂ reagent (150 μl)
After the reaction, 3 M sulfuric acid (50 μl) was added to stop the reaction, and the absorbance at 492 nm was measured (FIGS. 6 and 7).

From the dose-response curve, 10-1000 ng / m
1 could be measured in the HA concentration range. However, HA-P
Plates treated with a high concentration (3 μg / ml or more) solution of E tended to decrease the measurement sensitivity, and too low a concentration (0.1 μg / ml or less) tended to decrease the measured absorbance. Embodiment 7 FIG. Change in HA measured by HA-PE fixation depending on molecular weight HA-P was treated at a treatment concentration of 0.3 μg / ml in the same manner as in Example 6.
E (using HA having a weight-average molecular weight of 20,000) was immobilized on a plate on which hyaluronic acid (4,000, 10,000, 27,000, 53,000) having a different molecular weight was used.
00, 130,000, 300,000, 800,00
The solution with the changed concentration of 0) was used as a sample, and bHABP
(0.4 μg / ml) by the inhibition method (FIGS. 8 and 9).

As a result, a low molecular weight H having a molecular weight of 4,000
In the case of A, the reactivity tends to slightly decrease, but it is clear that even for a higher molecular weight of hyaluronic acid, there is almost no change in the reaction with respect to the concentration, and it is possible to measure HA in a wide molecular weight range. did.

Embodiment 8 FIG. HS-PE and Hep-PE
Reactivity with HS antibody HK249 Example 1 was used for each PE conjugate of heparan sulfate (HS (EHS)) derived from mouse EHS tumor, heparan sulfate (HS (PK)) derived from pig kidney, and heparin (Hep) derived from bovine small intestine. Was synthesized in the same manner as in Example 1, and fixed to a 96-well ELISA plate at a different concentration in the same manner as in Example 1, and blocked with 10 mg / ml BSA. Add 100 μl of anti-heparan sulfate monoclonal antibody HK249 (Seikagaku Corporation) to the plate
After the time treatment, the cells were washed with PBS-Tween, and then a peroxidase-conjugated sheep anti-rat immunoglobulin antibody (manufactured by Organon Technica) (100 μl) was added as a secondary antibody, and the cells were treated at room temperature for 1 hour, and then washed four times with PBS-Tween. Washed. OPA-H
_{The 2} O ₂ reagent was reacted at room temperature for 20 minutes and stopped with 3M sulfuric acid. Measure the absorbance at 492 nm with a microplate reader,
HK249 was a monoclonal antibody prepared using mouse EHS tumor-derived heparan sulfate proteoglycan as an antigen. The overall sulfate content was low and the N-sulfate residue of glucosamine (GlcN) was observed. Is an antibody that shows specificity for heparan sulfate,
Again, it showed high reactivity with EHS-derived HS-PE. Reactivity to HS-PE and Hep-PE derived from pig kidney was low.
It has been reported that HK249-positive heparan sulfate appears and increases in Alzheimer's and cancerous tissues, and the method of the present invention may be effective for the diagnosis thereof.

[0052]

The present invention relates to an L-GAG fixed to a carrier.
And the GAG binding substance,
Of blood, urine, cerebrospinal fluid,
GAG content in body fluids such as wound infiltration fluids can be measured. It is known that the amount of GAG in body fluids fluctuates in arthritis, hepatitis, nephritis, Alzheimer's, cancer, dermatitis, inflammation, parturition, and the like. Available to

In the present invention, the use of L-GAG, in which GAG of unknown or insufficient structure is used as a lipid conjugate, facilitates the adhesion to a plate and facilitates the adhesion to the plate.
By reacting GAG with a GAG-binding substance (anti-GAG antibody, GAG-binding protein, etc.), its GAG structure can be estimated. Furthermore, the L-GAG carrier used in the present invention can be a probe, and is a novel GAG-binding molecule.
It is used for searching for a physiologically active substance that has a binding property to the protein.

[Brief description of the drawings]

FIG. 1 shows the reaction curves of various GAG-PEs and monoclonal antibody MO225 in Example 1.

FIG. 2 shows CS-PE having different molecular weights in Example 2.
Fig. 4 shows a reaction curve between the antibody and the monoclonal antibody MO225.

FIG. 3 shows the reaction curves of CS and various antibodies after treating chondroitinase with varying concentrations on CS-PE-fixed plates in Example 3.

FIG. 4 shows a dose response curve for CS concentration measurement in Example 4.

FIG. 5 shows that in Example 5, immobilized HA-PE or free HA
FIG. 4 is a reaction curve showing the reactivity of a plate coated with and a hyaluronic acid binding protein.

FIG. 6 shows the response curve of the HA dose in the HA-PE-fixed plate prepared at various HA-PE coating concentrations in Example 6.

FIG. 7 shows the response curve of the HA dose in the HA-PE-fixed plate prepared at various HA-PE coating concentrations in Example 6.

FIG. 8 shows a response curve of an HA dose in an HA-PE-fixed plate using various HAs having different molecular weights in Example 7.

FIG. 9 shows a response curve of HA dose in an HA-PE-fixed plate using various HAs having different molecular weights in Example 7.

FIG. 10 shows the reaction curves of various GAG-PE and monoclonal antibody HK249 in Example 8.

Claims (11)

[Claims] 1. A method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule, comprising using a carrier having lipid-bound glycosaminoglycan immobilized on the surface of the carrier. 2. The glycosaminoglycan or glycosaminoglycan-binding property according to claim 1, wherein the lipid-bound glycosaminoglycan is covalently bound to a lipid at the reducing end of the glycosaminoglycan. How to measure molecules. 3. The glycosaminoglycan or glycosaminoglycan-binding property according to claim 1, wherein the glycosaminoglycan constituting the lipid-bound glycosaminoglycan is hyaluronic acid or chondroitin sulfate. How to measure molecules. 4. The glycosaminoglycan constituting the lipid-bound glycosaminoglycan has a molecular weight of 3 kDa to 200 k.
It is a hyaluronic acid of Da, The claim 1 characterized by the above-mentioned.
4. The method for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule according to any one of the above items 3. 5. The glycoprotein according to claim 1, further comprising a step of reacting the lipid-bound glycosaminoglycan fixed to the carrier with a glycosaminoglycan-binding molecule. A method for measuring a saminoglycan or a glycosaminoglycan-binding molecule. 6. The glycosaminoglycan or glycosaminoglycan according to claim 1, wherein the glycosaminoglycan binding molecule is an anti-glycosaminoglycan antibody or a glycosaminoglycan binding protein. A method for measuring a glycan-binding molecule. 7. The glycosaminoglycan-binding molecule according to claim 1, wherein the molecule is directly labeled with a labeling substance, or is indirectly labeled with a labeling substance. The method for measuring glycosaminoglycan or glycosaminoglycan binding molecule according to the above. 8. A kit for measuring glycosaminoglycan or a glycosaminoglycan-binding molecule, comprising a carrier having lipid-bound glycosaminoglycan fixed to the surface of the carrier. 9. A glycosaminoglycan measurement kit comprising at least a reagent for binding lipid to glycosaminoglycan to be measured and a carrier for fixing lipid-bound glycosaminoglycan. 10. The kit for measuring glycosaminoglycan according to claim 9, further comprising an antibody against chondroitin sulfate, heparan sulfate antibody, keratan sulfate, or a degradation product thereof. 11. A probe for analyzing or retrieving glycosaminoglycan-binding molecules, comprising a carrier having lipid-linked glycosaminoglycans fixed to the surface of the carrier.