JPH10153600A - Method for measuring ligand - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method, whereby two ligands in the same specimen of even a small amount can be measured simultaneously and simply with good sensitivity, and a solid phase body, and a measuring kit used in the method. SOLUTION: A solid phase body to which both a first receptor and a composite body of a second receptor, a first ligand and the first receptor adhere and, the composite body adheres via the first receptor, and a specimen including the first ligand and a second ligand are brought in touch with each other. A formation of a composite body of the first receptor and first ligand, and a formation of a composite body of the second receptor and second ligand are detected respectively. The first ligand and second ligand in the specimen are thus measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring two kinds of ligands, a solid phase and a kit used for the method.

[0002]

2. Description of the Related Art Conventionally, as a multi-item simultaneous immunoassay for simultaneously measuring various ligands, two types of antibodies are coated on the inner wall of a bead and a test tube in an enzyme immunoassay (EIA) using beads, respectively. , A method of separately measuring two kinds of ligands bound to beads and a test tube (Japanese Patent Publication No. 59-2346) by a sandwich method using a solid phase in which two or more kinds of antibodies are respectively directly fixed to a solid phase. A method for measuring various kinds of antigens in a sample (JP-A-4-232465) is known.

[0003] Also, various kinds of antibodies labeled with fluorescein isothiocyanate (FITC) were all used in anti-FITC.
A method of measuring the total amount of antigen in a sample by a sandwich method using a solid phase immobilized via an antibody is known. In this assay, antibodies are fixed to a solid phase via an anti-FITC antibody for the purpose of increasing the amount of various types of antibodies bound to the solid phase (JP-A-2-2285).
No. 61).

[0004] Keratan sulfate and hyaluronic acid are glycosaminoglycans constituting a proteoglycan aggregate which is one of the constituents of cartilage. In vivo, they are used as components of proteoglycans and components capable of binding to proteoglycans. Plays an important role.
These two components are closely related to each other. Therefore,
It is desired to simultaneously measure the concentrations of these two components as in vivo information indicating cartilage metabolism in joint diseases such as osteoarthritis and rheumatoid arthritis, or for clinical applications.

As a method for measuring hyaluronic acid, a labeled substance of a complex sandwiching hyaluronic acid between a hyaluronic acid-binding protein immobilized on a solid phase and a hyaluronic acid-binding protein labeled with a labeling substance is detected. For measuring hyaluronic acid (Japanese Patent Application Laid-Open No. 64-469)
No.), a method for measuring hyaluronic acid by competitively binding a labeled hyaluronic acid binding protein to hyaluronic acid bound to a solid phase and a sample hyaluronic acid to form a complex (Japanese Unexamined Patent Publication No. No. 150669), after reacting cartilage proteoglycan containing a hyaluronic acid binding region with hyaluronic acid in a sample, adding the proteoglycan not bound to the hyaluronic acid to a plate on which hyaluronic acid is fixed, A method for measuring hyaluronic acid by an inhibition method, which comprises measuring the fixed proteoglycan (Japanese Patent Application Laid-Open No. 64-29767); and immobilizing chondroitinase ABC-treated cartilage proteoglycan on a solid phase, and sandwiching the hyaluronic acid by a sandwich method. (JP-A-4-262797) is known.

As a method for measuring keratan sulfate, a sample containing keratan sulfate and keratan sulfate labeled with a labeling substance are added to a solid phase on which a monoclonal antibody which is an anti-keratan sulfate antibody is immobilized. Is competitively bound to the antibody, and the labeled substance of the conjugate of the antibody and keratan sulfate labeled with the labeled substance is detected to determine the keratan sulfate in the sample (Japanese Patent Application Laid-Open No. 60-22 / 1985).
No. 4065, Clinica. Chimica. Acta, 195 (1990) p17-
26), and a sandwich method using a monoclonal antibody of an anti-keratan sulfate antibody (WO 90/07120).

[0007]

However, 1) In the conventional method of binding various kinds of antigens to various kinds of antibodies directly fixed on the solid phase, it is necessary to simultaneously fix many kinds of antibodies directly on the solid phase. However, adsorption between a protein and a solid phase is highest when the pH is the pI value of the protein, and it is difficult to fix two or more types of antibody proteins to the solid phase under optimal conditions. Therefore, there were problems such as low measurement sensitivity or inconsistent measurement results.

2) A conventional method for measuring the total amount of antigen in a sample by a sandwich method using a solid phase in which all kinds of antibodies labeled with FITC are immobilized via an anti-FITC antibody, FIT for all kinds of antibodies
It had to be labeled with C, which was complicated. In addition, since the pIs of the various FITC-labeled antibodies are different from each other, it has been difficult to fix various FITC-labeled antibodies under optimal conditions.

In the conventional method for measuring the glycosaminoglycan hyaluronic acid and keratan sulfate using a hyaluronic acid-binding protein and an anti-keratan sulfate antibody, one type of glycosaminoglycan is used for one sample at a time. Because only a small amount can be measured, 1
In order to measure many kinds of glycosaminoglycans in a sample, even if the amount of the sample can be measured, or the problem that only one kind of glycosaminoglycan can be measured, each glycosaminoglycan must be measured using a different measurement system. In this case, there is a problem that the measurement is complicated. Therefore, two kinds of ligands in the same sample can be simultaneously and simply measured with high sensitivity, and a measurement method with stable measurement results has been desired.

[0010]

The present invention provides: 1) a solid phase body having both a first receptor and a second receptor-first ligand-first receptor complex fixed thereto; The solid body to which the complex is fixed via the first receptor is brought into contact with a sample containing the first ligand and the second ligand to form a complex between the first receptor and the first ligand, and A method for measuring the first ligand and the second ligand in the sample by detecting the formation of a complex between the two receptors and the second ligand, respectively; 2) a solid phase body to which the first receptor is fixed and a second receptor The body-first ligand-first receptor complex is immobilized via the first receptor, and the solid phase body is contacted with a sample containing the first ligand and the second ligand, and the first receptor and the first receptor are contacted. Formation of a complex with one ligand and formation of a complex of a second receptor and a second ligand Method of measuring the first ligand and second ligand in the sample by detecting respectively;

3) A solid phase in which both the first receptor and the second receptor-first ligand-first receptor complex are fixed, and the complex is formed via the first receptor. 4) The solid phase comprises an immobilized solid phase, a first receptor labeled with a first labeling substance, and a second receptor labeled with a second labeling substance.
A kit for measuring the first ligand and the second ligand in the sample; and 5) a solid phase body to which the first receptor is fixed, and the second receptor-first ligand-first receptor complex Measurement of a first ligand and a second ligand in a sample, comprising a solid body fixed via the first, a first receptor labeled with a first labeling substance and a second receptor labeled with a second labeling substance For kits.

In the present specification, the second receptor—the first ligand—of the first receptor complex—is formed by a covalent bond or a hydrogen bond, a bond by an electrostatic interaction, or a van der Waals force. A non-covalent bond such as a bond or a hydrophobic bond is meant.

The present invention will be described in more detail below. 1) The first receptor used in the present invention is a molecule capable of specifically binding a first ligand. For example, a molecule capable of binding with a specific affinity for the first ligand can be used. As the combination of the first ligand and the first receptor, in addition to the combination of the antigen and the antibody, sugar and lectin,
Examples thereof include a combination of a polysaccharide and a polysaccharide-binding protein, and a combination of an agonist and an agonist receptor.

Examples of the combination of an antigen and an antibody include a combination of glycosaminoglycan and an anti-glycosaminoglycan antibody. More specifically, for example, keratan sulfate and an anti-keratan sulfate antibody, and chondroitin sulfate and an anti-glycosaminoglycan antibody are used. Examples include chondroitin sulfate antibodies, heparan sulfate and anti-heparan sulfate antibodies.

The anti-keratan sulfate antibody, anti-chondroitin sulfate antibody and anti-heparan sulfate antibody are immunized with proteoglycan or the like containing keratan sulfate, chondroitin sulfate or heparan sulfate as antigens by a known method, and are immunized against these antigens by a known method. It can be obtained by producing polyclonal or monoclonal antibodies (for anti-keratan sulfate antibodies, see J. Biol. C
hem. 258 (1983) p8848-8854, for anti-chondroitin sulfate antibody, see J. Biol. Chem., 262 (1987) p4146-415.
2, For anti-heparan sulfate antibody, see J. Immunol., 13
7, (1986) p3900-3908).

A commercially available product can also be used. Commercially available products include anti-keratan sulfate (trade name) (clone name: 5-D-4, manufactured and sold by Seikagaku Corporation)
Chondroitin sulfate (Type-D) (trade name) (clone name: MO-225, manufactured and sold by Seikagaku Corporation),
Anti-heparan sulfate (trade name) (clone name: HepSS
-1, Seikagaku Corporation production and sales).

2) The second receptor of the present invention is a molecule capable of specifically binding a second ligand. As the combination of the second ligand and the second receptor, in addition to the combination of the antigen and the antibody, a sugar and a lectin, a polysaccharide and a polysaccharide-binding protein (eg, glycosaminoglycan and a glycosaminoglycan-binding protein, etc.) And combinations of agonists and agonist receptors.

3) Examples of the solid phase material used in the present invention include microplate wells, tubes, membranes, beads, and gels. These may be used in combination. Examples of the material include polystyrene, polypropylene, polyvinyl chloride, nitrocellulose, polyallomer, polyethylene, nylon, glass, agarose, and the like.

4) a first receptor used in the present invention;
A solid phase to which both the second receptor-first ligand-first receptor complex is anchored, wherein the complex is anchored via the first receptor; and The solid-phase body to which the receptor-first ligand-first receptor complex is fixed via the first receptor is, for example, brought into contact with the solid-phase body and the first receptor to form the first solid-phase on the solid phase. After fixing the receptor, the first ligand is further contacted with the second receptor to which the first ligand is bound, and the first receptor fixed to the solid phase and the first ligand bound to the second receptor are separated. By binding with a specific affinity, it can be produced by fixing the second receptor-first ligand-first receptor complex to the solid phase via the first receptor. It is preferable that the obtained solid phase is further contacted with a first receptor to block a portion of the first ligand which is not bound to the first receptor fixed to the solid phase.

As a method for fixing the first receptor to the solid phase, a general method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bonding method, an entrapment method ("Immobilized enzyme", 1975)
Yearly publication, see Kodansha Co., Ltd., pp. 9-75). In particular, the physical adsorption method is preferred in terms of simplicity. Preferably, the surface of the solid phase to which the first receptor is not fixed is blocked by serum albumin, gelatin or the like.

Further, in the case of the above-mentioned solid body in which both the first receptor and the second receptor-first ligand-first receptor complex are fixed, the first receptor fixed to the solid body is used. A portion of the body must remain unbound with the second receptor to which the first ligand has bound. That is, when the first receptor is an antibody, the antigen binding site of some antibodies must not be saturated. For this reason, the amounts of the first receptor and the second receptor to which the first ligand is brought into contact with the solid phase must be determined in advance by study, but this can be easily performed by those skilled in the art. Can be determined.

In a preferred embodiment of the solid phase used in the present invention, the first ligand and the second ligand are different glycosaminoglycans, and the first receptor is a glycosaminoglycan having the first ligand. The solid phase body is a protein capable of specifically binding glycan, and the second receptor is a protein capable of specifically binding glycosaminoglycan as the second ligand. More specifically, a solid phase in which the first receptor is an antibody against glycosaminoglycan and the second receptor is a glycosaminoglycan binding protein.

As a method for obtaining the second receptor to which the first ligand is bound used in the present invention, the first ligand and the second receptor can be physically bound or chemically bound by a known method. And a method of obtaining the second receptor to which the first ligand is bound from a natural product or the like. As the second receptor to which the first ligand is bound,
When the second receptor is a polysaccharide binding protein, more specifically a glycosaminoglycan binding protein, for example,
Examples include a hyaluronic acid-binding protein to which keratan sulfate is bound, a hyaluronic acid-binding protein to which chondroitin sulfate is bound, and a hyaluronic acid-binding protein to which heparan sulfate is bound.

Examples of the hyaluronic acid-binding protein to which keratan sulfate is bound include cartilage-derived proteoglycans (eg, aggrecan (PG-H)), chondrosarcoma-derived proteoglycans, and keratan sulfate-bound hyaluronic acid-binding regions. And a fusion protein. Further, in some cases, chondroitin sulfate is bound to these proteins, and the chondroitin sulfate is a chondroitin sulfate degrading enzyme (for example, chondroitinase ABC).
) Is preferably removed in advance.

The hyaluronic acid-binding protein to which keratan sulfate is bound can be prepared by a known method (JP-A-4-262797).
No., Adv. Drug Dev. Rev. (1991) p257-264, Biochem.
Biophys. Acta. (1979) p281-289).

Also, commercially available products can be used. Commercially available products include proteoglycan monomers (bovine nasal cartilage or rat cartilage species) (trade name, manufactured by ICN Biomedical Co., Ltd., sold by Seikagaku Corporation). When this commercial product is used, it is preferable to remove chondroitin sulfate in advance by a method such as the action of a chondroitin sulfate degrading enzyme (eg, chondroitinase ABC).

Examples of the hyaluronic acid-binding protein to which chondroitin sulfate is bound include cartilage-derived proteoglycans (eg, aggrecan (PG-H), etc.), PG-M
(Versican), neurocan, chondrosarcoma-derived proteoglycan, chondroitin sulfate bound C
D44 and chondroitin sulfate are bound thereto, and examples thereof include a part of these proteins containing a hyaluronic acid binding region or a fusion protein. Keratan sulfate or heparan sulfate may be bound to these proteins, and the keratan sulfate or heparan sulfate is a keratan sulfate degrading enzyme (for example, keratanase or keratanase II) or a heparan sulfate degrading enzyme (heparitinase,
It is preferable to remove in advance by a method such as the action of heparinase.

The hyaluronic acid-binding protein to which chondroitin sulfate is bound can be obtained by a known method (Adv. Drug Dev. Rev. (1991) p257-264, Glycobiolog).
y, 2 (1992) p93-97, Cell Engineering Separate Volume / Handbook Series "Adhesion Molecule Handbook", Shujunsha, No. 298-3076
P., 1994). For example, aggrecan can be extracted from cartilage such as bovine, porcine, deer or human by solubilizing cartilage components with 4-6 M guanidine hydrochloride and then extracting under conditions under which proteoglycans associate (eg, in a 0.5 M guanidine hydrochloride solution). ) To perform high density sedimentation (approximately 1.
(6 g / ml or more), and subjected to sedimentation equilibrium centrifugation under a condition in which proteoglycan does not associate (for example, in a 4 to 6 M guanidine hydrochloride solution) to obtain a high density (about 1.5 g / ml).
g / ml or more) fraction, which can be dialyzed to give aggrecan.

A commercially available product can also be used. Commercially available products include proteoglycan monomers (bovine nasal cartilage or rat cartilage species) (trade name, manufactured by ICN Biomedical Co., Ltd., sold by Seikagaku Corporation). When this commercial product is used, it is preferable to remove keratan sulfate in advance by a method such as the action of a keratan sulfate degrading enzyme (eg, keratanase, keratanase II).

The hyaluronic acid-binding protein to which heparan sulfate is bound includes CD4 to which heparan sulfate is bound.
4 and heparan sulfate bound thereto and a part of these proteins containing a hyaluronic acid binding region or a fusion protein. In some cases, chondroitin sulfate is bound to these proteins, and it is preferable that the chondroitin sulfate be removed in advance by a method such as the action of a chondroitin sulfate-degrading enzyme (for example, chondroitinase ABC).

The hyaluronic acid-binding protein to which heparan sulfate is bound can be obtained by a known method (Glyc
obiology, 2 (1992) p93-97, Cell Engineering Separate Volume / Handbook Series “Adhesion Molecule Handbook”, published by Shujunsha, No.
298-3076, published in 1994).

5) The measuring method of the present invention comprises the solid phase of the present invention,
Or a solid body to which the first receptor is fixed and a second receptor-
The first ligand-first receptor complex is immobilized via the first receptor, the solid phase body is contacted with a sample containing the first ligand and the second ligand, the first ligand in the sample and The first ligand and the second ligand in the sample are measured by detecting the formation of the complex with the first receptor and the formation of the complex between the second ligand and the second receptor in the sample, respectively. Is the way.

The first and second ligands measured by the method of the present invention include, for example, polysaccharides (eg, glycosaminoglycans), oligosaccharides, proteins, peptides, agonists and the like. Further, when the ligand is glycosaminoglycan, a combination of two kinds of ligands such as keratan sulfate and hyaluronic acid, chondroitin sulfate and hyaluronic acid, and heparan sulfate and hyaluronic acid are exemplified.

One of the preferable embodiments of the measuring method of the present invention is as follows.
In this method, the first ligand and the second ligand are different glycosaminoglycans, and the first receptor and the second receptor are proteins capable of specifically binding them. More specifically, a measurement method in which the first receptor is an antibody against glycosaminoglycan and the second receptor is a glycosaminoglycan binding protein is mentioned.

In this case, the combination of the first receptor and the second receptor includes an anti-keratan sulfate antibody and a hyaluronic acid binding protein, an anti-chondroitin sulfate antibody and a hyaluronic acid binding protein, or an anti-heparan sulfate antibody. Combination with a hyaluronic acid-binding protein, and the like. In these cases, it is possible to measure keratan sulfate and hyaluronic acid, chondroitin sulfate and hyaluronic acid, or heparan sulfate and hyaluronic acid.

The above-mentioned complex formation and detection methods include, in addition to ordinary immunoassays using antigens or antibodies, affinity assays using sugars and lectins, polysaccharides and polysaccharide binding proteins, agonists and agonist receptors, and the like. The method used can be used.

These measuring methods are known methods (Sai Irie, edited by "Sequence Radioimmunoassay", Kodansha, 1979; Eiji Ishikawa et al., "Enzyme Immunoassay", 2nd edition, Medical Shoin, 1982). "Enzyme Immunoassay", Tokyo Chemical Dojin, 1989, P. Tijssen, Eiji Ishikawa, translation;
"Protein Nucleic Acid Enzyme" separate volume No. 31, enzyme immunoassay,
Published by Kyoritsu Shuppan Co., Ltd., 1987, p13-26, JP-A-4-2
No. 62797, Anal. Biochem., 149, 555-565 (1985),
WO90 / 07120, JP-A-60-224065, etc.). For example, a sandwich method or a competitive method can be used.

In the sandwich method, the order of forming the above complex can be any of so-called forward, reverse, and simultaneous (see “Protein Nucleic Acid Enzyme”, Supplement No. 31,
Enzyme immunoassay, published by Kyoritsu Shuppan Co., Ltd., 1987, p13-26
reference). As a more specific method of the sandwich method, for example, each of the first receptor and the second receptor of the solid phase used in the present invention, the first ligand and the second ligand in the sample are reacted, Next, after the first receptor labeled with the first labeling substance and the second receptor labeled with the second labeling substance are simultaneously or sequentially contacted, the solid phase and the liquid phase are separated (BF separation). A method of detecting the first labeling substance and the second labeling substance in the same phase simultaneously or sequentially.

6) The first labeling substance and the second labeling substance used in the present invention are not particularly limited as long as they generate a detectable signal at a level correlated with the amount of the measurement component in the sample. . Further, a labeling substance capable of simultaneously detecting the first labeling substance and the second labeling substance can be used in combination.

As a labeling substance, enzymes (alkaline phosphatase, β-galactosidase, peroxidase,
Glucose oxidase, etc.), fluorescent substances (FITC,
Phycoerythrin, europium, phycocyanin, allophycocyanin, rhodamine, Texas red, umbelliferone, tricolor, cyanine, 7-amino-4
-, methyl coumarin-3-acetic acid (AMCA)), a radioactive isotope ^{(125 I, 131 I, 3} H), biotin, avidin (e.g., streptavidin), and the like.

As a combination of the first labeling substance and the second labeling substance, for example, as a combination of a fluorescent substance and an enzyme,
Examples include europium, FITC, phycoerythrin, phycocyanin, allophycocyanin, rhodamine or Texas Red as a fluorescent substance, alkaline phosphatase as an enzyme, and p-nitrophenyl phosphate as a substrate.

A preferable combination of the labeling substances when the first labeling substance and the second labeling substance are simultaneously measured is a combination of the fluorescent substances in a case where the excitation wavelengths are close to each other and the fluorescence wavelengths are so far as not to interfere with each other. For example, a combination of AMCA and europium chelate, a combination of FITC and phycoerythrin or tricolor, and the like are particularly preferable, and a combination of AMCA and europium chelate or FITC and tricolor is preferable.

The labeling method of the labeling substance may be a known method suitable for the labeling substance (see Seismic Chemistry Experiment Course 2 “Protein Chemistry (Lower)”, published by Tokyo Kagaku Dojin, 1987). For example, when biotin is used, a method using a hydrazide derivative of biotin (Avidin-Biotin Ch
emistry: A Handbook, p57-63, PIERCE CHEMICAL COMPANY,
(See 1994 issue.) When using FITC,
The method can be appropriately selected from the methods described in 3-17843 and the like.

7) Samples include, for example, serum, plasma,
Body fluids such as urine and joint fluid, culture supernatants of cells or bacteria, and the like can be used.

8) In the measurement method of the present invention, whether the solid phase body of the present invention, the first receptor labeled with or capable of being labeled with the first labeling substance, and the first receptor labeled with the second labeling substance are used. Alternatively, a kit comprising a second receptor that can be labeled can be used.

Further, a solid body to which the first receptor is fixed, a solid body to which the second receptor-first ligand-first receptor complex is fixed via the first receptor, a first label A kit consisting of a first receptor that is or can be labeled with a substance and a second receptor that is labeled or can be labeled with a second labeling substance can also be used.

Preferred kits include: (1) the first receptor is an anti-keratan sulfate antibody, and the second receptor-first ligand-first receptor is hyaluronic acid-binding protein-keratan sulfate-anti-keratan sulfate The solid phase body, which is an antibody,
A kit comprising an anti-keratan sulfate antibody labeled with a first labeling substance, and a hyaluronic acid binding protein labeled with a second labeling substance, (2) the first receptor is an anti-chondroitin sulfate antibody, and the second receptor -The first ligand-the first receptor is a hyaluronic acid binding protein-chondroitin sulfate-anti-chondroitin sulfate antibody, the solid phase body, an anti-chondroitin sulfate antibody labeled with a first labeling substance, and a second labeling substance. A kit comprising a labeled hyaluronic acid-binding protein, (3) the first receptor is an anti-heparan sulfate antibody, and the second receptor-first ligand-first receptor is hyaluronic acid-binding protein-heparan sulfate- The above-mentioned solid body which is an anti-heparan sulfate antibody, an anti-heparan sulfate antibody labeled with a first labeling substance, and hyaluron labeled with a second labeling substance Such a kit consisting of binding proteins.

Examples of the hyaluronic acid-binding protein as a raw material of the hyaluronic acid-binding protein labeled with the second labeling substance include cartilage-derived proteoglycans (eg, aggrecan (PG-H)) and PG-M (versican) , Neurocan, chondrosalcoma-derived proteoglycan, CD44, and a part or fusion protein of these proteins containing a hyaluronic acid binding region. In addition, chondroitin sulfate, keratan sulfate, heparan sulfate and the like may be bound to these proteins, and these may be chondroitin sulfate degrading enzymes (eg, chondroitinase ABC), keratan sulfate degrading enzymes (eg, keratanase, keratanase). II) or heparan sulfate degrading enzyme (for example, heparitinase, heparinase, etc.).

FIG. 1 is a schematic diagram illustrating a method for measuring the first ligand and the second ligand in a sample. Here, the solid phase to which the first receptor is fixed and the solid phase to which the second receptor-first ligand-first receptor complex is fixed are different even if they are the same solid phase. You may.

[0050]

EXAMPLES The reagents and materials used in the examples are shown below. 1) Ascites containing anti-keratan sulfate antibody (1/20 / 5-D-4) 0.5 ml of pristane (manufactured by Sigma) was intraperitoneally administered to mice. Two weeks after pristane administration, 1 × 10
0.5 ml of a cell suspension containing ⁷ hybridomas was administered. When the abdomen was enlarged 10 days after administration, a 20G needle was inserted into the abdomen to collect ascites. (See Monoclonal Antibody Experiment Manual, edited by Sakuji Tominaga et al., 1987, Kodansha.) The above hybridoma is described in Caterson et al. (J. Biol. Chem., 258, (1983) p8848-8854). The hybridoma produced by the method was obtained by culturing.

2) Buffer (1) Buffer A: 0.15 M containing 0.03% NaN ₃
Phosphate buffer (pH 7.2) (2) Buffer B: 0.05% Tween
0.15 M phosphate buffer (pH 7.2) containing 20 and 0.03% NaN ₃

3) Chondroitinase ABC-treated proteoglycan derived from bovine nasal cartilage Proteoglycan monomer (bovine nasal cartilage) (manufactured by ICN Biomedical, sold by Seikagaku Corporation) 3.8
mg was dissolved in 0.5 ml of Tris-HCl buffer (pH 8.0) containing 0.3 M sodium acetate. 0.02M
In 0.1 ml of Tris-HCl buffer (pH 8.0), chondroitinase ABC 1.0 unit (protease-free,
20 μl of a solution of Seikagaku Kogyo Co., Ltd.) was added and incubated at 37 ° C. for 4 hours. Then, this was dialyzed for 3 days at 4 ° C. using distilled external solution as distilled water, and chondroitinase ABC of bovine nasal cartilage-derived proteoglycan was used.
An inner dialysis solution containing the processed product was obtained.

4) Preparation of biotinylated anti-keratan sulfate antibody 1 ml of ascites containing the anti-keratan sulfate antibody prepared in 1) above
Was diluted to 4 volumes with 0.1 M phosphate buffer (pH 8.0), and then protein A cellulofine (Seikagaku Corporation) equilibrated with 0.1 M phosphate buffer (pH 8.0) in advance. )) Column (2.0 ml of gel). After washing with 25 volumes of 0.1 M phosphate buffer (pH 8.0) of the column, the antibody was eluted with 0.1 M citrate buffer (pH 3.0). The eluate was immediately neutralized with 0.1 M Tris-HCl buffer (pH 9.0). This solution was dialyzed against 0.1 M sodium carbonate (pH 8.2) at 4 ° C for 1 day.
The dialysate was diluted to give a diluent with an antibody concentration of 1 mg / ml.
To 400 µl of this diluted solution, add biotin-N-hydroxysuccinimide ester (Biotin-N-hydroxysuccinimide).
Ester) (manufactured by Pierce) was dissolved in dimethyl sulfoxide to a concentration of 1 mg / ml, and 24 μl of the solution was added, followed by incubation at room temperature for 4 hours. This reaction solution was added to phosphate buffered saline (PBS) containing 0.1% NaN ₃ at 4 ° C. for 1 hour.
After dialysis for one day, 755 μg / ml of a dialysis solution containing a biotinylated anti-keratan sulfate antibody was obtained.

5) Substrate buffer 1 M aqueous diethanolamine solution containing 0.03% NaN ₃ and 1 mM MgCl ₂ (pH 9.8)

6) Preparation of Biotinylated Hyaluronic Acid Binding Protein Hyaluronic acid binding protein (HABP) was obtained from Tengbl
ad et al. (Biochem. Biophys. Acta 578 (1979) p281-2).
It was prepared according to the method described in 89). HABP obtained
30 mg of hyaluronic acid and 30 mg of hyaluronic acid were dissolved in 5 ml of 0.05 M phosphate buffer (pH 7.0) and stored at 4 ° C. overnight. 0.
1 M NaHCO ₃ (pH 8.2) was added to make up to 30 ml.
Biotin-N-hydroxysuccinimide ester (Bi
otin-N-Hydroxysuccinimide ester) (Pierce) 30
mg was dissolved in 15 ml of dimethyl sulfoxide.
The reaction was performed at room temperature for 4 hours. 0.02M phosphate buffer pH
It was dialyzed against 7.0 at 4 ° C. for 24 hours. Final concentration of 4
Guanidine hydrochloride was added to give M. The same volume of hyaluronic acid-bound spherical cellulose equilibrated with a 4M guanidine hydrochloride solution was added thereto and mixed. Set this to 0.
Dialysis was performed against a 5 M sodium acetate solution (pH 5.8). The components in the dialysis were packed into a column, and 0.1 M sodium acetate buffer (pH 5.8), 0.1 M sodium acetate buffer (pH 5.8) containing 1 M NaCl, and 0.1 M sodium acetate buffer (pH 5.8) containing 3 M NaCl were added.
Washing was performed sequentially with a 1M sodium acetate buffer (pH 5.8). Biotinylated HABP was eluted with 4M guanidine hydrochloride solution. This was dialyzed against deionized water at 4 ° C. for 1 day. Then, concentration was performed using an ultrafiltration membrane having a molecular weight cut off of 10,000 to obtain biotinylated HABP. The hyaluronic acid-bound spherical cellulose was prepared by the method described in JP-A-4-262797.

Example 1 Immunoplate (trade name: Nunc immunoplate, number: 439454, material: polystyrene, manufactured by Nunc)
To each well, 100 μl of a solution obtained by diluting ascites containing an anti-keratan sulfate antibody 400-fold with 2 buffer A was added, and the mixture was incubated at 37 ° C. for 1.5 hours. The solution on the plate was removed, 150 μl of a 1% solution of bovine serum albumin (for enzyme immunoassay, manufactured by Bayer) in buffer A was added to each well, and the mixture was incubated at 37 ° C. for 1 hour to perform blocking. Thereafter, the solution on the plate was removed, and the plate was washed with buffer B. Chondroitinase ABC-treated proteoglycan from bovine nasal cartilage
100 μl of a solution dissolved in BS to a concentration of 0.5 μg / ml was added to each well, and incubated at 37 ° C. for 1.5 hours. After the wells were washed with buffer B, 100 μl of ascites containing anti-keratan sulfate antibody diluted 800-fold with buffer A was added, and the mixture was incubated at 37 ° C. for 1 hour. After washing the wells with buffer B, buffer A
100 μl of a standard sample containing keratan sulfate (derived from bovine nasal cartilage, average molecular weight of 5,000) and hyaluronic acid (average molecular weight of 84,000) as the diluent solvent in Table 1.
l was added to each well and incubated for 1.5 hours at 37 ° C.

[0057]

[Table 1]

After the wells were washed with the buffer B, 100 μl of the biotinylated anti-keratan sulfate antibody diluted 1,000 times with the buffer A was added to the wells and incubated at 37 ° C. for 1 hour. After the wells were washed with buffer B, streptavidin-conjugated europium (trade name: Eu)
ro Flow STM Tracer (Cyber Flow, Inc.) diluted 51 times with buffer A according to the attached manual.
Each μl was added to each well and incubated at 37 ° C. for 30 minutes with shaking on a plate shaker.
After removing the liquid from the plate, the plate was air-dried, and the excitation wavelength was 337 nm and the fluorescence wavelength was 61 with an immunoplate reader for fluorescence.
Keratan sulfate was measured at 5 nm.

After washing the plate with buffer B,
Dissolve biotinylated HABP in buffer A and add 350 ng / ml
Add 100 μl of the solution at a concentration of
Incubated at 37 ° C. for 1.5 hours. After the wells were washed with buffer B, 100 μl of a 1,000-fold dilution of streptavidin-conjugated alkaline phosphatase (manufactured by Sigma) diluted 1,000 times with buffer A was added to each well, and incubated at 37 ° C. for 1 hour. After the wells were washed with buffer B, p-nitrophenyl phosphate (Sigma) was used.
Was dissolved in a substrate buffer, and 100 μl of a 1 mg / ml solution was added to each well, followed by incubation at 37 ° C. for 20 minutes. 405nm wavelength with immunoplate reader for absorbance
(Control wavelength: 630 nm) was measured. A calibration curve of keratan sulfate was prepared by plotting the measured fluorescence intensity against the concentration of keratan sulfate, and a calibration curve of hyaluronic acid was prepared by plotting the absorbance measured against the concentration of hyaluronic acid. . The results are shown in FIG.

From these results, keratan sulfate and hyaluronic acid can be measured simultaneously with high sensitivity and high sensitivity.
It was shown that two kinds of ligands can be measured from a sample.

Example 2 A sample containing keratan sulfate and hyaluronic acid at the concentrations shown in Table 2 was prepared in the same manner as in Example 1.

[0062]

[Table 2]

For these samples, the biotinylated HABP concentration added to the wells of the plate was 1,750 ng.
Keratan sulfate and hyaluronic acid were measured in the same manner as in Example 1 except that the amount was changed to / ml, and the concentration of keratan sulfate and fluorescence intensity, and the concentration of hyaluronic acid and absorbance were plotted. The results are shown in FIG. According to this result, keratan sulfate and hyaluronic acid can be measured with high sensitivity, and each gives a calibration curve that does not affect each other. Therefore, in the method of the present invention, two kinds of ligands can be measured with one sample. It was shown to be.

[0064]

According to the solid phase used in the present invention, only the first receptor is directly immobilized on the solid phase, and the optimal conditions for immobilization are first determined. Selection can be based on the receptor. Further, by using the second receptor to which the first ligand is bound at an appropriate concentration, the second receptor can be specifically prepared at a required concentration without using a complicated operation. It can be immobilized on the same solid phase via a receptor. Therefore, the first receptor and the second receptor can be stably immobilized, so that the first ligand and the second ligand in the same sample can be measured with high sensitivity and simultaneously.

Furthermore, since two kinds of ligands can be measured simultaneously, the amount of a sample can be reduced, the operation is simple, the time can be reduced, and the consumption of materials such as plates to be used can be reduced. There are advantages.

Further, according to the method of the present invention, two kinds of glycosaminoglycans are simultaneously measured with high sensitivity, whereby a plurality of diseases related to glycosaminoglycans (for example,
Primary screening for diseases associated with cartilage abnormalities such as osteoarthritis and rheumatoid arthritis; diseases associated with corneal tissue abnormalities; inflammation such as rheumatism; cancer; liver diseases; mucopolysaccharidosis such as morphosis and Hurler's disease It can be done easily.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a method for measuring a first ligand and a second ligand in a sample.

FIG. 2 is a calibration curve of keratan sulfate and hyaluronic acid.

FIG. 3 shows a measurement example of keratan sulfate and hyaluronic acid.

Claims (10)

[Claims] 1. A solid phase to which both a first receptor and a second receptor-first ligand-first receptor complex are fixed, wherein the complex is bound via the first receptor. The immobilized solid body is brought into contact with a sample containing the first ligand and the second ligand to form a complex between the first receptor and the first ligand and to form a complex between the second receptor and the second ligand. A method for measuring a first ligand and a second ligand in a sample by detecting body formation, respectively. 2. A solid phase to which a first receptor is fixed and a solid phase to which a second receptor-first ligand-first receptor complex is fixed via a first receptor. A sample containing a ligand and a second ligand is brought into contact with the sample, and the formation of a complex between the first receptor and the first ligand and the formation of a complex between the second receptor and the second ligand are detected. A method for measuring the first ligand and the second ligand. 3. The formation of a complex between the first receptor and the first ligand is detected by a first receptor labeled with a first labeling substance,
The method according to claim 1 or 2, wherein the formation of a complex between the second receptor and the second ligand is detected by a second receptor labeled with a second labeling substance. 4. The method according to claim 1, wherein the first ligand and the second ligand are different glycosaminoglycans, and the first receptor and the second receptor are proteins capable of specifically binding them. Measurement method. 5. The method according to claim 4, wherein the first receptor is an antibody and the second receptor is a glycosaminoglycan binding protein. 6. The method of claim 1, wherein the first ligand is keratan sulfate, the second ligand is hyaluronic acid, the first receptor is an anti-keratan sulfate antibody, and the second receptor is a hyaluronic acid binding protein. 5. The measuring method according to 5. 7. A solid phase to which both a first receptor and a second receptor-first ligand-first receptor complex are fixed, wherein the complex is bound via the first receptor. A solid phase that is fixed. 8. The first ligand and the second ligand are different glycosaminoglycans, and the first receptor is a protein capable of specifically binding to the first ligand, glycosaminoglycan; The solid phase body according to claim 7, wherein the receptor is a protein capable of specifically binding glycosaminoglycan as the second ligand. 9. A first ligand in a sample, comprising the solid phase body according to claim 7, a first receptor labeled with a first labeling substance, and a second receptor labeled with a second labeling substance. Kit for measuring second ligand. 10. A solid phase to which a first receptor is fixed, a solid phase to which a second receptor-first ligand-first receptor complex is fixed via a first receptor, a first label A kit for measuring a first ligand and a second ligand in a sample, comprising a first receptor labeled with a substance and a second receptor labeled with a second labeling substance.