JPH09229930A - Measuring method of hyaluronic acid and measuring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly-sensitive and speedy method and a kit for measuring hyaluronic acids. SOLUTION: A complex having the structure of [CD44-hyaluronic acid- hyaluronic acid-bonding protein or CD44] is formed by bringing a sample containing hyaluronic acids into contact with hyaluronic acid-bonding protein marked with CD44 and a marking substance or CD44. And hyaluronic acids in the sample is measured by detecting the marking substance in the above- mentioned complex. Or by bringing the sample containing hyaluronic acids into contact with hyaluronic acids marked with a marking substance and CD44, a complex with the hyaluronic acids marked with the marking substance and CD44 is formed. And, hyaluronic acids in the sample is measured by detecting the marking substance in this complex.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a CD44 or a CD.
The present invention relates to a highly sensitive and rapid method for measuring hyaluronan using a partial protein or a fusion protein containing 44 hyaluronan-binding regions.

[0002]

2. Description of the Related Art Hyaluronic acid is an acidic mucopolysaccharide obtained by linearly polymerizing a unit in which N-acetylglucosamine and glucuronic acid are linked by β-1,4. Hyaluronic acid is widely distributed in systemic tissues such as cartilage, synovial fluid of joint space, umbilical cord, serum, urine, and vitreous body of the eye, and contributes to stabilization of tissue construction as one of extracellular matrix components. It is known that the concentration of hyaluronic acid in blood is increased in diseases associated with destruction of tissue structure or deterioration of metabolic function of hyaluronic acid, such as rheumatoid arthritis, cancer and liver disease. Therefore, quantifying hyaluronic acid in body fluids such as blood and synovial fluid is said to be useful for diagnosing these diseases or diagnosing the recovery process after surgery.

At the cellular level, almost all cells in the living body have the function of synthesizing hyaluronic acid, and hyaluronic acid has come to be considered as an important substance for living organisms.

The following methods are known as conventional methods for measuring hyaluronic acid. As the sandwich method, 1) a hyaluronic acid-binding protein obtained by trypsin treatment of cartilage proteoglycan is immobilized on a solid phase, and then a sample hyaluronic acid is added to bind to the protein, and a labeled hyaluronic acid-binding protein is further added. Hyaluronic acid is measured by sandwiching the protein immobilized on a solid phase with the labeled protein to form a sandwich-shaped conjugate, and measuring the labeling substance of the conjugate. Method (Japanese Patent Publication 6-41952, Clin.Chim.Acta., 181,317-327 (198
9)), 2) A sample containing a sample hyaluronic acid is added to a chondroitinase ABC-treated product of cartilage proteoglycan adhered to a solid phase, the treated product and the sample hyaluronic acid are allowed to bind to each other, and a labeled substance is prepared in advance. , A hyaluronic acid-binding protein obtained by tryptic treatment of cartilage proteoglycan was added, and a sample hyaluronic acid was sandwiched between the treated product and the labeled protein to form a sandwich-shaped conjugate, There is known a method for quantifying a sample hyaluronic acid using a labeling substance of a linear conjugate (Japanese Patent Laid-Open No. 4-262797).

Further, as an inhibition method (competitive method), hyaluronic acid as a sample, hyaluronic acid bound to a solid phase, and hyaluronic acid-binding protein labeled with radioactive iodine or unlabeled hyaluronectin are reacted, and solidified. Phase-bound hyaluronic acid and the labeled hyaluronic acid-binding protein or the hyaluronectin are bound to form a complex, and radioactive iodine in the complex is detected,
Alternatively, a method for measuring hyaluronic acid by detecting a complex of hyaluronic acid and hyaluronectin bound to a solid phase with an enzyme-labeled anti-hyaluronectin antibody is known (Japanese Patent Laid-Open No. 63-150669).

However, the conventional measuring methods have drawbacks such as low sensitivity and long reaction time.

[0007]

The present invention has high sensitivity and
An object of the present invention is to provide a method and a kit for rapid measurement of hyaluronic acid.

[0008]

Means for Solving the Problems The inventors of the present invention have made extensive studies in view of the above problems, and as a result, formed a complex with hyaluronic acid using a specific protein, thereby enabling highly sensitive and rapid measurement of hyaluronic acid. The present invention has been completed by finding that the above can be performed.

That is, the present invention relates to a method for measuring hyaluronic acid in a sample, which comprises a step of reacting CD44 with hyaluronic acid in the sample to form a complex of CD44 and hyaluronic acid.

Preferably, the present invention relates to a method for measuring hyaluronic acid, wherein the complex of CD44 and hyaluronic acid is a complex represented by the following formula (I).

[0011]

Embedded image In the formula (I), A is CD44, B is hyaluronic acid,
C represents hyaluronic acid binding protein or CD44. )

More preferably, after reacting CD44 with hyaluronic acid in the sample and optionally with a hyaluronic acid-binding protein to form a complex represented by the above formula (I), the complex is unreacted. Separated from the reacted CD44 or hyaluronan-binding protein, the complex or unreacted C
The present invention relates to the method for measuring hyaluronic acid, which comprises detecting D44 or a hyaluronic acid-binding protein.

Further preferably, hyaluronic acid in the sample is reacted with CD44 fixed to the solid phase and a hyaluronic acid-binding protein or CD44 labeled with a labeling substance, or with hyaluronic acid in the sample. By reacting CD44 labeled with a labeling substance with a hyaluronic acid-binding protein fixed to a solid phase, a complex represented by the formula (I) is formed, and the complex is reacted with the unreacted Separation from labeled hyaluronan-binding protein or labeled CD44, and detection of labeling substance in the complex or unreacted labeled hyaluronan-binding protein or labeled substance of labeled CD44 The present invention also relates to the method for measuring hyaluronic acid.

Further, the measuring method of the present invention comprises reacting CD44 with hyaluronic acid in a sample and competitive hyaluronic acid, and binding CD44 competitively to hyaluronic acid in the sample and competitive hyaluronic acid. , CD4
The method for measuring hyaluronic acid comprises the step of forming a complex between 4 and hyaluronic acid in a sample and a complex between CD44 and competing hyaluronic acid.

The term "competitive hyaluronic acid" as used herein refers to hyaluronic acid added to a measurement system in order to compete with hyaluronic acid in a sample, for example, hyaluronic acid labeled with a labeling substance or solid phase Examples include fixed hyaluronic acid.

Further, as a more preferable measuring method of the present invention, a method having the following features 1) to 4) can be mentioned.

1) By contacting a specimen containing hyaluronic acid with CD44 immobilized on a solid phase and then with hyaluronic acid-binding protein labeled with a labeling substance or CD44, the compound of the above formula (I) The complex represented is formed, and the complex is separated from the unreacted labeled hyaluronic acid-binding protein or CD44, and the labeling substance in the complex or the unreacted labeled hyaluronic acid-binding protein It is characterized in that hyaluronic acid in a sample is measured by detecting a labeling substance of protein or CD44.

2) Contacting a sample containing hyaluronic acid with a hyaluronic acid-binding protein fixed to a solid phase body,
Then, by contacting with CD44 labeled with a labeling substance, a complex represented by the formula (I) is formed, and the complex is separated from the unreacted labeled CD44, The hyaluronic acid in the sample is measured by detecting the labeling substance of or the unreacted labeling substance of the labeled CD44.

3) By contacting a specimen containing hyaluronic acid, hyaluronic acid labeled with a labeling substance, and CD44 fixed or capable of being fixed to a solid phase, the composite of CD44 and hyaluronic acid in the specimen Body and a complex of the CD44 and a hyaluronic acid labeled with a labeling substance are formed, the complex is separated from the unreacted labeled hyaluronic acid, and CD44 and a hyaluronic acid labeled with the labeling substance are separated. It is characterized in that the hyaluronic acid in the sample is measured by detecting the labeling substance in the complex or the unreacted labeling substance of the labeled hyaluronic acid.

4) By contacting a specimen containing hyaluronic acid, hyaluronic acid fixed to a solid-phase body, and CD44 labeled or capable of being labeled with a labeling substance, the CD44 and hyaluronic acid in the specimen are brought into contact with each other. In a sample by forming a complex and a complex between the CD44 and hyaluronic acid fixed to a solid phase and detecting the CD44 in the complex between the CD44 and hyaluronic acid fixed to a solid phase It is characterized by measuring the hyaluronic acid of.

The present invention also provides a kit for use in a method for measuring hyaluronic acid by forming a complex between CD44 and hyaluronic acid, which comprises C as a constituent component.
The present invention relates to a kit for measuring hyaluronic acid, which contains D44.

Preferably, the kit for measuring hyaluronic acid of the present invention is mainly composed of CD44 and a hyaluronic acid-binding protein, and the CD44 or hyaluronic acid-binding protein is or can be labeled with a labeling substance. And more preferably mainly CD
It is composed of a solid phase body to which 44 is fixed and a hyaluronic acid-binding protein labeled with a labeling substance.

The hyaluronic acid measuring kit of the present invention mainly comprises a solid phase having CD44 fixed thereon and hyaluronic acid labeled with a labeling substance.
In addition, the kit for measuring hyaluronic acid of the present invention is mainly labeled with a labeling substance or can be labeled with a CD.
44 and a solid phase body to which hyaluronic acid is fixed.

The CD44 referred to in the present invention is a molecule different from the hyaluronan-binding protein. That is,
In the present invention, CD44 means (i) CD44 itself (C
A complete protein containing all of the extracellular region of D44, the transmembrane region and the intracytoplasmic region), (ii) a partial protein containing the hyaluronic acid binding region of CD44, and (iii) C
It refers to a fusion protein containing the hyaluronic acid binding region of D44. On the other hand, the hyaluronic acid-binding protein in the present invention means, as described below, proteoglycan, core protein of proteoglycan, partial proteins thereof,
Link protein, hyaluronectin, etc.

Hereinafter, the present invention will be described in detail.

(1) Method for measuring hyaluronic acid The method for measuring hyaluronic acid of the present invention is characterized by including at least the step of forming a complex of CD44 and hyaluronic acid.

CD44 is one of leukocyte antigens, is an adhesion molecule having hyaluronic acid as a ligand, and anti-C.
It is defined by the reactivity with D44 antibody. CD
44 is a transmembrane protein, and it is known that many isomers (isoforms) exist. For example, human standard CD44 (CD44H) has 24
It is known to consist of an extracellular region consisting of 8 amino acid residues, a transmembrane region consisting of 21 amino acid residues and an intracytoplasmic region consisting of 72 amino acid residues, and it is known that the molecular weight is about 85 to 90 KDa. The extracellular region of CD44 is homologous to the cartilage proteoglycan core protein and the link protein on the amino terminal side (for example, in the case of CD44H,
There is a hyaluronic acid binding region (for example, in the case of CD44H, a region consisting of amino acid residues 12 to 101 from the amino terminus) having about 30% homology) (hereinafter, referred to as “C
D44 hyaluronic acid binding region ". ), And further chondroitin sulfate in the membrane proximal region on the carboxyl terminal side,
Those to which glycosaminoglycans such as heparan sulfate or oligosaccharides are bound are also known. In addition, the membrane proximal region is composed of 10 exons of alternative splicing (alternative splicing).
licing) may result in insertion of a mutated portion, which also results in a large number of isomers.

CD44 is a cell-cell or cell-cell matrix (hyaluronic acid, collagen type I, collagen IV).
It is known that it is expressed on the cell membrane of leukocytes in general, fibroblasts, epithelial cells, etc. CD44 has various functions such as fibronectin and collagen type I and collagen type IV receptor functions and involvement in cancer metastasis, and one of them is the function of hyaluronic acid receptor. CD44 on the cell membrane is normally in an inactive form for binding to hyaluronan, and can acquire the ability to bind to hyaluronan by being activated by phorbol myristate acetate (PMA) or the like. It has been known.

CD44 is Pgp-1, ECMR
III, Hermes antigen, gp90HERMES, H
-CAM, ln (Lu) -related antig
en, Hutch-1, ln (Lu) -related
It is also called p80, p80A3D8, p85, etc. (Cell, 61, 1303-1313 (1990), J. Exp. Med., 172, 69-75.
(1990), Cell Engineering Separate Volume, Handbook Series, "Adhesion Molecule Handbook", pages 298-3076, published in 1994).

On the other hand, C of human, baboon, mouse, rat, etc.
The cDNA encoding D44 is known, and the amino terminus of the extracellular region of CD44 obtained by genetic engineering and the Fc fragment of human IgG1 (that is, the hinge region, the C _H 2 portion of the constant region and the C _H 3 portion). ) Is known to be fused to the carboxyl terminus (Cell, 61, 1303-1313 (1990)).

Further, using a culture supernatant containing the above fusion protein and a fluorescein-conjugated sheep anti-human IgG antibody, a tissue immunostaining report was obtained on a tissue section of rat intestine, lung, kidney, bone, or skin as a specimen, and as a specimen. Of the binding of the fusion protein to rat lymph node high endothelial cells by flow cytometry, and the effect on the binding between the fusion protein and the specimen by the treatment with hyaluronidase or chondroitin ABC lyase of the specimen and the endothelial cells The result of detecting the binding between the fusion protein and the fusion protein in the presence of hyaluronic acid or chondroitin sulfate shows that the fusion protein recognizes hyaluronic acid on a solid sample such as tissue or cell, and is weaker than hyaluronic acid. However, it has been reported that it also binds to chondroitin sulfate. (Cell, 61,1303-1313 (1990)).

Examples of the CD44 itself used in the present invention include standard type CD44 (CD44H) and CD44.
Mutant molecule (CD44V), CD44E, soluble CD44
(SCD44) and the like. There are cases where glycosaminoglycans and oligosaccharides such as chondroitin sulfate and heparan sulfate are bound to these CD44s themselves, and these glycosaminoglycans and oligosaccharides are
It may be removed by a method such as allowing a glycolytic enzyme (eg, chondroitinase ABC, heparitinase, N-glycosidase, O-glycosidase, etc.) to act in an appropriate step of separating and purifying 44 itself.

CD44 itself is leukocytes, fibroblasts, epithelial cells, etc., which are naturally expressed or genetically engineered using a DNA encoding the complete protein of CD44, preferably cultured cell lines thereof. Cell (eg MOLT4, JURKAT, U937, etc.)
And the like, it can be obtained using a known method for separating and purifying a membrane-bound protein.

As CD44 used in the present invention, in addition to the above-mentioned CD44 itself, a protein containing the hyaluronic acid binding region of CD44 may be used. The protein is not particularly limited as long as it has the ability to bind hyaluronic acid, but it is a partial protein of CD44 containing the hyaluronic acid binding region of CD44 (eg, the entire extracellular region of CD44 or a part thereof). Or a fusion protein of CD44, a partial protein of CD44 or a protein having the amino acid sequence of the hyaluronic acid binding region of CD44 and a protein of a different type from CD44.

The protein of a different type from CD44 forming the fusion protein is not particularly limited as long as it does not prevent the binding between the hyaluronic acid binding region of the formed fusion protein and hyaluronic acid. Those having properties and functions such as a property of being fixed to a solid phase and a property of preventing the hyaluronic acid binding region of the fusion protein from being buried in the solid phase are preferable. Specific examples of such a protein include immunoglobulin (eg, IgG), glutathione S-transferase, and the like, and further include immunoglobulin partial proteins (Fc portion, etc.) and the like.

Preferred fusion proteins include, for example, those in which the carboxyl terminus of the extracellular region of CD44 and the amino terminus of immunoglobulin are bound, and more preferably, the carboxyl terminus of the extracellular region of CD44 and Fc of IgG. Examples include fusion proteins in which the amino terminus of the fragment is linked.

The fusion protein may form a multimer (for example, a dimer) as long as it has a binding ability with hyaluronic acid. For example, the above-mentioned fusion protein containing IgG or an Fc fragment thereof can usually form an IgG-like dimer by disulfide bond under non-reducing conditions.

As a method for producing a partial protein or a fusion protein containing the hyaluronic acid binding region of CD44,
For example, the following methods 1) to 3) can be exemplified.

1) CD44 is decomposed by a known means such as digestion with a proteolytic enzyme (eg, trypsin, chymotrypsin, pepsin, etc.), and then it has the ability to bind hyaluronic acid using a carrier to which hyaluronic acid is bound. A method for separating and purifying partial proteins.

2) In the operation of 1) above, the sugar chain is partially or completely removed by a method such as the action of a glycolytic enzyme (eg chondroitinase ABC, heparitinase, N-glycosidase, O-glycosidase, etc.). A method for obtaining a broken partial protein.

3) A method for obtaining a fusion protein using a known fusion method (for example, a genetic engineering method or a method for obtaining a hybrid protein). Here, as the genetic engineering method, for example, CD44 containing a hyaluronic acid binding region is used.
CDNA encoding a partial protein of E. coli (eg, extracellular region of CD44) and a protein of a different type from CD44 (eg, immunoglobulin, more preferably IgG)
A cDNA prepared by ligating a cDNA encoding a cDNA encoding the Fc fragment etc. of CDM8) into a vector such as CDM8 to prepare a plasmid.
OS cells or the like) or a host organism (for example, silkworm or the like) is transferred by a known method to obtain a transformant. The fusion protein can be obtained by culturing and breeding this transformant by a method suitable for the transformant. The desired fusion protein can be obtained from the culture or breeding product of this transformant by protein isolation and purification methods generally known in the fields of genetic engineering and protein chemistry. For example, by crushing the culture or recovering the culture supernatant, a fraction containing the fusion protein is obtained. When the fusion protein contains the Fc fragment of IgG, affinity chromatography using a protein A-binding carrier or the like Examples of the method include a method of obtaining the fusion protein by a method.

The method for measuring hyaluronic acid in a sample of the present invention is characterized by comprising the step of reacting CD44 with hyaluronic acid in a sample to form a complex of CD44 and hyaluronic acid. There is no particular limitation as long as it is a measurement method utilizing the formation of such a complex, but preferably, as a complex of CD44 and hyaluronic acid, as represented by the following formula (I), There is a so-called sandwich method in which a complex having a sandwich-like binding structure sandwiching hyaluronic acid in a sample to be measured is formed.

[0043]

Embedded image In the formula (I), A is CD44, B is hyaluronic acid,
C represents hyaluronic acid binding protein or CD44. )

The line connecting A, B and C in the above formula (I) means binding based on affinity,
It does not mean a covalent bond. Examples of the hyaluronic acid-binding protein used herein include proteoglycans (eg, cartilage proteoglycans, chondroitinase ABC treated products of cartilage proteoglycans, etc.), proteoglycan core proteins (eg, cartilage proteoglycan core proteins, etc.), and parts thereof. protein,
Examples include link protein and hyaluronectin.

As a method for producing hyaluronic acid-binding protein, a known method (Leurent et al, Anal. Biochem., 1
09, p.386-394 (1980), JP-A-4-262797, Anal.Bioche
m., 149, 555-565 (1985)). For example, a partial protein of cartilage proteoglycan can be prepared according to the method of Leurent et al., The method described in JP-A-4-262797, and the like. That is, for example, proteoglycan is solubilized and extracted from cartilage using guanidine hydrochloride or the like, and the dialyzed product is caused to act on a proteolytic enzyme (trypsin or the like) to decompose the core protein, and a hyaluronic acid-binding carrier (for example, hyaluronic acid Examples thereof include a method of obtaining the above-mentioned partial protein by affinity chromatography using a bound spherical cellulose or the like).

(A) A particularly preferred example of specific examples of sandwich method As a measuring method by the sandwich method utilizing the formation of the complex represented by the formula (I), specifically, a sample containing hyaluronic acid is used. By contacting with CD44 immobilized on a solid phase and then with a hyaluronic acid-binding protein labeled with a labeling substance or CD44,
Hyaluronic acid in a sample is reacted with the CD44 and a hyaluronic acid-binding protein labeled with the labeling substance to form a complex represented by the formula (I), and the complex is labeled with the hyaluronic acid. Examples include a method in which hyaluronic acid in a sample is measured by separating from acid-binding protein and detecting a labeling substance in the complex. This method is preferable because it has extremely high measurement sensitivity for hyaluronic acid.

In the above-mentioned sandwich method, either a hyaluronic acid-binding protein labeled with a labeling substance or CD44 labeled with a labeling substance can be used, but a hyaluronic acid binding labeled with a labeling substance can be used. It is preferable to use a sex protein, as compared with the case where the CD44 is used, because there is no possibility that the background will increase due to the interaction between the same proteins.

(B) Another example of specific examples of sandwich method Further, the following method can also be used as a measuring method by the sandwich method. That is, a specimen containing hyaluronic acid is brought into contact with a hyaluronic acid-binding protein fixed to a solid phase body, and then C labeled with a labeling substance is added.
It is a method of measuring hyaluronic acid in a sample by forming a complex represented by the formula (I) by contacting it with D44 and detecting a labeling substance in the complex.

In the above-mentioned sandwich method, the complex represented by the above formula (I) can be formed in any of the so-called forward, reverse and simultaneous (“Protein Nucleic Acid Enzyme” Supplement No. 31, Enzyme-linked immunosorbent assay, published by Kyoritsu Shuppan Co., Ltd., 1987, p13-26).

In the measuring method of the present invention by these sandwich methods, examples of the solid phase body to which CD44 or hyaluronic acid-binding protein is immobilized include plates, tubes, beads, membranes and gels. As the material, polystyrene, polypropylene, nylon, polyacrylamide and the like are preferable.

As a method for immobilizing CD44 or hyaluronic acid-binding protein on these solid phase bodies, a general method (immobilized enzyme) such as physical adsorption method, covalent bond method, entrapment method, etc. , Kodansha, 1975, pp. 9-75). In particular, the physical adsorption method is preferable because the operation is simple.

On the surface of the solid phase body to which CD44 or hyaluronic acid-binding protein is adhered, there may be a surface portion where these are not adhered, and hyaluronic acid or other If the molecular species of (3) are fixed, accurate measurement results may not be obtained. Therefore, it is preferable to add a blocking substance to cover the portion to which CD44 or hyaluronic acid-binding protein is not fixed before contacting the sample with the solid phase body. Examples of such blocking substances include serum albumin, casein, milk protein and the like, which can be collected from bovine and the like.

In addition, after binding hyaluronic acid in the sample to CD44 or hyaluronic acid-binding protein fixed to the solid phase, the surface of the solid phase is washed with a washing solution to remove non-specific adsorbates. Is preferred. As a cleaning liquid,
For example, a buffer solution (eg, phosphate buffer solution, Tris-hydrochloric acid buffer solution) to which a surfactant such as Tween-based surfactant is added can be used.

Hyaluronic acid binding protein or CD4
Labeling substances used for labeling 4 include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), isotopes ( ¹²⁵ I, ¹³¹ I, ³ H, etc.), fluorescent dyes (fluorescein isothiocyanate, Umbelliferone, etc.), chemiluminescent substances (luminol, etc.), haptens, biotin, avidin (eg, streptavidin, etc.) and the like, but not particularly limited as long as they can be used for labeling proteins in general.

Hyaluronic acid binding protein or CD4
The labeling method of 4 is a known method suitable for a labeling substance, for example, when labeling an enzyme, a glutaraldehyde method, a periodate crosslinking method, a maleimide crosslinking method, a carbodiimide method,
When labeling with radioactive isotopes, such as the activated ester method, the chloramine T method, the lactoperoxidase method, etc. (see Biochemistry Laboratory 2 "Protein Chemistry (below)", Tokyo Kagaku Dojin, 1987) You can choose.

The method of detecting the labeling substance varies depending on the labeling substance to be used. For example, when biotin is used as the labeling substance, an enzyme to which streptavidin or the like is bound is added, and peroxidase is added via the streptavidin or the like. Enzyme such as biotin as a labeling substance is bound to a complex containing biotin, a color-developing substrate such as tetramethylbenzidine and hydrogen peroxide solution are added as a substrate for the enzyme, and the degree of color development of the product by the enzymatic reaction is measured by absorbance. A method etc. can be mentioned. Further, for example, when a fluorescent substance or a chemiluminescent substance is used as the labeling substance, a method of measuring the fluorescence or luminescence of the solution after the reaction can be mentioned.

Further, as a measuring method utilizing the formation of a complex of CD44 and hyaluronic acid of the present invention, for example, there is a method called a so-called inhibition method (competitive method) using the following competitive hyaluronic acid. it can.

(C) Example of Inhibition Method As an example of the inhibition method, a sample containing CD44, hyaluronic acid, and hyaluronic acid labeled with a labeling substance are brought into contact with each other, and the CD44, hyaluronic acid in the sample and the labeled substance are labeled. To form a complex of the CD44 with hyaluronic acid in the sample and a complex of the CD44 with the labeled hyaluronic acid, and the CD44 with the labeled hyaluronic acid. A method of indirectly quantifying hyaluronic acid in a sample by detecting the labeling substance in the complex of is mentioned. This utilizes the fact that the binding between CD44 and labeled hyaluronic acid is inhibited depending on the concentration of hyaluronic acid in the sample.

As a method of contacting CD44, a specimen containing hyaluronic acid, and hyaluronic acid labeled with a labeling substance, a method of simultaneously contacting these three components, or C
CD44 by contacting D44 with a specimen containing hyaluronic acid
4 may be bound to the hyaluronic acid, and then CD44 not bound to the hyaluronic acid may be contacted with hyaluronic acid labeled with a labeling substance.

Further, it is preferable that the CD44 is fixed to the solid phase body in advance as in the sandwich method. Examples of the solid phase to which CD44 is fixed include plates, tubes, beads, membranes, gels, etc., as described above. As the material, polystyrene, polypropylene, nylon, polyacrylamide and the like are preferable. As a method for fixing CD44 to these solid phase bodies, a general method as a method for preparing an immobilized enzyme such as the physical adsorption method, the covalent bond method, or the entrapment method described above can be applied. The method is preferable because it is simple.

The hyaluronic acid labeled with the labeling substance used here can be obtained by binding the labeling substance to hyaluronic acid by a known means. The hyaluronic acid used is not particularly limited, but has an average molecular weight of 200.
The thing of 0-2 million is mentioned. As the labeling substance used for labeling the hyaluronan, radioisotopes ^{(12 5}
I, ¹³¹ I, ³ H, etc.), fluorescent dyes (fluorescein isothiocyanate, umbelliferone, etc.), chemiluminescent substances (luminol, etc.), enzymes (peroxidase, alkaline phosphatase, β-galactosidase, etc.), or other substances (biotin) , Avidin, etc.) and the like. As a method for labeling hyaluronic acid, a known method suitable for a labeling substance may be used. For example, when biotin is used, a method using a hydrazide derivative of biotin (Av
idin-Biotin Chemistry: A Handbook, p57-63, PIERCE CHE
MICAL COMPANY, 1994), and when fluorescein isothiocyanate is used, it can be appropriately selected from the methods described in JP-B-63-17843.

(D) Another Example of Inhibition Method Further, as another method of such an inhibition method, for example, hyaluronic acid fixed to a solid phase, a specimen containing hyaluronic acid and CD44 are brought into contact with CD44. Hyaluronic acid in the sample and hyaluronic acid fixed to the solid phase are reacted to form a complex of the CD44 and hyaluronic acid in the sample and the CD44 and hyaluronic acid fixed to the solid phase. An example is a method of indirectly quantifying hyaluronic acid in a sample by forming a complex and detecting the CD44 in the complex of CD44 and hyaluronic acid fixed to the solid phase. Here, as a method for detecting CD44,
Examples thereof include a method of using CD44 labeled with a labeling substance in advance to detect the labeling substance, a method of using an antibody of CD44, and the like.

As a method of contacting hyaluronic acid, a specimen containing hyaluronic acid, and CD44 fixed to a solid phase, a method of simultaneously contacting these three components or a CD
44 and the specimen containing hyaluronic acid are brought into contact with each other to produce the CD4
4 may be combined with hyaluronic acid in the sample, and then CD44 that is not bonded to hyaluronic acid in the sample may be contacted with hyaluronic acid fixed to the solid phase.

The hyaluronic acid adhered to the solid phase body used in this alternative method is applied to a solid phase body such as a plate, a tube, a bead, a membrane or a gel by a physical adsorption method, an ionic bonding method, a covalent bonding method, A general method for preparing immobilized enzymes such as the inclusion method (immobilized enzyme, 1975, published by Kodansha,
It can be obtained by fixing hyaluronic acid by applying (see pages 9 to 75). In particular, the physical adsorption method is preferred in terms of simplicity. As the material of the solid phase body, polystyrene, polypropylene, nylon, polyacrylamide and the like are preferable, and those having an amino group introduced on the surface of the solid phase body are also preferable. The hyaluronic acid fixed to the solid phase is not particularly limited, but has an average molecular weight of 2000 to 20.
One of them is about 100,000.

In the measuring method of the present invention, the concentration of hyaluronic acid in the sample was prepared in advance by using a standard solution of hyaluronic acid having a known concentration to prepare a calibration curve for the relationship between the concentration of hyaluronic acid and the detection result of the labeled substance. By a method or the like using the detection result and the calibration curve for a sample of unknown concentration,
It can be quantified.

According to the measuring method of the present invention, human, bovine,
Hyaluronic acid can be quantified in a liquid sample such as synovial fluid of a rat or the like, body fluid such as serum or urine, or a culture solution of cells or microorganisms that produce hyaluronic acid.

The measuring method of the present invention uses a measurement range of 0.
It is a measuring method with high reactivity to hyaluronic acid having an average molecular weight of 80,000 or more at 1 to 1000 ng / ml. The sample containing hyaluronic acid used in the measuring method of the present invention does not need to be purified in advance. That is, even if collagen or fibronectin or the like is contained in the sample in addition to hyaluronic acid, hyaluronic acid can be selectively measured, and therefore the measurement result is not affected by them. Further, even if the sample contains glycosaminoglycan other than hyaluronic acid, there is no cross-reaction with them and the measurement results are not affected by these.

(2) Hyaluronic Acid Measuring Kit The hyaluronic acid measuring kit of the present invention contains CD44 as a constituent, and when used in the above-mentioned sandwich method, it is mainly labeled with CD44 and a labeling substance. Or a hyaluronic acid-binding protein that can be labeled. In such a kit, if the CD44 previously fixed to the solid phase body is used, the operation of fixing to the solid phase body can be omitted in the above-described measurement method.

When used in the inhibition method, it is preferably composed mainly of a solid phase having CD44 adhered thereto and hyaluronic acid labeled with a labeling substance. It is composed of a solid phase body to which hyaluronic acid is fixed.

The kit further contains hyaluronic acid (average molecular weight of 8 to 8) as a standard for preparing a calibration curve.
2 million range), a detection reagent for labeling substances,
CD4, a reagent for labeling hyaluronan-binding protein
A reagent (for example, a labeled antibody) that detects 4 can be added. Further, in addition to these components, the blocking substance, the washing solution, the sample diluent, the enzyme reaction stopping solution, and the like may be contained.

Further, these components can be housed in separate containers and stored as a kit which can be used according to the above formulation at the time of use. The method for measuring hyaluronic acid of the present invention is very simple in operation, and C
By using D44, hyaluronic acid can be specifically measured, and even a very small amount of hyaluronic acid of 0.1 ng / ml can be measured, and the sensitivity is extremely high. Further, since the reaction time with hyaluronic acid is short, this is a measuring method with a short measuring time. Further, it has high reactivity with hyaluronic acid having an average molecular weight of 80,000 or more, and it is possible to measure hyaluronic acid having a higher molecular weight than the conventional method. Further, the fluctuation of the measured value is extremely small, and the measuring method is excellent in reproducibility.

As a protein containing the hyaluronic acid binding region of CD44, C particularly produced by genetic engineering
By using D44, it is possible to obtain a constant quality of the protein, and therefore it is possible to provide a stable measurement method and kit with little fluctuation in measurement results and good reproducibility. Therefore, the method of the present invention, for example, by detecting a minute change in the concentration of hyaluronic acid in the diagnosis of inflammation such as rheumatism or the diagnosis of cancer or liver disease, etc. It is an extremely effective method that is expected to be accurately detected, and it is also possible to measure trace amounts of hyaluronic acid produced by cultured cells, microorganisms, etc., which can be used for process control and has a high technical value. .

[0073]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.

(1) Sandwich Method A particularly preferable mode of the measuring method by the sandwich method of the present invention will be described below. The case of using CD44 fixed to the solid phase and the hyaluronic acid-binding protein labeled with a labeling substance will be described here. In the measurement method of the present invention, as described above, the CD44 fixed to the solid phase is fixed. The hyaluronic acid-binding protein thus prepared and CD44 labeled with a labeling substance can also be used.

First, CD44 is fixed (coated) on the solid phase body. As a fixing method, for example, CD44 p
It is dissolved in a buffer solution (for example, phosphate buffer solution, carbonate buffer solution, etc.) of about H7-9 and added to the solid phase body, and stored at 37 ° C for 1-2 hours or at 4 ° C overnight. Examples of the method of fixing may be mentioned.

After the above fixation, a blocking substance is added and stored at about 37 ° C. for 30 minutes to 2 hours or at room temperature for 1 hour.
It is preferable to store it for 2 hours to cover the portion where the CD44 is not fixed. Then, a sample containing hyaluronic acid is added to the solid phase to which CD44 is fixed,
For example, the mixture is left standing or stirred at 37 ° C. for an optimum time of 20 to 80 minutes to bind hyaluronic acid to the CD44.
Then, the solid phase is added to a buffer solution (for example, phosphate buffer solution, Tris-HCl buffer solution, etc.) to which a tween-based surfactant or the like is added.
Wash with a cleaning solution such as. Furthermore, a hyaluronic acid-binding protein labeled with a labeling substance is added to the solid phase body, and the protein is allowed to bind to hyaluronic acid by allowing to stand still or stirring at 37 ° C. for an optimum time of 20 to 80 minutes. . By this operation, [CD44-hyaluronic acid-
Hyaluronic acid-binding protein]. Next, the labeling substance of the complex is detected to measure hyaluronic acid.

In the operation of reacting hyaluronic acid in the sample with CD44 and the operation of reacting hyaluronic acid bound to CD44 with the protein, it is preferable to carry out stirring because the reaction time can be shortened.

The method for detecting the labeling substance of the complex is, for example, when biotin is used as the labeling substance, for example, an enzyme to which streptavidin is bound is added to the complex, or hyaluronic acid labeled with biotin is added. When the binding protein is added, the enzyme bound with streptavidin is also added at the same time, and the enzymatic reaction of the enzyme bound to the complex via streptavidin is measured. The method of simultaneously adding the hyaluronic acid-binding protein labeled with the labeling substance and the reagent for detecting the labeling substance is preferable in that the measurement time can be shortened.

Then, a calibration curve is prepared for the relationship between the hyaluronic acid concentration and the detection result of the labeled substance, and hyaluronic acid in the unknown sample is quantified using the detection result of the unknown sample and the calibration curve.

(2) Inhibition Method A preferred embodiment of the inhibition method (competitive method) of the present invention will be described below. First, CD44 is fixed to the solid phase body, and then a blocking substance is preferably added to perform blocking. The fixing method is the same as that described in the above sandwich method, but it is preferable that the amount of CD44 fixed to the solid phase saturated with an appropriate amount of standard hyaluronic acid is preliminarily studied.

Then, the specimen containing hyaluronic acid was added to the solid phase body to which CD44 was fixed, and the mixture was allowed to stand or stir at 25 to 40 ° C. for 20 to 90 minutes for the optimum time to bind hyaluronic acid to CD44. Let Further, add hyaluronic acid labeled with a labeling substance, and add 2 at 25-40 ° C.
Stir or stir for an optimum time of 0 to 90 minutes, and
Hyaluronic acid labeled with a labeling substance is bound to D44. After this, it is preferable to wash the solid phase with, for example, a buffer solution (eg, phosphate buffer solution, Tris-HCl buffer solution, etc.) to which a surfactant (eg, Tween-based surfactant etc.) is added. Next, the hyaluronic acid in the sample is indirectly measured by detecting the labeling substance of hyaluronic acid labeled with the labeling substance bound to the CD44.

As another preferred form of the inhibition method, first, competitive hyaluronic acid is fixed (coated) on the solid phase. As a fixing method, for example, hyaluronic acid is dissolved in a buffer solution having a pH of about 7.0 to 9.2 (for example, a phosphate buffer solution, a carbonate buffer solution, etc.) and added to the solid-phase body for 25 to 25 times.
Store at 40 ° C for 1-2 hours or at 4 ° C for 14-18
Examples include a method of storing for about a time and fixing. After the above fixation, a blocking substance is added to
It is preferable to store at 40 ° C. for 1 to 2 hours or at room temperature for about 1 to 2 hours to coat the part to which the hyaluronic acid is not fixed. As a blocking substance,
Examples thereof include serum albumin, casein, milk protein and the like that can be collected from bovine and the like.

A specimen containing hyaluronic acid was added to the solid phase body to which the competitive hyaluronic acid was fixed, and then CD44 labeled with a labeling substance was added to the solid phase body at 25 to 40 ° C. for 2 hours.
The sample is hyaluronic acid fixed to the solid phase and bound to the labeled CD44 while being allowed to stand still or stirred for an optimum time of 0 to 90 minutes and being inhibited in the presence of hyaluronic acid as a sample. After that, it is preferable to wash the solid phase with, for example, a buffer solution (for example, a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, etc.) to which a surfactant (such as the Tween-based surfactant, etc.) is added. Next, the hyaluronic acid in the sample is indirectly measured by detecting the labeled CD44 labeling substance bound to the hyaluronic acid fixed to the solid phase body. This method is preferable because it can be carried out in one step reaction and is very simple.

The method for detecting the labeled substance in these inhibition methods is the same as the above-mentioned sandwich method, and it is possible to quantify hyaluronic acid in an unknown sample by preparing a calibration curve for the inhibition behavior by the standard hyaluronic acid concentration. it can.

[0085]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited thereto.

[0086]

[Preparation Example 1] <Preparation of fusion protein> A fusion protein of the extracellular region of CD44 and an Fc fragment of immunoglobulin (hereinafter, may be simply referred to as “fusion protein”) was prepared by the method of B. Seed et al. (Cell, 61,1303-1313 (1990))
Prepared according to. That is, the cDNA encoding the extracellular region of CD44, the cDNA encoding the Fc fragment of human IgG1 on the intracellular region side (carboxyl terminal side) and the cDNA encoding the signal sequence of CD5 on the amino terminal side are linked. The cDNA fragment thus prepared was incorporated into a CDM8 vector and transferred into COS cells by a known method using DEAE dextran to transiently express the fusion protein in COS cells. The COS cells were cultured in a serum-free medium, and the culture supernatant containing the extracellularly secreted fusion protein was collected. Purification of the fusion protein
It was carried out by affinity column chromatography using a protein A-binding resin by utilizing the immunoglobulin site in the molecule.

[0087]

Preparation Example 2 <Preparation of hyaluronic acid-binding protein> A hyaluronic acid-binding protein was prepared according to the production example (hyaluronic acid-binding protein) described in JP-A-4-262797. That is, an extraction operation was performed using a 4M guanidine hydrochloride solution from bovine nose septal cartilage, and then a supernatant of the extract was collected.
This was dialyzed against deionized water and freeze-dried to obtain a crude extract. This crude extract was digested with trypsin and then purified by affinity chromatography using a hyaluronic acid-binding resin.

[0088]

[Preparation Example 3] <Preparation of hyaluronic acid-binding protein labeled with a labeling substance> A biotin-labeled hyaluronic acid-binding protein was prepared as follows. That is, the purified hyaluronan-binding protein obtained in Preparation Example 2 (1 mg /
100 ml of NHS-LC-Biotin (product name: PIERCE) 1 mg / ml in 1 ml.
In addition, the mixture was reacted at room temperature for 4 hours and labeled with biotin. As the reaction buffer, 0.1 M carbonate buffer (pH 8.5) was used. After the reaction, phosphate buffered saline (pH
7.2-7.5: hereinafter, abbreviated as PBS) was dialyzed at 4 ° C. overnight to remove free biotin to obtain a biotin-labeled hyaluronic acid-binding protein.

[0089]

[Preparation Example 4] <Preparation of biotin-labeled hyaluronic acid> Hyaluronic acid (average molecular weight 40,000) was dissolved in 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.5). Biotin in 1 ml of 10 mg / ml solution
-Solution 2 in which LC-Hydrazide (trade name, manufactured by Pierce) was dissolved in dimethyl sulfoxide to 50 mM.
5 μl was added, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was further added to 0.1 M
Dissolved in MES buffer (pH 5.5) at 100 mg /
12.5 μl of a solution in ml was added, and the mixture was reacted at 20 ° C. for 16 hours with stirring.

Thereafter, dialysis was carried out using a dialysis membrane having a molecular weight of 3500 as the dialysis external solution for 2 hours at 20 ° C., and dialysis was carried out 3 times by changing the dialysis external solution. Then, the dialyzing solution containing biotin-labeled hyaluronic acid was adjusted to 5 mg / ml with distilled water and stored at -20 ° C.

[0091]

Preparation Example 5 <Preparation of fusion protein labeled with labeling substance> A biotin-labeled fusion protein was prepared as follows. That is, the purified fusion protein (1
mg / ml) 1 ml, NHS-LC-Biotin
100 μl of 1 mg / ml (trade name: manufactured by Pierce) was added, reacted at room temperature for 4 hours and labeled with biotin. As the reaction buffer, 0.1 M carbonate buffer (pH 8.5) was used. After completion of the reaction, dialysis was carried out against PBS at 4 ° C. overnight to remove free biotin to obtain a biotin-labeled fusion protein.

[0092]

[Comparative Example 1] <Measurement of hyaluronic acid using a commercially available kit for measuring hyaluronic acid> Measurement of hyaluronic acid was performed using a commercially available kit for measuring hyaluronic acid (trade name: hyaluronic acid "Chugai", manufactured by Chugai Pharmaceutical Co., Ltd.). went. The assay method of this kit is as follows: Beads bound with a partial peptide containing the hyaluronan-binding region of cartilage proteoglycan [described in the package insert of the kit as hyaluronan-binding protein (hereinafter abbreviated as HABP)] (It is described as binding protein beads in the document) and biotinylated HA
It is a method for measuring hyaluronic acid by a sandwich method using BP, and detection of biotinylated HABP is a method by an enzymatic reaction after reacting a peroxidase-labeled avidin.

As a sample, hyaluronic acid having an average molecular weight of 800,000 to 1.2 million (manufactured by Seikagaku Corporation), an average molecular weight of 1
Million, 380,000, 80,40, 40,000 and 10,000 hyaluronic acid,
Also, the standard solution of the kit for measuring hyaluronic acid was dissolved in the reaction buffer solution attached to the kit to prepare a solution of hyaluronic acid having a concentration of 50 to 800 ng / ml of each molecular weight, and the solution was measured according to the protocol of the kit.

As described in the package insert of the kit, the operation time was 90 minutes for reacting the sample hyaluronic acid with the binding protein beads, and the reaction was performed by adding biotinylated HABP and peroxidase-labeled avidin. The operation was carried out for 60 minutes and the enzymatic reaction of peroxidase was carried out for 30 minutes, for a total of 3 hours.
In addition, in addition to this, the package insert shall allow the reaction to stand (stand). Simultaneous reproducibility
Coefficient of variation of measured values (CV value) when measured simultaneously 0 times
Is 15% or less, the measurement range is 10 to 800 ng
/ Ml.

The method for detecting the labeling substance is in accordance with the protocol of the kit, and O is used as a substrate for peroxidase.
-Phenylenediamine dihydrochloride and hydrogen peroxide water were added to develop color, and after color development, the reaction was stopped with 3N sulfuric acid, and the absorbance at a wavelength of 492 nm (control wavelength 630 nm) was measured with an absorptiometer.

The obtained results are shown in FIG. From this result, it was found that this method for detecting the formation of HABP-hyaluronic acid-biotinylated HABP complex bound to beads is a highly reactive assay method for hyaluronic acid having an average molecular weight of 50,000 or more.

[0097]

Example 1 <Quantification of hyaluronic acid (sandwich method)> A solution of the fusion protein purified in Preparation Example 1 was treated with phosphate buffered saline (pH 7.2 to 7.5, containing no divalent ion) (hereinafter , P
Dilute to 600 ng / ml with BS (-) and add 50 μl (30 ng / well) of this solution to each well of Nunc Immuno Plate (trade name: Maxisorp, manufactured by Nunc) and store at 4 ° C for 16 hours. The coating was performed uniformly.

Then, this plate was diluted with PBS (-) to 2 times.
After washing twice, 3% bovine serum albumin (BSA) (sold by Seikagaku Corporation) as a blocking substance in order to coat the portion of the well not coated with the fusion protein
A PBS (-) solution containing was added and left standing at room temperature for 2 hours.

Then, the plate was washed with a washing solution (0.05
After washing 3 times with PBS (-) containing% Tween 20,
Hyaluronic acid standard solution of each concentration (concentration 0.1 to 50 ng /
50 μl of each concentration of ml, average molecular weight of 800,000 to 1.2 million, manufactured by Seikagaku Kogyo Co., Ltd. was added to the plate and allowed to react at 37 ° C. for 60 minutes. As the solvent for the hyaluronic acid standard solution, PBS (−) containing 1% BSA (hereinafter, referred to as “reaction diluent”) was used.

After that, this plate was washed with the washing solution described above.
After washing twice, 25 μl of a solution of the biotin-labeled hyaluronic acid-binding protein described in Preparation Example 3 diluted to 0.05 μg / ml with a reaction diluent was added, and in order to detect biotin in the obtained sandwich-like complex, Peroxidase-conjugated streptavidin (sold by Seikagaku Corporation)
25 μl of a solution obtained by diluting 2500 times with the reaction diluent was added, and the reaction was allowed to stand at 37 ° C. for 60 minutes. Further, this plate was washed three times with the above washing solution, and a tetramethylbenzidine solution (manufactured by Moss: as a substrate for peroxidase).
Hereinafter, 50 μl of TMB) was added and reacted at 37 ° C. for 15 minutes to develop color.

After color development, 50 μl of 1N HCl was applied to the plate.
The reaction was stopped by the addition of 1 and the absorbance of the coloring solution at a wavelength of 450 nm (control wavelength 630 nm) due to the decomposition of TMB was measured by a well reader (sold by Seikagaku Corporation). FIG. 2 shows the obtained results.

From this, according to the measuring method of the present invention,
It can be seen that 0.1 ng / ml of hyaluronic acid can be quantified. Since the sensitivity of the conventional measuring method is 0.5 ng / ml (see JP-A-4-262797) and 10 ng / ml (see JP-B-6-41952 and the commercially available kit described in Comparative Example 1 above), It was found that the measuring method is an extremely sensitive hyaluronic acid measuring method.

[0103]

[Example 2] In Example 1, instead of adding hyaluronic acid standard solution to the plate and allowing the plate to stand at 37 ° C for 1 hour to react, the plate was mixed for 30 minutes with a plate mixer (trade name: Micro Tube Mixer, sold by Titec). After reacting with stirring, a biotin-labeled hyaluronic acid-binding protein solution and a peroxidase-bound streptavidin (manufactured by Jackson Co., Ltd., sold by Seikagaku Corporation) solution were added, and the mixture was allowed to stand at 37 ° C for 60 minutes. The same measurement was performed except that the plate was stirred for 30 minutes instead of being reacted, and the TMB solution was added and the mixture was stirred at 37 ° C. for 15 minutes to be reacted.

The results are shown in FIG. It can be seen that the sensitivity was higher because the absorbance value was higher than that in Example 1. In addition, the time required for the measurement after preparing the plate to which the fusion protein is fixed is about 3 hours in the method described in Comparative Example 1, while it is 2 hours 1 in Example 1.
It is shortened to about 5 minutes, and is 1 in this embodiment in which a stirring operation is added.
The time was significantly reduced to about 15 minutes.

[0105]

Example 3 <Quantification of hyaluronic acid (inhibition method)> A solution of the fusion protein purified in Preparation Example 1 was treated with phosphate buffered saline (pH).
7.2-7.5: PBS) to 2 μg / ml, and 50 μl of this diluted solution was added to each well of the Nunc Immuno Plate and uniformly stored by storing at 4 ° C. for 16 hours.

Then, the plate was added to 200 μl of PBS.
After twice washing with PBS, PBS (-) solution 200μ containing 3% BSA (sold by Seikagaku Corporation) as a blocking substance
1 was added to each well and left at room temperature for 2 hours.

Then, the plate was washed three times with 200 μl of a washing solution (PBS containing 0.05% Tween 20),
Hyaluronic acid standard solution (each concentration of 0.1 to 1000 ng / ml, average molecular weight 1, 4, 8, 38, 1,000,000) was prepared using a reaction diluent (PBS (-) containing 1% BSA). 50 μl of each solution was added to the plate at a concentration of 37 ° C.
For 60 minutes.

Thereafter, 10 μl of a solution prepared by dissolving the biotin-labeled hyaluronic acid of Preparation Example 4 in the reaction diluent to 100 ng / ml was added to each well and reacted at 37 ° C. for 60 minutes.

Then, after washing three times with 200 μl of the washing solution,
A solution of peroxidase-bound streptavidin (sold by Seikagaku Corporation) diluted 5000-fold with a reaction diluent was added in an amount of 50 μl and reacted at 37 ° C. for 30 minutes.

After that, the plate was washed three times with a washing solution, 50 μl of TMB solution (manufactured by Moss) was added, and the mixture was reacted at 37 ° C. for 15 minutes to develop color. After the color development, 50 μl of 1N hydrochloric acid was added to stop the reaction, and the wavelength of the coloring liquid by the decomposition of TMB was 4
The absorbance at 50 nm (control wavelength 630 nm) was measured using a well reader (sold by Seikagaku Corporation). FIG. 4 shows the obtained results. In addition, the vertical axis of FIG. 4 shows the difference in absorbance between the absorbance in the control and the absorbance in the hyaluronic acid standard solution at each concentration.

As a result, C fixed to the solid phase of the present invention
According to the inhibition method using D44, it is possible to measure hyaluronic acid of about 0.1 ng / ml, and the reactivity is high for the sample hyaluronic acid having an average molecular weight of about 40,000 or more, particularly about 80,000 or more. It has been shown.

[0112]

Example 4 <Quantification of Hyaluronic Acid (Inhibition Method)> Hyaluronic acid (average molecular weight 800,000 to 1.2 million, manufactured by Seikagaku Corporation) was used as PB.
50 μl of a solution dissolved in S and adjusted to 100 μg / ml was added to each well of the Nunc Immunoplate, and stored uniformly at 4 ° C. for 16 hours for uniform coating.

Then, this plate was added to 200 μl of PBS.
After twice washing with PBS, PBS (-) solution 200μ containing 3% BSA (sold by Seikagaku Corporation) as a blocking substance
1 was added to each well and left at room temperature for 2 hours.

Then, the plate was washed 3 times with 200 μl of a washing solution (PBS containing 0.05% Tween 20),
Hyaluronic acid standard solution (each concentration of 0.1 to 1000 ng / ml, average molecular weight 1, 4, 8, 38, 1,000,000) was prepared using a reaction diluent (PBS (-) containing 1% BSA). With respect to the solution having the concentration and the reaction diluent as a control, 25 μl of each solution was added to the plate, and further 25 μl of the solution prepared by dissolving the biotin-labeled fusion protein described in Preparation Example 5 in the reaction diluent to 5 μg / ml was added, and added.
The reaction was carried out at 7 ° C for 60 minutes.

Then, after washing three times with 200 μl of the washing solution,
A solution of peroxidase-conjugated streptavidin (sold by Seikagaku Corporation) diluted 5000 times with a reaction diluent was added in an amount of 50 μl each, and reacted at 37 ° C. for 30 minutes.

After that, the plate was washed three times with a washing solution, and 50 μl of TMB solution (manufactured by Moss Co., Ltd.) was added, and the mixture was reacted at 37 ° C. for 15 minutes to develop color. After the color development, 50 μl of 1N hydrochloric acid was added to stop the reaction, and the wavelength of the coloring liquid by the decomposition of TMB was 4
The absorbance at 50 nm (control wavelength 630 nm) was measured using a well reader (sold by Seikagaku Corporation). The results obtained are shown in FIG. In addition, the vertical axis of FIG. 5 shows the difference in absorbance between the absorbance in the control and the absorbance in the hyaluronic acid standard solution at each concentration.

Thus, according to the inhibition method using hyaluronic acid fixed to the solid phase body of the present invention, 0.1 ng / m 2 was obtained.
It was shown that hyaluronic acid of about 1 can be measured, and the reactivity of the sample hyaluronic acid having an average molecular weight of about 80,000 or more is high.

[0118]

Example 5 <Relationship Between Molecular Weight of Hyaluronic Acid and Reactivity> The relationship between the molecular weight of hyaluronic acid and reactivity was examined in the measurement method of Example 1. Hyaluronic acid having an average molecular weight of 800,000 to 1.2 million (manufactured by Seikagaku Corporation), and an average molecular weight of 10
The amount of hyaluronic acid of 0,000, 380,000, 80,000, 40,000 was investigated. Concentration of hyaluronic acid of each molecular weight 0.1-10
It was prepared by a reaction diluent so that each concentration was 00 ng / ml, and measured by the method of Example 1. The obtained results are shown in FIG.

From this, it can be seen that the present method strongly reacts with hyaluronic acid having an average molecular weight of 80,000 or more and shows highly sensitive detectability, while the reaction with hyaluronic acid having an average molecular weight of 80,000 or less is weak. In the sandwich method in which hyaluronic acid is sandwiched between HABPs as in Comparative Example 1, since the reactivity with hyaluronic acid having an average molecular weight of 50,000 or more is high, the method by the sandwich method of the present invention is more effective than the conventional measurement method. It was found that high molecular weight hyaluronic acid was measured with high sensitivity.

[0120]

Example 6 <Influence on Measurement of Glycosaminoglycans Other Than Hyaluronic Acid> Cross-reactivity to glycosaminoglycans other than hyaluronic acid and influence on measurement of glycosaminoglycans in the method of Example 1 were examined. It was

Absorbance was measured by the method of Example 1 using 0.1 to 1000 ng / ml of various glycosaminoglycans (chondroitin sulfates A, B, C, D, E, keratan sulfate, and heparan sulfate) as samples, The cross-reactivity with the glycosaminoglycan was examined and the results are shown in FIG. 7.

Further, 1 μg of the glycosaminoglycan other than hyaluronic acid was added to 50 ng / ml of the sample hyaluronic acid.
/ Ml (20 times the amount of hyaluronic acid) was made to coexist, and the sample was measured by the method of Example 1 to examine the influence on the measurement results. That is, the absorbance of a sample in which these substances coexist was measured by the same method as in Example 1, and the ratio (%) to the absorbance of the sample of Example 1 (glycosaminoglycan-free system containing only hyaluronic acid) was determined. I asked. The obtained results are shown in FIG.

A fusion protein of the extracellular region of CD44 and the Fc fragment of human IgG1 which is a fusion protein similar to the fusion protein used in the present invention recognizes hyaluronic acid on a solid sample such as a tissue surface or a cell surface. Moreover, since it is known that it has a binding property to chondroitin sulfate although it is weaker than hyaluronic acid (Cell, 61, 1303-1313 (1990)), when CD44 is used for hyaluronic acid measurement, it has specificity. There was a risk of problems. However, according to the method of the present invention, from the above results, it is possible to measure hyaluronic acid in a liquid sample, and there is no cross-reaction (binding) with glycosaminoglycans other than hyaluronic acid, It can be seen that the presence of glycosaminoglycans does not affect the measurement of hyaluronic acid.

[0124]

Example 7 <Influence on the measurement of collagen type I, collagen type IV and fibronectin> Sample hyaluronic acid 100 ng / m
A sample in which 0.02 to 200 μg / ml of collagen type I, collagen type IV or fibronectin was coexistent in 1 was measured by the method of Example 1, and the influence on the measurement result was examined. That is, the absorbance of the sample in which these coexist was measured by the same method as in Example 1, and the ratio (%) to the absorbance of the sample of Example 1 (non-coexisting system) was determined. The results obtained are shown in FIG.

From these results, according to the assay method of the present invention, collagen type I (CL1 in the figure), type IV (CL4 in the figure),
It was shown that hyaluronic acid can be selectively measured without being affected by fibronectin (FN in the figure).

[0126]

Example 8 <Hyaluronic Acid Addition Recovery Test in Diluted Serum> In the method of Example 1, a control serum (human serum) was used as a sample.
When using the undiluted solution of human urine and its diluted solution,
The measured values tended to be slightly lower in the undiluted solution, probably due to reaction inhibition, but almost theoretical measured values were obtained in the diluted solution.

Therefore, in order to further investigate whether the method of Example 1 can specifically measure hyaluronic acid in serum,
An addition recovery test was performed by adding a known concentration of hyaluronic acid to the diluted serum. A known amount of hyaluronic acid (average molecular weight 800,000 to 1,200,000, manufactured by Seikagaku Corporation) was added to the control serum to a final concentration of 10 ng / ml, and finally diluted 3-fold with a reaction diluent. did. The hyaluronic acid concentration of each sample was calculated from the standard curve and compared with the added hyaluronic acid concentration, and as a result, a recovery rate of 96% was obtained.

Accordingly, according to the measuring method of the present invention,
It can be seen that hyaluronic acid in serum can be specifically measured.

[0129]

[Example 9] <Evaluation of reproducibility> In the method of Example 1, the CV value was determined by measuring 5 times simultaneously for a sample having a hyaluronic acid concentration of 10 ng / ml. It is said that the CV value is 15% or less using 10 ng / ml), while the method of the present invention shows an excellent value of about 5%, and this method is extremely excellent in reproducibility. It was shown that

[0130]

The hyaluronic acid measuring method of the present invention is very easy to operate and has a short measuring time. In addition, hyaluronic acid can be specifically measured, and even a very small amount can be measured, which is a highly sensitive measurement method. In addition, higher molecular weight hyaluronic acid can be measured than in the conventional method, and the reproducibility is excellent.

The measuring method of the present invention is, for example, for detecting a slight change in the concentration of hyaluronic acid in diagnosing inflammation such as rheumatism or diagnosing cancer or liver disease, etc. It is effective for measurement.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between hyaluronic acid concentration and absorbance, which was obtained using a commercially available hyaluronic acid measurement kit in Comparative Example 1.

FIG. 2 is a graph showing the relationship between hyaluronic acid concentration and absorbance obtained by the method for measuring hyaluronic acid of Example 1 (sandwich method).

FIG. 3 is a graph showing the relationship between hyaluronic acid concentration and absorbance obtained by the method for measuring hyaluronic acid of Example 2 (sandwich method).

FIG. 4 is a graph showing the relationship between the absorbance and the molecular weight and concentration of hyaluronic acid obtained by the method for measuring hyaluronic acid (inhibition method) in Example 3.

FIG. 5 is a graph showing the relationship between the molecular weight and concentration of hyaluronic acid and the absorbance, which was obtained by the method for measuring hyaluronic acid (inhibition method) in Example 4.

FIG. 6 is a graph showing the relationship between the molecular weight of hyaluronic acid and the absorbance obtained by the method for measuring hyaluronic acid of Example 5.

FIG. 7 is a graph showing the results of a cross-reactivity test with a glycosaminoglycan other than hyaluronic acid in Example 6.

FIG. 8 is a graph showing the influence of glucosaminoglycans other than hyaluronic acid on the measurement in Example 6.

FIG. 9 is a graph showing the influence of collagen type I, collagen type IV, and fibronectin on the measurement in Example 7.

[Explanation of symbols]

1 ... Measurement curve of hyaluronic acid having an average molecular weight of 800,000 to 1.2 million 2 ... Measurement curve of hyaluronic acid having an average molecular weight of 1,000,000 3 ... Measurement curve of hyaluronic acid having an average molecular weight of 380,000 4 ... Average Measurement curve of hyaluronic acid with a molecular weight of 80,000 5 ... Measurement curve of hyaluronic acid with an average molecular weight of 40,000 6 ... Measurement curve of hyaluronic acid with an average molecular weight of 10,000 7 ... Measurement of hyaluronic acid standard solution in a commercial kit Curve 8 ... Measurement curve when agitating and reacting 9 ... Measurement curve when agitating and reacting (Example 1
Results: 10 ... Chondroitin sulfate A measurement curve 11 ... Chondroitin sulfate B measurement curve 12 ... Chondroitin sulfate C measurement curve 13 ... Chondroitin sulfate D measurement curve 14 ... Chondroitin sulfate E Measurement curve 15 ... Keratan sulfate measurement curve 16 ... Heparan sulfate measurement curve 17 ... Hyaluronic acid measurement curve obtained from pig skin 18 ... Hyaluronic acid measurement obtained from human umbilical cord Curve A ... Measurement result when glycosaminoglycan containing only hyaluronic acid is not added B ... Measurement result when heparan sulfate coexists C ... Measurement result when keratan sulfate coexists D ... Measurement results when chondroitin sulfate E coexisted E ... Measurement results when chondroitin sulfate D coexisted F ... Measurement results in the presence of chondroitin sulfate C G ... Measurement results in the presence of chondroitin sulfate B H ... Measurement results in the presence of chondroitin sulfate A CL4 ... Collagen type IV CL1 ... Measurement results when collagen type I coexisted FN ... Measurement results when fibronectin coexisted

Claims (14)

[Claims] 1. A method for measuring hyaluronic acid in a sample, which comprises the step of reacting CD44 with hyaluronic acid in the sample to form a complex of CD44 and hyaluronic acid. 2. A complex of CD44 and hyaluronic acid,
The measuring method according to claim 1, which is a complex represented by the following formula (I). Embedded image In the formula (I), A is CD44, B is hyaluronic acid,
C represents hyaluronic acid binding protein or CD44. ) 3. After reacting CD44 with hyaluronic acid in a sample and optionally with a hyaluronic acid-binding protein to form a complex represented by the formula (I), the complex is reacted with The method according to claim 2, wherein the complex or unreacted CD44 or hyaluronan-binding protein is detected by separating from CD44 or hyaluronan-binding protein. 4. Reacting hyaluronic acid in a sample with CD44 fixed to a solid phase and a hyaluronic acid-binding protein or CD44 labeled with a labeling substance, or by labeling hyaluronic acid in a sample with a labeling substance By reacting the CD44 thus prepared with a hyaluronan-binding protein fixed to a solid phase, a complex represented by the formula (I) is formed, and the complex is reacted with the unreacted labeled hyalurone. Separated from the acid-binding protein or the labeled CD44, the labeling substance in the complex or the unreacted labeled hyaluronic acid-binding protein or the labeled C
The measuring method according to claim 2, wherein the labeling substance of D44 is detected. 5. The method according to claim 2, wherein the hyaluronic acid-binding protein is selected from the group consisting of proteoglycan, link protein, and hyaluronectin. 6. CD44 and hyaluronic acid in a sample and competitive hyaluronic acid are reacted to bind CD44 competitively to hyaluronic acid in the sample and competitive hyaluronic acid, whereby CD44 and hyaluronic acid in the sample. The measuring method according to claim 1, comprising the step of forming a complex with an acid and a complex with CD44 and a competitive hyaluronic acid. 7. The measuring method according to any one of claims 1 to 6, wherein CD44 is a partial protein or a fusion protein containing a hyaluronic acid binding region of CD44. 8. A fusion protein containing the hyaluronan-binding domain of CD44 is a fusion protein consisting of CD44 or a partial protein containing the hyaluronan-binding domain of CD44 and an immunoglobulin or a partial protein of immunoglobulin. The measuring method according to claim 7. 9. A kit for use in a method for measuring hyaluronic acid in a sample by forming a complex between CD44 and hyaluronic acid in the sample, comprising CD44 as a component. Hyaluronic acid measurement kit. 10. The hyaluronic acid according to claim 9, which is mainly composed of CD44 and a hyaluronic acid-binding protein, and wherein the CD44 or the hyaluronic acid-binding protein is or can be labeled with a labeling substance. Acid measurement kit. 11. The kit for measuring hyaluronic acid according to claim 9, which is mainly composed of a solid phase having CD44 fixed thereon and a hyaluronic acid-binding protein labeled with a labeling substance. 12. The kit for measuring hyaluronic acid according to claim 9, which is mainly composed of a solid phase having CD44 fixed thereon and hyaluronic acid labeled with a labeling substance. 13. The kit for measuring hyaluronic acid according to claim 9, which is mainly composed of CD44 which is or can be labeled with a labeling substance and a solid phase body to which hyaluronic acid is fixed. 14. The kit for measuring hyaluronic acid according to any one of claims 9 to 13, wherein CD44 is a partial protein or a fusion protein containing a hyaluronic acid-binding region of CD44.