JP2000111553A - Measuring method for normal aggrecan and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an extremely practical measuring method for normal aggrecan and to provide an extremely practical detection method for the anomaly of an articular cartilage substrate. SOLUTION: A specimen is brought into contact with a solid phase to which one or two or more antibodies selected from a group of antibodies composed of an antikeratan sulfate antibody (e.g. 5D4) and an antichondroitin sulfate antibody (e.g. CS56, LY111) are stuck, Thereby, a complex which is composed of 'a solid phase-stuck antibody-normal aggrecan-hyaluronic acid' is formed. Then, a proten which has a bonding capability to hyaluronic acid (preferably a protein labeled with a marker substance, e.g. a protein derived from cartilage proteoglycan core protein) is brought into contact with the solid phase. Thereby, a sandwich-shaped complex which is composed of 'a solid phase-stuck antibody- normal aggrecan-hyaluronic acid-a protein having a bonding capability to hyaluronic acid' is formed. When this method is used, the anomality of an articular cartilage substrate can be detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring aggrecan (normal aggrecan) capable of associating with hyaluronic acid, and more particularly, to a method for immunoassay of normal aggrecan and its application (detecting abnormalities in articular cartilage matrix). Method and a kit for measuring normal aggrecan).

[0002]

2. Description of the Related Art Proteoglycans in cartilage are called "aggrecans" and chondroitin sulfate (hereinafter sometimes referred to as "CS") and keratan sulfate (hereinafter sometimes referred to as "KS") as core proteins. ) Has a bonded structure. Aggrecan usually associates with hyaluronic acid (hereinafter sometimes referred to as “HA”) in the cartilage matrix to form a large aggregate. It is known that the ability of aggrecan to associate with HA decreases with the degradation and denaturation of the cartilage matrix. Degradation products of aggrecan are also present in synovial fluid, and it is also known that a decrease in the ability of cartilage or aggrecan in synovial fluid to associate with HA is useful as an indicator of cartilage matrix abnormality (WO 9).
No. 5/22765).

[0003] In the present specification, aggrecan having the ability to associate with HA is referred to as "normal aggrecan", aggrecan not having the ability to associate with HA is referred to as "abnormal aggrecan", and normal aggrecan and abnormal aggrecan are collectively referred to as "aggrecan". .
In this specification, the degradation product of aggrecan present in synovial fluid is also included in the term “aggrecan”. Less than,
The technique closest to the present invention will be described with reference to the document names.

[0004] US Patent No. 5,185,245 includes anti-K
S antibody or anti-CS antibody is immobilized on the solid phase,
(Joint fluid), and further contacted with a labeled anti-KS antibody or anti-CS antibody, whereby “anti-CS antibody (or anti-KS antibody) -proteoglycan-anti-CS antibody (anti-K
S antibody)) to form a sandwich-like complex
A method for detecting proteoglycan in synovial fluid by detecting this, a method for detecting joint destruction, and the like are described. In addition, a kit for detecting proteoglycan in a body fluid, which contains, as a component, a solid phase to which an antibody against a glycosaminoglycan component of proteoglycan is fixed is described. However, the idea of measuring normal aggrecan separately from abnormal aggrecan, using a protein capable of binding to HA, and using a specific antibody (5D4, CS
No mention is made of using a solid phase to which one or more antibodies selected from the group consisting of 56 and LY111) are immobilized.

[0005] WO95 / 22765 discloses that HA associated with normal aggrecan and free HA in a sample are decomposed or removed with hyaluronidase or the like, and the resulting treated sample is subjected to a solid phase (eg, a solid phase capable of binding or adsorbing normal aggrecan). A method of detecting normal aggrecan bound or adsorbed to a solid phase (HA-bound solid phase) is described. In addition, for the detection of normal aggrecan bound or adsorbed to a solid phase, an anti-CS antibody or an anti-KS antibody can be used.
S-56, MO-225, MC21C, S54C, 3B
3, and specifically 5 as an anti-KS antibody.
D4 is described. It is also described that the pathology of arthritis can be grasped by measuring normal aggrecan by this method.

However, specific antibodies (5D4, CS
There is no disclosure of using a solid phase to which one or more antibodies selected from the group consisting of 56 and LY111 are immobilized, and using a protein capable of binding to hyaluronic acid. In this method, a reagent such as hyaluronidase is required, and a step of hyaluronidase treatment is required.

[0007]

SUMMARY OF THE INVENTION Normal aggrecan can be synthesized with specificity, high sensitivity, high quantitativeness and reproducibility without using many devices and reagents (eg, hyaluronidase).
If a simple, quick and inexpensive method could be provided,
It is extremely practical as a method for measuring normal aggrecan, and it is extremely easy to detect abnormalities in the articular cartilage matrix. Therefore, such a measuring method and a measuring kit have been desired. That is, an object of the present invention is to provide an extremely practical method for measuring normal aggrecan and an extremely practical method for detecting abnormalities in the articular cartilage matrix.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that normal aggrecan can be made extremely specific and highly sensitive by using a solid phase to which a specific antibody is fixed. The present inventors have found a method that can easily, rapidly and inexpensively measure with good quantitative and reproducibility, and have found that this method can detect abnormalities in the articular cartilage matrix extremely easily, thereby completing the present invention.

That is, the gist of the present invention is a method of measuring normal aggrecan in a sample, which comprises at least the following steps (hereinafter, may be abbreviated as “the measuring method of the present invention”). Step 1: A sample is brought into contact with a solid phase to which one or more antibodies selected from an antibody group consisting of an anti-keratan sulfate antibody and an anti-chondroitin sulfate antibody are fixed, and “solid-phase fixed antibody-normal aggrecan-hyaluronic acid” Forming a composite. Step 2: A protein having a binding ability with hyaluronic acid is further contacted with the solid phase to form a sandwich-like complex composed of "solid phase-immobilized antibody-normal aggrecan-hyaluronic acid-protein capable of binding with hyaluronic acid" Process to make it. Step 3: a step of detecting the sandwich-like complex formed in step 2.

The above-mentioned anti-keratan sulfate antibody is preferably 5D4, and the above-mentioned anti-chondroitin sulfate antibody is preferably one or two or more antibodies selected from the group consisting of CS56 and LY111. Preferably, step 1 above comprises 5D4, CS56 and LY11
A step of contacting a sample with a solid phase to which one or more antibodies selected from the group consisting of 1 are fixed to form a complex consisting of “solid phase fixed antibody-normal aggrecan-hyaluronic acid” The gist is the characteristic measuring method of the present invention.

[0011] The above-mentioned protein capable of binding to hyaluronic acid is labeled with a labeling substance,
In the present invention, preferably, in the above step 2, the solid phase is further contacted with a protein having a binding ability with hyaluronic acid labeled with a labeling substance, and “solid-phase-immobilized antibody-normal aggrecan-hyaluronic acid-hyaluronic acid” And a protein having a binding ability. The protein capable of binding to the hyaluronic acid is a protein derived from the cartilage proteoglycan core protein.
Is further contacted with the solid phase, a protein capable of binding to hyaluronic acid derived from cartilage proteoglycan core protein or from a cartilage proteoglycan core protein labeled with a labeling substance.
The subject of the present invention is a method for forming a sandwich-like complex comprising "normal aggrecan-hyaluronic acid-protein capable of binding to hyaluronic acid".

Further, the present invention provides a method for detecting an abnormality of an articular cartilage matrix, which comprises at least the following steps. (Hereinafter, also referred to as “the detection method of the present invention”). Step 1: Normal aggrecan in joint fluid is measured by the above-described measurement method of the present invention. Step 2: a step of associating the amount of normal aggrecan in the synovial fluid obtained in Step 1 with the abnormality of the articular cartilage matrix.

The present invention also provides a kit for measuring normal aggrecan (hereinafter sometimes abbreviated as "the kit of the present invention") characterized by containing at least the following (A) and (B). I have. (A) A solid phase to which one or more antibodies selected from the group consisting of an anti-keratan sulfate antibody and an anti-chondroitin sulfate antibody are fixed. (B) a protein labeled with a labeling substance and capable of binding to hyaluronic acid. The kit for measuring normal aggrecan described above is a kit for detecting abnormalities in articular cartilage matrix, and the present invention provides a kit for detecting abnormalities in articular cartilage matrix comprising the measurement kit of the present invention (hereinafter, the “detection kit of the present invention”). May be abbreviated as ".").

[0014]

Embodiments of the present invention will be described below. <1> Measurement method of the present invention The measurement method of the present invention is a method for measuring normal aggrecan in a sample, comprising at least the following steps. Step 1: a step of bringing a sample into contact with a solid phase to which one or more antibodies selected from the following antibody group are fixed to form a complex consisting of “solid phase-fixed antibody-normal aggrecan-HA”. Antibody group: anti-KS antibody, anti-CS antibody Step 2: The solid phase is further contacted with a protein capable of binding to HA, and the “solid-phase fixed antibody-normal aggrecan-
Forming a sandwich-like complex consisting of “a protein capable of binding to HA-HA”. Step 3: a step of detecting the sandwich-like complex formed in step 2.

Hereinafter, the measuring method of the present invention will be described in detail for each step. (1) Step 1 (1) -1 Anti-KS antibody and anti-CS antibody (1) -1-1 Anti-KS antibody The anti-KS antibody is not particularly limited as long as it is an antibody that reacts with KS. The antibody may be an antibody or a polyclonal antibody, but is preferably a monoclonal antibody, and among them, the following monoclonal antibody is preferred. (A) 5D4 5D4 [J. Biol. Chem., 258, 8848- (1
983), Eur. J. Biochem., 157, 385-
391 (1986)] is a monoclonal antibody obtained by immunizing a mouse with a proteoglycan core protein obtained by digesting a proteoglycan monomer of human articular cartilage with chondroitinase ABC and immunizing a mouse with the following reactivity. Reacts with corneal and cartilage KS; It reacts with proteoglycan monomers prepared from human, monkey, bovine, sheep, chicken and shark hyaline cartilage, but also dermatan sulfate (hereinafter sometimes referred to as “DS”) and heparin (hereinafter also referred to as “Hep”). Does not react with heparan sulfate (hereinafter, also referred to as “HS”) or HA. 5D4 is sold by Seikagaku Corporation.

(1) -1-2 Anti-CS Antibody The anti-CS antibody is not particularly limited as long as it reacts with CS, and may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies are preferable, and among them, the following monoclonal antibodies are preferable. (B) CS56 CS56 [Exp. Cell Res., 158, 321-
(1985), J. Cell Biol., 91, 205-.
(1981), Biochem. Biophys. Ac
ta., 694, 375- (1982), J. Biol. Ch.
em., 255, 7102- (1980), Int. Re.
v. Cytol., 53, 65- (1978)] is a monoclonal antibody obtained by immunizing mice with chicken gizzard fibroblasts as an antigen and having the following reactivity; native CS proteoglycan Specific for glycosaminoglycan moieties, chondroitin sulfate A (C
SA) reacts with chondroitin sulfate C (CS-C), but does not react with DS. CS56 is sold by Seikagaku Corporation. (C) LY111 LY111 is a monoclonal antibody obtained by immunizing mice with CS-A as an antigen. LY1
11 is sold by Seikagaku Corporation.

(1) -2 Solid phase The solid phase on which the anti-KS antibody or anti-CS antibody is immobilized is not particularly limited as long as the antibody can be immobilized and the solid phase is insoluble in water, a sample or a reaction solution for measurement. . Examples of the shape of the solid phase include a plate (for example, a well of a microplate), a tube, beads, a membrane, and a gel. Examples of the material of the solid phase include polystyrene, polypropylene, nylon, and polyacrylamide. Among these, a plate made of polystyrene is preferable. As a method for immobilizing an anti-KS antibody and / or an anti-CS antibody on these solid phases, a general method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bonding method, and an inclusive method (immobilized enzyme, 1975) Year, Kodansha, pp. 9-75) can be applied. Among them, the physical adsorption method is preferred because the operation is simple and frequently used. Specific examples of the physical adsorption method include the following methods; anti-KS antibody and / or anti-CS antibody are buffered with a buffer of about pH 7 to 9 (eg, phosphate buffer, phosphate buffered saline ( PBS),
Carbonate buffer, etc.) and add it to a solid phase (eg, microplate) and store at about 37 ° C for 1-2 hours or 4 ° C
Store overnight and allow to stick. In addition, the surface of the solid phase to which the anti-KS antibody and / or the anti-CS antibody has been immobilized may sometimes have a surface portion on which the anti-KS antibody and / or anti-CS antibody are not immobilized. If it adheres, accurate measurement results may not be obtained. Therefore, it is preferable to add a blocking substance to cover the portion where the anti-KS antibody and / or anti-CS antibody is not fixed before bringing the sample into contact with the solid phase. Examples of such a blocking substance include serum albumin, casein, skim milk, gelatin and the like, and commercially available blocking substances can also be used. Specific examples of the blocking method include the following methods; adding a blocking substance (serum albumin, casein, skim milk, gelatin, etc.) and storing at about 37 ° C. for 30 minutes to 2 hours; Store at room temperature (15-25 ° C) for 1-2 hours.

(1) -3 Specimen The specimen is not particularly limited as long as it contains normal aggrecan, which is a substance to be measured, or is likely to contain normal aggrecan.
Also, the sample need not be purified in advance. Specific examples of the specimen include joint fluid, cartilage extract, blood, serum, plasma, urine, and the like. A method for contacting a sample with a solid phase to which an anti-KS antibody and / or an anti-CS antibody is fixed is as follows:
There is no particular limitation as long as the solid phase comes into contact with the sample. For example, a sample may be added to and brought into contact with a solid phase to which an anti-KS antibody and / or an anti-CS antibody is immobilized, and a solid phase to which an anti-KS antibody and / or an anti-CS antibody is immobilized may be added to the sample. You may make it contact. After contacting these two components, in order to sufficiently bind the anti-KS antibody and / or anti-CS antibody to the normal aggrecan in the sample, the reaction is carried out at, for example, 0 to 45 ° C., preferably 37 ° C. for about 1 hour. Is preferred. After the reaction, sufficient separation of the solid phase and the liquid phase or washing of the solid phase, for example, washing the surface of the solid phase with a washing solution to remove non-specifically adsorbed substances and components in the unreacted sample can be performed. preferable. As the cleaning liquid, for example,
Buffers to which a nonionic surfactant such as Tween-based surfactant is added (for example, phosphate buffer, P
BS, Tris-HCl buffer, etc.).
By bringing the sample into contact with the solid phase to which the anti-KS antibody and / or anti-CS antibody is immobilized, aggrecan contained in the sample and the anti-KS antibody and / or anti-CS antibody form a complex. It should be noted that normal aggrecan has an ability to associate with HA, and usually exists as a complex consisting of “normal aggrecan-HA” in a specimen such as synovial fluid, so that anti-KS antibody and / or anti-CS antibody are By bringing the sample into contact with the fixed solid phase, the “solid-phase fixed antibody-
A complex consisting of "normal aggrecan-HA" is formed.
Also, in this step, “solid phase fixed antibody-abnormal aggrecan”
Are also formed. In addition, when the normal aggrecan in the sample is not present as a complex consisting of “normal aggrecan-HA”, the sample contains H having a molecular weight of several hundred thousand or more.
It is necessary to add A to form a complex consisting of “normal aggrecan-HA”.

(2) Step 2 (2) -1 Protein capable of binding to HA The protein capable of binding to HA is not particularly limited as long as it is a protein capable of binding to HA. Proteoglycans (eg, cartilage proteoglycan, trypsin digest of cartilage proteoglycan, chondroitinase A of cartilage proteoglycan
BC digest), proteoglycan core protein (eg, cartilage proteoglycan core protein, etc.), link protein, hyaluronectin, CD4
4. Partial proteins containing the HA binding site of these proteins, or fusion proteins of the partial proteins with other proteins, and the like. Particularly, tryptic digest of cartilage proteoglycan, and further tryptic digestion of bovine nasal cartilage proteoglycan Are preferred. In addition,
The trypsin digest of bovine nasal cartilage proteoglycan is sold by Seikagaku Corporation as "hyaluronic acid binding protein". Also, H
It is preferable that the protein capable of binding to A is labeled with a labeling substance, since the detection in step 3 is easy. Labeling substances used for labeling a protein capable of binding to HA include enzymes (peroxidase,
Alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), fluorescent dye (fluorescein isothiocyanate (FITC))
Etc.), chemiluminescent substances (such as luminol), biotin,
Avidin (including streptavidin) and the like can be mentioned, but are not particularly limited as long as they can be normally used for labeling proteins. Labeling methods include known methods suitable for labeling substances, for example, a glutaraldehyde method, a periodic acid crosslinking method, a maleimide crosslinking method, a carbodiimide method, an activated ester method, etc. [“Protein Chemistry (Lower)”, Tokyo Chemical Dojin , Issued in 1987]. For example, when biotin is used as a labeling substance, a method using a hydrazide derivative of biotin (Avi
din-Biotin Chemistry: A Han
dbook, p57-63, PIERCE CHEMIC
AL Company, published in 1994), and when fluorescein isothiocyanate is used, it can be appropriately selected from the methods described in JP-B-63-17843. A tryptic digest of bovine nasal cartilage proteoglycan labeled with biotin is marketed by Seikagaku Corporation as “biotin-labeled hyaluronic acid binding protein”. By bringing a protein having a binding ability with HA into contact with the solid phase (having a complex formed of “solid phase-fixed antibody-normal aggrecan-HA”), “solid-phase fixed antibody-normal aggrecan-HA- A sandwich-like complex consisting of "a protein capable of binding to HA" is formed. Such a sandwich-like complex is not formed in the complex consisting of “solid phase-immobilized antibody-abnormal aggrecan” because a protein having a binding ability with HA does not bind. After the two are brought into contact with each other, in order to sufficiently bind the complex consisting of “solid phase-immobilized antibody-normal aggrecan-HA” and a protein capable of binding to HA, for example, 0 to 45 ° C., preferably 3 ° C.
The reaction is preferably performed at 7 ° C. for about 1 hour. After the reaction, it is preferable to sufficiently separate the solid phase and the liquid phase or wash the solid phase, for example, wash the surface of the solid phase with a washing liquid to remove non-specifically adsorbed substances and the unreacted proteins. As the cleaning liquid, for example, Tween (Tween)
n) It is preferable to use a buffer to which a nonionic surfactant such as a surfactant is added. In addition, when using a protein to which KS or CS is bound, such as cartilage proteoglycan, as a protein capable of binding to HA, a complex consisting of “solid phase-fixed antibody-normal aggrecan-HA” was formed. Thereafter, it is preferable to contact CS and / or KS with the solid phase to block unreacted antibodies (antibodies that do not form a complex with normal aggrecan).

(3) Step 3 Step 3 is a step of detecting the sandwich-like complex formed in step 2. The detection may be performed using an antibody that specifically reacts with a protein capable of binding to HA, and when a protein capable of binding to HA is labeled with a labeling substance in step 2, Those skilled in the art can appropriately select a detection method according to the labeling substance used. For example, when using biotin as a labeling substance, an enzyme to which streptavidin or the like is bound is added, and an enzyme such as peroxidase is bound to a complex containing biotin as a labeling substance via the streptavidin or the like, A method of adding a coloring substrate such as tetramethylbenzidine or the like and a hydrogen peroxide solution as a substrate of the enzyme and measuring the degree of coloring of a product by the enzymatic reaction by a change in absorbance can be mentioned. When a fluorescent substance or a chemiluminescent substance is used, a method of measuring the fluorescence or luminescence of the solution after the reaction may be used. The phrase "a protein capable of binding to hyaluronic acid is labeled with a labeling substance" does not only mean that the protein is directly labeled, but also does not mean that the protein or antibody is labeled as described above. It also means that it can be finally labeled with avidin or the like. In the measurement method of the present invention, the concentration of aggrecan in a sample is determined by preparing a calibration curve for the relationship between the aggrecan concentration and the detection result (eg, absorbance) of a labeled substance using an aggrecan standard solution having a known concentration in advance. Can be obtained by using the detection result of the sample and the calibration curve. According to the method of the present invention, by using an anti-KS antibody and / or an anti-CS antibody and a protein capable of binding to hyaluronic acid, aggrecan capable of binding to hyaluronic acid (normal aggrecan) is separated from abnormal aggrecan. And can be detected. In the measurement method of the present invention, as is clear from the examples described later, since normal aggrecan can be measured without using a reagent such as hyaluronidase or a device such as high-performance liquid chromatography, it is simple, quick and inexpensive. Can be measured.
In addition, the measurement method of the present invention can measure normal aggrecan specifically, with high sensitivity, and with good quantitativeness and reproducibility.

<2> Detection Method of the Present Invention The detection method of the present invention is a method for detecting abnormalities of articular cartilage matrix, which includes at least the following steps. Step 1: Normal aggrecan in synovial fluid is measured by the measuring method according to any one of claims 1 to 5. Step 2: a step of associating the amount of normal aggrecan in the synovial fluid obtained in Step 1 with the abnormality of the articular cartilage matrix. Hereinafter, the detection method of the present invention will be described in detail for each step.

(1) Step 1 Step 1 is exactly the same as the measurement method of the present invention <1> above, but in the detection method of the present invention, synovial fluid is used as a specimen. Note that an extract of cartilage may be used. (2) Step 2 Step 2 is a step of associating the normal aggrecan amount in the synovial fluid obtained in Step 1 with the abnormality of the articular cartilage matrix. The “normal aggrecan amount” may be the measured value of the normal aggrecan amount itself measured in step 1, or the ratio of the normal aggrecan amount measured in step 1 to the aggrecan amount in the synovial fluid. You may. The amount of aggrecan in the synovial fluid can be determined, for example, in US Pat.
It can be measured by the method described in No. 245 or the like. The abnormality of the articular cartilage matrix is not particularly limited as long as it is an abnormality accompanied by a decrease in the amount of normal aggrecan in the synovial fluid, and for example, a disease involving degeneration or destruction of articular cartilage, more specifically deformability Arthropathy, rheumatoid arthritis,
Examples include traumatic arthritis, gout and the like. In such a disease, the amount of normal aggrecan in the synovial fluid is significantly reduced as compared with the amount of normal aggrecan in the synovial fluid of healthy humans (humans without abnormalities in the articular cartilage matrix). If the amount of normal aggrecan is lower than the amount of normal aggrecan in the synovial fluid of healthy subjects, there is a high possibility that "there is an abnormality in the articular cartilage matrix" or "there is an abnormality in the articular cartilage matrix" ]. If the amount of normal aggrecan measured in step 1 is equal to the amount of normal aggrecan in the synovial fluid of a healthy person, there is no possibility that “there is no abnormality in the articular cartilage matrix” or “ Low ". In the detection method of the present invention, not only the presence or absence of abnormalities in the articular cartilage matrix,
Detection of the degree of the abnormality is also included. For example, the amount of normal aggrecan in the synovial fluid of an individual is periodically measured in step 1, and if the amount of normal aggrecan is decreasing, the abnormalities of the articular cartilage matrix are progressing. It is highly probable that abnormalities are progressing. " When the amount of normal aggrecan measured in step 1 is on the increase, it is highly likely that “the abnormality of the articular cartilage matrix is in the direction of improvement” or “the abnormality of the articular cartilage matrix is in the direction of improvement” ]. When there is no change in the amount of normal aggrecan measured in step 1, “there is no change in the abnormality (normality) of the articular cartilage matrix” or “the change in the abnormality (normality) of the articular cartilage matrix” It is likely that there is not. " The normal aggrecan amount as a detection standard for the abnormality of the articular cartilage matrix may be the normal aggrecan concentration obtained using a calibration curve created for the relationship between the concentration of the normal aggrecan standard and the detection result of the labeling substance. Alternatively, the ratio may be a ratio to the normal aggrecan amount in the synovial fluid of a healthy human (a human having no abnormality in the articular cartilage matrix) without using the calibration curve.

<3> Measurement Kit of the Present Invention The measurement kit of the present invention is a kit for measuring normal aggrecan, including at least the following components. (A) a solid-phase antibody group to which one or more antibodies selected from the following antibody groups are fixed: anti-keratan sulfate antibody, anti-chondroitin sulfate antibody (B) labeled with a labeling substance and capable of binding hyaluronic acid Description of the solid phase of the protein (A) and the protein of the (B) is the same as in <1> the measurement method of the present invention. The measurement kit of the present invention is not particularly limited as long as it contains at least the above (A) and (B), and further includes a standard concentration of a normal aggrecan standard, a detection reagent for a labeling substance, and HA as a standard for preparing a calibration curve. Reagent for labeling a protein having binding ability, or a reagent for detecting a protein having binding ability to HA (antibody to the labeled protein, etc.)
Etc. can be added as a configuration. Further, in addition to these components, the blocking substance, the washing solution, the sample diluting solution, the enzyme reaction stopping solution, and the like may be included. These configurations can be stored in separate containers, respectively, and stored as a kit that can be used according to the measurement method of the present invention at the time of use. The measurement of normal aggrecan using the measurement kit of the present invention can be performed according to the measurement method of the present invention of <1>.

<4> Detection Kit of the Present Invention The detection kit of the present invention is a kit for detecting abnormalities in the articular cartilage matrix, comprising the measurement kit of the present invention. The description of the configuration and the like of the detection kit of the present invention is the same as that of the measurement kit of the present invention <3>. Detection of abnormalities in the articular cartilage matrix using the detection kit of the present invention can be performed according to the detection method of the present invention described in the above <2>.

[0025]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.
The antibodies, proteins capable of binding to HA, and normal aggrecan used in this example will be described. (1) Antibody (1-1) 5D4 was used as an anti-KS antibody. The characteristics and the like of 5D4 have been described above. (1-2) CS56, LY111,
MO-225 and MC21C were used. The characteristics of CS56 and LY111 have been described above. MO-225
And the characteristics of MC21C will be described below. MO-225: MO-225 [J. Biol. Chem.,
262, 4146-4152 (1987), J. Bio
Chem., 264, 8012-8018 (198
9)] is a monoclonal antibody obtained by immunizing a mouse with a chicken embryo limb bud proteoglycan as an antigen and having the following reactivity; a determinant (D-glucuronic acid-2) on an intact CS chain -Sulfuric acid (β1 → 3) -N-
Recognition of the disaccharide structure of acetylgalactosamine-6-sulfate (D-unit)), and those in which the repeating structure of D-units are linked react more strongly. MO-225 is
Sold by Seikagaku Corporation. MC21C: MC21C [Dev. Biol., 133, 4
75-488 (1989), Differentiat
ion, 43, 37-50 (1990), Int. Dev.
Biol., 34, 191-204 (1990)] is a monoclonal antibody obtained by immunizing a mouse with a mature rat bone protein as an antigen and having the following reactivity: rat, mouse, chicken, shark. Reacts with bovine intact CS-C and does not react with DS, heparan sulfate (hereinafter sometimes referred to as "HS") or KS. MC21C is sold by Seikagaku Corporation. (1-3) 2-B-1 was used as an anti-proteoglycan core protein antibody. The characteristics of 2-B-1 will be described below. 2-B-1 [Histochemicala
1 J., 21, 455-459 (1989)] is a monoclonal antibody obtained by immunizing a mouse with proteoglycan prepared from a human Yorksack tumor as an antigen and having the following reactivity; proteoglycan Recognizes the core protein of In adult tissues, the intima and media of the aorta are stained, and weakly react to perivascular and perimus connective tissues, while in the fetus, all tissues are strongly stained by stroma. In cancer tissues, it reacts with all stromal fiber components such as stomach cancer, rectal cancer, breast cancer, uterine cancer, and ovarian cancer, and shows a cancer-specific stromal image. 2-B-1 is sold by Seikagaku Corporation.

(2) Protein having HA binding ability "Biotin-labeled hyaluronic acid-binding protein" sold by Seikagaku Corporation was used. this is,
Guanidine hydrochloride extracted core protein from bovine nasal septum cartilage was digested with trypsin, purified by affinity chromatography, and labeled with N-hydroxysuccinimide biotin.

(3) Normal aggrecan As a standard of normal aggrecan, bovine cartilage proteoglycan (normal bovine aggrecan) manufactured by Seikagaku Corporation
It was used.

Embodiment 1Measurement of normal aggrecan by the measurement method of the present invention  (1) Phosphate buffered saline (pH 7.2-
7.5, not containing divalent ions) [hereinafter referred to as "PBS (-)"
There is also a saying. ] To 20 μg / mL.
50 μL (1 μg / well) of each solution was added to 96 well
Each plate of Noplate (made by Nunc Corporation; trade name: Maxi Soap)
Well by storing at 4 ° C for 16 hours.
Was fixed. (2) Wash this plate twice with PBS (-),
3% bovine serum albumin (BSA;
PBS (-) solution containing Seikagaku Corporation
And allowed to stand at room temperature for 2 hours. Then this plate
Cleaning solution [0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.)
After washing 3 times with PBS (-) containing 1% BS
A containing PBS (-) (hereinafter referred to as "reaction diluent")
U. ) Diluted bovine normal aggrecan 100,10,
At a concentration of 1, 0.1 or 0.01 μg / ml (hereinafter
Below, it may be referred to as “normal aggrecan standard solution”. )
Was added and reacted at 37 ° C. for 1 hour. Remove the above solution
Remove the plate and attach the reaction diluent to the plate on which the anti-KS antibody is fixed.
5 μg / ml of shark fin-derived keratan sulfate diluted with 50
Add μl to the wells and add the anti-CS antibody
0, 1, 5 or 10 μg diluted with reaction diluent
/ ml chondroitin sulfate D (Seikagaku Corporation)
Add 50 μl to the well and leave at 37 ° C. for 60 minutes
By doing so, unreacted antibodies were blocked. This
Wash the plate 3 times with the above washing solution, and then use the reaction diluent.
Diluted biotin-labeled-hyaluronic acid binding protein
(Sold by Seikagaku Corporation) at a final concentration of 1 μg / ml
The mixture was allowed to stand at 37 ° C. for 60 minutes to react. This press
The plate is washed three times with the above-mentioned washing solution, and 5,000 times with the reaction diluent.
1: 2 diluted peroxidase-labeled streptavidin
(Manufactured by Jackson, sold by Seikagaku Corporation) 50
μl was added, and allowed to stand at 37 ° C. for 30 minutes to react. Step
After washing the rate three times with the washing solution, the peroxidase
Tetramethylbenzidine solution (produced by Moss:
Below, it is abbreviated as “TMB”. ) At 37 ° C
The reaction was allowed to proceed for 15 minutes to develop color. After coloring, add 1 plate
The reaction was stopped by adding 50 μl of N-HC1,
Absorbance at 450 nm wavelength of colored liquid due to decomposition (control wavelength
630 nm) (A450 / 630)
It was measured by K601 (available from Seikagaku Corporation). 5
The result of D4 is shown in FIG. 1, the result of CS56 is shown in FIG.
11 and FIG. 4 show the results of MO-225 and M, respectively.
The result of C21C is shown in FIG. 5, and the result of 2-B-1 is shown in FIG.
Shown respectively. From the results of FIGS. 1 to 3, 5D4, CS56
When using a solid phase to which LY111 and
A dose curve was obtained, and the concentration was determined in the range of about 0.1 to 10 μg / mL.
It turns out that quantities are possible. From this result, the present invention
According to the method, quantitative and reproducible normal aggrecan with high sensitivity
It was shown that the measurement could be performed well. In contrast, FIGS.
As can be seen from FIG. 6, the anti-CS antibody MO-225
And MC21C and anti-proteoglycan core protein
Using a solid phase to which 2-B-1 which is a protein antibody is fixed
If no calibration curve is obtained, the
Was not suitable for measurement of 1 to 3
As can be seen from the results, the calibration curve for normal aggrecan is
To the concentration of chondroitin sulfate D used for blocking
Therefore it was not affected.

Embodiment 2Hyaluronic acid, keratan sulfate, hyaluronidase and
Effect of keratanase  Various concentrations of HA and KS diluted with the reaction diluent were
Each was measured by the method of Example 1 (5D4 was used for the antibody), and
The specificity of the invention measurement method was examined. The method of the first embodiment
And a normal aggrecan standard solution and 10 mU / well
Hyaluronidase SD (sold by Seikagaku Corporation)
Is 5mU / well Keratanase II (Seikagaku Corporation)
Was added to the wells and reacted at 37 ° C. for 60 minutes.
The influence on the measurement method of the present invention was examined. Result of HA measurement
Fig. 7 shows the results, and Fig. 8 shows the results of KS measurement.
FIG. 9 shows the results of examining the effects of Dase SD.
The results of investigating the effects of the enzyme II are shown in FIG.
You. Based on these results, the measurement method of the present invention is HA or K
S alone did not react, nor did hyaluronidase, kerata
Since KS is inhibited by the addition of RNase,
Specifically, normal aggrecan associated with HA
It was shown that it was measuring. In the method of the present invention,
For luronidase treatment, high-performance liquid chromatography, etc.
Simple, quick and inexpensive
Aggrecan can always be measured.

Embodiment 3Determination of normal aggrecan in articular cartilage  Healthy rabbits (10 cases), papain-induced arthritis rabbits (1
0 case) in the left and right femur and left and right tibia articular cartilage
The method described in Example 1 for the quantification of normal aggrecan present
The method was performed. After freeze-drying the articular cartilage, guanidine hydrochloride
The extract was treated with The results are shown in FIG. FIG.
From Figure 1, normal agriculture in papain-induced arthritis rabbit cartilage
The average value of the can amount is significantly lower than that of healthy rabbits.
did. In addition, healthy rabbits and papain-induced arthritis
Normal aggrecan in the joint fluid of barley was measured in the same manner.
Of course, similar results were obtained.

Embodiment 4Measurement kit of the present invention and detection kit of the present invention  The measurement kit of the present invention and the detection of the present invention comprising the following constitutions
A kit was created. 1. 1. One 96-well immunoplate to which CS56 is fixed Normal aggrecan standard solution 1 set 3. 3. One biotin-labeled-hyaluronic acid binding protein 4. One peroxidase-labeled streptavidin 5. One TMB solution 1 stop solution (1N HCl) In addition, 5D4 was fixed to the immunoplate of 1 above.
96-well immunoplate or LY111 fixed
Of the present invention, which was replaced with a 96-well immunoplate.
A detection kit and a detection kit of the present invention were prepared.

[0032]

The measurement method of the present invention is a method that can measure normal aggrecan with specificity, high sensitivity, good quantitativeness and reproducibility, and is simple, quick and inexpensive, and is very practical. In addition, the detection method of the present invention is an application of the measurement method of the present invention, and is extremely practical because abnormalities of the articular cartilage matrix can be detected extremely easily. Since the measurement kit of the present invention and the detection kit of the present invention are kits utilizing the measurement method of the present invention and the detection method of the present invention, normal aggrecan can be specifically and highly sensitively, quantitatively and reproducibly well, easily and quickly. It can be measured and can be provided at low cost. INDUSTRIAL APPLICABILITY The present invention is useful not only for grasping the state of cartilage disease and the like, but also as a useful evaluation method for drug development relating to joint diseases.

[Brief description of the drawings]

FIG. 1 shows the results of measurement of normal aggrecan using a plate to which 5D4 is immobilized.

FIG. 2 shows the results of measurement of normal aggrecan when using a plate to which CS56 is fixed.

FIG. 3 shows a measurement result of normal aggrecan when a plate to which LY111 is fixed is used.

FIG. 4 shows the results of measurement of normal aggrecan when using a plate to which MO-225 is fixed.

FIG. 5 shows a measurement result of normal aggrecan when a plate to which MC21C is fixed is used.

FIG. 6 shows the results of measurement of normal aggrecan when using a plate to which 2-B-1 is fixed.

FIG. 7 shows measurement results of HA using the measurement method of the present invention.

FIG. 8 shows the results of KS measurement using the measurement method of the present invention.

FIG. 9 shows the effect of hyaluronidase on the measurement method of the present invention.

FIG. 10 shows the effect of keratanase on the measurement method of the present invention.

FIG. 11 shows the concentration of normal aggrecan in a healthy rabbit cartilage extract and a papain-induced arthritis articular cartilage extract.

Claims (8)

[Claims] 1. A method for measuring normal aggrecan in a specimen, comprising at least the following steps: Step 1: A sample is brought into contact with a solid phase to which one or more antibodies selected from an antibody group consisting of an anti-keratan sulfate antibody and an anti-chondroitin sulfate antibody are fixed, and “solid-phase fixed antibody-normal aggrecan-hyaluronic acid” Forming a composite. Step 2: A protein having a binding ability with hyaluronic acid is further contacted with the solid phase to form a sandwich-like complex composed of "solid phase-immobilized antibody-normal aggrecan-hyaluronic acid-protein capable of binding with hyaluronic acid" Process to make it. Step 3: a step of detecting the sandwich-like complex formed in step 2. 2. The method according to claim 1, wherein the anti-keratan sulfate antibody is 5D4. 3. The method according to claim 1, wherein the anti-chondroitin sulfate antibody is C-type.
1 selected from the group consisting of S56 and LY111
3. The method according to claim 1, wherein the antibody is at least two antibodies. 4. The method according to claim 1, wherein the protein capable of binding to hyaluronic acid is labeled with a labeling substance. 5. The method according to claim 1, wherein the protein capable of binding to hyaluronic acid is a protein derived from cartilage proteoglycan core protein. 6. A method for detecting an abnormality of an articular cartilage matrix, which comprises at least the following steps. Step 1: According to any one of claims 1 to 5,
Normal aggrecan in joint fluid is measured. Step 2: a step of associating the amount of normal aggrecan in the synovial fluid obtained in Step 1 with the abnormality of the articular cartilage matrix. 7. A kit for measuring a normal aggrecan, comprising at least the following (A) and (B): (A) A solid phase to which one or more antibodies selected from the group consisting of an anti-keratan sulfate antibody and an anti-chondroitin sulfate antibody are fixed. (B) a protein labeled with a labeling substance and capable of binding to hyaluronic acid 8. The kit for measuring normal aggrecan according to claim 7, which is a kit for detecting abnormalities in the articular cartilage matrix.