JP4124526B2 - Normal aggrecan assay and its application - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring aggrecan having a capability of associating with hyaluronic acid (normal aggrecan), more specifically, a method for immunoassay of normal aggrecan and its application (method for detecting abnormality of articular cartilage matrix and kit for measuring normal aggrecan) )
[0002]
[Prior art]
Proteoglycan in cartilage is called “aggrecan”, and is a structure in which chondroitin sulfate (hereinafter also referred to as “CS”) and keratan sulfate (hereinafter also referred to as “KS”) are bound to a core protein. have.
Aggrecan usually associates with hyaluronic acid (hereinafter sometimes referred to as “HA”) in the cartilage matrix to form a huge aggregate. It is known that the ability of aggrecan to associate with HA decreases with cartilage matrix degradation and degeneration.
It is also known that degradation products of aggrecan are also present in synovial fluid, and a decrease in the ability of aggrecan in the cartilage or synovial fluid to associate with HA is useful as an indicator of cartilage matrix abnormality (WO95). / 22765 gazette).
[0003]
In the present specification, an aggrecan having an ability to associate with HA is referred to as “normal aggrecan”, an aggrecan having no ability to associate with HA is referred to as “abnormal aggrecan”, and a normal aggrecan and abnormal aggrecan are simply referred to as “aggrecan”. In the present specification, the degradation product of aggrecan present in joint fluid is also encompassed by the term “aggrecan”.
Hereinafter, the technique closest to the present invention will be described with reference to literature names.
[0004]
In US Pat. No. 5,185,245, an anti-KS antibody or anti-CS antibody is fixed to a solid phase, a specimen (joint fluid) is brought into contact therewith, and a labeled anti-KS antibody or anti-CS antibody is further brought into contact therewith. A method for detecting proteoglycan in synovial fluid by forming a sandwich-like complex consisting of "anti-CS antibody (or anti-KS antibody) -proteoglycan-anti-CS antibody (anti-KS antibody)", In addition, a method for detecting joint destruction is described. In addition, a detection kit for proteoglycans in body fluids is described that includes a solid phase to which an antibody against the glycosaminoglycan component of proteoglycan is fixed as a constituent component. However, the idea of measuring normal aggrecan separately from abnormal aggrecan, using a protein capable of binding to HA, and a specific antibody (one or more antibodies selected from the group consisting of 5D4, CS56 and LY111) are fixed There is no mention of using a prepared solid phase.
[0005]
In WO95 / 22765, a solid phase (for example, HA is bound) to which normal aggrecan is bound or adsorbed by decomposing or removing HA associated with normal aggrecan and free HA in the sample with hyaluronidase or the like. A normal aggrecan bound to or adsorbed to the solid phase is described. In addition, for detection of normal aggrecan bound or adsorbed to the solid phase, anti-CS antibody or anti-KS antibody can be used, and specific examples of anti-CS antibodies include CS-56, MO-225, MC21C, S54C and 3B3 are described, and specifically, 5D4 is described as the anti-KS antibody. In addition, it is described that the condition of arthritis can be grasped by measuring normal aggrecan by this method.
[0006]
However, there is disclosure about using a solid phase to which a specific antibody (one or two or more antibodies selected from the group consisting of 5D4, CS56 and LY111) is fixed, and using a protein capable of binding to hyaluronic acid. Absent. In this method, a reagent such as hyaluronidase is necessary, and a hyaluronidase treatment step and the like are necessary.
[0007]
[Problems to be solved by the invention]
Measurement of normal aggrecan can be achieved by providing a method that can measure normal aggrecan in a specific, highly sensitive, quantitative and reproducible, simple, rapid and inexpensive manner without using many devices and reagents (eg, hyaluronidase). The method is extremely practical and the detection of abnormalities of the articular cartilage matrix becomes extremely easy, and such a measurement method and measurement kit have been desired.
That is, an object of the present invention is to provide a very practical method for measuring normal aggrecan and a very practical method for detecting abnormalities of articular cartilage matrix.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the inventor of the present invention uses a solid phase to which a specific antibody is fixed, so that normal aggrecan is extremely specific, highly sensitive, quantitative and reproducible, and simple. The present inventors have found a method that can be measured quickly and inexpensively, and found that the abnormality of the articular cartilage matrix can be detected very simply by this method, thereby completing the present invention.
[0009]
That is, the gist of the present invention is a method for measuring normal aggrecan in a specimen (hereinafter also abbreviated as “the measurement method of the present invention”), characterized by including at least the following steps.
Step 1: A specimen is brought into contact with a solid phase to which one or two or more antibodies selected from an antibody group consisting of an anti-keratan sulfate antibody and an anti-chondroitin sulfate antibody are fixed, and from “solid phase fixed antibody-normal aggrecan-hyaluronic acid” Forming a complex.
Step 2: A protein having binding ability to hyaluronic acid is further brought into contact with the solid phase to form a sandwich complex composed of “solid-phase-fixed antibody-normal aggrecan-hyaluronic acid-protein having binding ability to hyaluronic acid”. Process.
Step 3: A step of detecting the sandwich complex formed in Step 2.
[0010]
The anti-keratan sulfate antibody is preferably 5D4, and the anti-chondroitin sulfate antibody is preferably one or more antibodies selected from the antibody group consisting of CS56 and LY111. In step 1, the sample is brought into contact with a solid phase on which one or more antibodies selected from the group consisting of 5D4, CS56 and LY111 are immobilized, and a complex comprising “solid phase immobilized antibody-normal aggrecan-hyaluronic acid” The gist of the measurement method of the present invention is characterized by being a step of forming a film.
[0011]
The protein having the ability to bind to hyaluronic acid is labeled with a labeling substance, and the present invention is preferably hyaluronic acid in which the above step 2 is further labeled with the labeling substance on the solid phase. And a protein having binding ability to contact with each other to form a sandwich complex consisting of “solid-phase-fixed antibody-normal aggrecan-hyaluronic acid-hyaluronic acid and binding protein”. The gist of the invention measurement method.
The protein having the ability to bind to hyaluronic acid is a protein derived from cartilage proteoglycan core protein. In the present invention, preferably, the above step 2 is performed on the solid phase and further derived from cartilage proteoglycan core protein or a labeling substance. A sandwich-like complex composed of “solid-phase-adhered antibody-normal aggrecan-hyaluronic acid-hyaluronic acid-binding protein” is brought into contact with the labeled cartilage proteoglycan core protein-derived hyaluronic acid. The gist of the measurement method of the present invention is characterized by being a step of forming.
[0012]
The present invention also includes a method for detecting an abnormality of an articular cartilage matrix, comprising 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 joint fluid obtained in Step 1 with the abnormality of the articular cartilage matrix.
[0013]
The gist of the present invention is a normal aggrecan measurement kit (hereinafter sometimes abbreviated as “the measurement kit of the present invention”) characterized in that it contains 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.
The kit for measuring normal aggrecan is a detection kit for abnormalities of articular cartilage matrix, and the present invention is a kit for detecting abnormalities of articular cartilage matrix (hereinafter referred to as “the detection kit of the present invention”). It may be abbreviated as “)”.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described.
<1> Measurement method of the present invention
The measurement method of the present invention is a method for measuring normal aggrecan in a specimen, comprising at least the following steps.
Step 1: A step of bringing a specimen into contact with a solid phase to which one or two 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: A step of bringing a protein having binding ability with HA into contact with the solid phase to form a sandwich complex composed of “solid phase-fixed antibody-normal aggrecan-HA-HA and binding protein”.
Step 3: A step of detecting the sandwich complex formed in Step 2.
[0015]
Hereinafter, the measurement method of the present invention will be described in detail for each step.
(1) Process 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, and may be a monoclonal antibody or a polyclonal antibody, and is preferably a monoclonal antibody. It is preferably an antibody.
(A) 5D4
5D4 [J. Biol. Chem., 258, 8848- (1983), Eur. J. Biochem., 157, 385-391 (1986)] was obtained by digesting human articular cartilage proteoglycan monomer with chondroitinase ABC. It is a monoclonal antibody obtained by immunizing mice with the proteoglycan core protein obtained as an antigen, and has the following reactivity; it reacts with KS of cornea and cartilage. It also reacts with proteoglycan monomers prepared from hyaline cartilage of humans, monkeys, cows, sheep, chickens, and sharks, but dermatan sulfate (hereinafter sometimes referred to as “DS”), heparin (hereinafter also referred to as “Hep”). Does not react with heparan sulfate (hereinafter sometimes referred to as “HS”) and HA.
5D4 is sold by Seikagaku Corporation.
[0016]
(1) -1-2 anti-CS antibody
The anti-CS antibody is not particularly limited as long as it is an antibody that reacts with CS, and may be a monoclonal antibody or a polyclonal antibody, and is preferably a monoclonal antibody. It is preferably an antibody.
(B) CS56
CS56 [Exp. Cell Res., 158, 321- (1985), J.
Cell Biol., 91, 205- (1981), Biochem.
Biophys. Acta., 694, 375- (1982), J. Biol.
Chem., 255, 7102- (1980), Int. Rev. Cytol., 53, 65- (1978)] is a monoclonal antibody obtained by immunizing mice with chicken gizzard fibroblasts as an antigen. Specific for the glycosaminoglycan part of native CS proteoglycan, reacts with chondroitin sulfate A (CS-A) and chondroitin sulfate C (CS-C), but not 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.
LY111 is sold by Seikagaku Corporation.
[0017]
(1) -2 Solid phase
The solid phase to which the anti-KS antibody or the anti-CS antibody is fixed is not particularly limited as long as the antibody can be fixed and is insoluble in water, a specimen, or a measurement reaction solution. Examples of the shape of the solid phase include a plate (for example, a well of a microplate), a tube, a bead, a membrane, a gel, and the like. Examples of the solid phase material include polystyrene, polypropylene, nylon, and polyacrylamide.
Among these, a plate made of polystyrene is preferable.
As a method for fixing an anti-KS antibody and / or an anti-CS antibody to these solid phases, a general method (immobilized enzyme, 1975) as a method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bond method, and a comprehensive method. Year, published by Kodansha, see pages 9-75).
Among these, the physical adsorption method is preferable because the operation is simple and frequently used.
Specific examples of the physical adsorption method include the following methods;
An anti-KS antibody and / or anti-CS antibody is dissolved in a buffer solution (eg, phosphate buffer, phosphate buffered saline (PBS), carbonate buffer, etc.) having a pH of about 7 to 9, and is dissolved in a solid phase (eg, a microplate). In addition, it is stored at about 37 ° C. for 1 to 2 hours, or is stored at about 4 ° C. overnight and fixed.
In addition, on the surface of the solid phase to which the anti-KS antibody and / or anti-CS antibody is fixed, there may be a case where a surface portion to which these are not fixed remains, and there are aggrecan and other molecular species in the specimen. If it adheres, accurate measurement results may not be obtained. Therefore, it is preferable to add a blocking substance before the specimen is brought into contact with the solid phase so as to cover the portion where the anti-KS antibody and / or the anti-CS antibody is not fixed. Examples of such blocking substances include serum albumin, casein, skim milk, gelatin, and the like, and commercially available blocking substances can also be used.
Specific examples of the blocking method include the following method; a blocking substance (serum albumin, casein, skim milk, gelatin, etc.) is added and stored at about 37 ° C. for 30 minutes to 2 hours, Store at room temperature (15-25 ° C.) for 1-2 hours.
[0018]
(1) -3 Sample
The specimen is not particularly limited as long as it contains a normal aggrecan as a measurement target substance or a specimen that may contain a normal aggrecan. The specimen may not be purified in advance. Specific examples of the specimen include joint fluid, cartilage extract, blood, serum, plasma, urine and the like.
The method of contacting the specimen with the solid phase to which the anti-KS antibody and / or anti-CS antibody is fixed is not particularly limited as long as the solid phase and the specimen are contacted. For example, the sample may be added to and contacted with the solid phase to which the anti-KS antibody and / or anti-CS antibody is fixed, or the solid phase to which the anti-KS antibody and / or anti-CS antibody is fixed is added to the sample. You may make it contact.
In addition, after making these both contact, in order to sufficiently bind the anti-KS antibody and / or anti-CS antibody and normal aggrecan in the specimen, for example, react at 0 to 45 ° C., preferably 37 ° C. for about 1 hour. Is preferred. In addition, after the reaction, the solid phase and the liquid phase can be sufficiently separated or the solid phase can be washed, for example, the surface of the solid phase can be washed with a washing solution to remove non-specifically adsorbed substances and components in the sample that have not reacted. preferable.
As the cleaning solution, for example, a buffer solution (for example, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) to which a nonionic surfactant such as a Tween surfactant is added is preferably used.
By contacting the specimen with a solid phase to which the anti-KS antibody and / or anti-CS antibody is fixed, the aggrecan contained in the specimen and the anti-KS antibody and / or anti-CS antibody form a complex. In addition, normal aggrecan has the ability to associate with HA, and is usually present as a complex consisting of “normal aggrecan-HA” in specimens such as joint fluid. Therefore, anti-KS antibody and / or anti-CS antibody is By bringing the specimen into contact with the fixed solid phase, a complex consisting of “solid phase-fixed antibody-normal aggrecan-HA” is formed. In this step, a complex composed of “solid-phase-fixed antibody-abnormal aggrecan” is also formed. In addition, when normal aggrecan in the sample does not exist as a complex composed of “normal aggrecan-HA”, a complex composed of “normal aggrecan-HA” is added by adding HA having a molecular weight of several hundred thousand or more to the sample. It needs to be formed.
[0019]
(2) Process 2
(2) -1 Protein having binding ability to HA
The protein having the ability to bind to HA is not particularly limited as long as it is a protein having the ability to bind to HA, but HA-binding proteoglycan (for example, cartilage proteoglycan, tryptic digest of cartilage proteoglycan, chondroitinase of cartilage proteoglycan ABC digest, etc.), proteoglycan core protein (eg, cartilage proteoglycan core protein), link protein, hyaluronectin, CD44, partial protein containing HA binding site of these proteins, or the partial protein and other proteins Among them, a tryptic digest of cartilage proteoglycan, and a trypsin digest of bovine nasal cartilage proteoglycan are particularly preferable. The tryptic digest of bovine nasal cartilage proteoglycan is sold as “hyaluronic acid binding protein” by Seikagaku Corporation.
In addition, it is preferable that the protein having the ability to bind to HA is labeled with a labeling substance because detection in Step 3 is easy.
Labeling substances used for labeling a protein having binding ability with HA include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), fluorescent dyes (fluorescein isothiocyanate (FITC), etc.), chemiluminescence Examples include substances (such as luminol), biotin, avidin (including streptavidin), etc., but are not particularly limited as long as they are usually capable of labeling proteins. For example, the glutaraldehyde method, the periodic acid crosslinking method, the maleimide crosslinking method, the carbodiimide method, the activated ester method, etc. ["Protein Chemistry (below)", Tokyo Chemical Dojin, published in 1987) can be selected as appropriate. For example, bio as a labeling substance When using emissions, a method of using a hydrazide derivative of biotin (Avidin-
Biotin Chemistry: A Handbook, p57-63,
In the case of using fluorescein isothiocyanate, the method described in JP-B-63-17843 can be appropriately selected.
A tryptic digest of bovine nasal cartilage proteoglycan labeled with biotin is sold as “Biotin-labeled hyaluronic acid binding protein” by Seikagaku Corporation.
By contacting a protein having a binding ability with HA to the solid phase (in which a complex composed of “solid-phase-fixed antibody-normal aggrecan-HA” is formed), “solid-phase-fixed antibody-normal aggrecan-HA— A sandwich complex consisting of “a protein capable of binding to HA” is formed. Since a protein composed of “solid-phase-fixed antibody-abnormal aggrecan” does not bind to a protein capable of binding to HA, such a sandwich-like complex is not formed.
In order to sufficiently bind the complex consisting of “solid-phase-fixed antibody-normal aggrecan-HA” and the protein capable of binding to HA, for example, 0 to 45 ° C., preferably 37 It is preferable to react at about 1 hour for about 1 hour. Further, after the reaction, it is preferable to sufficiently separate the solid phase and the liquid phase or to wash the solid phase, for example, to wash the surface of the solid phase with a washing solution to remove non-specifically adsorbed substances and the protein that has not reacted. As the cleaning liquid, for example, a buffer solution to which a nonionic surfactant such as a Tween surfactant is added is preferably used.
When a protein having KS or CS binding, such as cartilage proteoglycan, is used as the protein capable of binding to HA, a complex consisting of “solid-phase-fixed antibody-normal aggrecan-HA” was formed. It is preferable that CS and / or KS is subsequently brought into contact with the solid phase to block unreacted antibodies (antibodies not forming a complex with normal aggrecan).
[0020]
(3) Process 3
Step 3 is a step of detecting the sandwich complex formed in Step 2. Detection may be performed using an antibody that specifically reacts with a protein capable of binding to HA, and when a protein labeled with a labeling substance is used in step 2 with a protein capable of binding to HA, A person 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 conjugated with streptavidin or the like is added, and an enzyme such as peroxidase is bound to a complex containing biotin as a labeling substance via this streptavidin or the like, Examples include a method in which a coloring substrate such as tetramethylbenzidine and a hydrogen peroxide solution are added as a substrate of the enzyme, and the degree of coloring of the product due to the enzyme reaction is measured by a change in absorbance. Moreover, when using a fluorescent substance and a chemiluminescent substance, the method etc. which measure the fluorescence and light emission of the solution after reaction are mentioned. Note that “the protein having the ability to bind hyaluronic acid is labeled with a labeling substance” does not mean that the protein is directly labeled, but the labeled antibody or streptoid 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 the sample is determined in advance by preparing a calibration curve for the relationship between the aggrecan concentration and the detection result (for example, absorbance) of the labeled substance using an aggrecan standard solution with a known concentration. This 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. Can be detected.
In the measurement method of the present invention, as will be apparent from the examples described later, normal aggrecan can be measured without using a reagent such as hyaluronidase or an apparatus such as high performance liquid chromatography. Can be measured. In addition, the measurement method of the present invention can measure normal aggrecan with high specificity and reproducibility with high sensitivity.
[0021]
<2> Detection method of the present invention
The detection method of the present invention is a method for detecting an abnormality of an articular cartilage matrix including at least the following steps.
Step 1: Normal aggrecan in joint fluid is measured by the measurement method according to any one of claims 1 to 5.
Step 2: A step of associating the amount of normal aggrecan in the joint 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.
[0022]
(1) Process 1
Step 1 is exactly the same as the measurement method of the present invention described in <1> above, but joint fluid is used as a specimen in the detection method of the present invention. An extract of cartilage may be used.
(2) Process 2
Step 2 is a step of associating the amount of normal aggrecan in the joint fluid obtained in Step 1 with the abnormality of the articular cartilage matrix.
The “normal aggrecan amount” may be the measurement value of the normal aggrecan amount measured in step 1 or the ratio of the normal aggrecan amount measured in step 1 to the aggrecan amount in joint fluid. May be. The amount of aggrecan in the synovial fluid can be measured by, for example, the method described in US Pat. No. 5,185,245.
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. For example, a disease involving degeneration or destruction of articular cartilage, more specifically, deformability Examples include arthropathy, rheumatoid arthritis, traumatic arthritis, gout and the like. In such diseases, the amount of normal aggrecan in synovial fluid is significantly reduced compared to the amount of normal aggrecan in the synovial fluid of healthy humans (humans with no abnormalities of articular cartilage matrix). If the amount of normal aggrecan produced is small compared to the amount of normal aggrecan in healthy human joint fluid, there is a high possibility that "articular cartilage matrix is abnormal" or "articular cartilage matrix is abnormal Can be associated. If the amount of normal aggrecan measured in step 1 is equivalent to the amount of normal aggrecan in healthy human joint fluid, there is a possibility that “articular cartilage matrix is not abnormal” or “articular cartilage matrix is abnormal. Can be associated with “low”.
The detection method of the present invention includes not only the presence or absence of abnormality of the articular cartilage matrix but also detection of the degree of abnormality. For example, when the amount of normal aggrecan in an individual's joint fluid is regularly measured in step 1, and the amount of normal aggrecan tends to decrease, “the abnormality of the articular cartilage matrix is progressing” or “articular cartilage matrix” It is highly possible that the abnormality is progressing. " In addition, when the amount of normal aggrecan measured in Step 1 tends to increase, “the abnormality of the articular cartilage matrix is in an improvement direction” or “the abnormality of the articular cartilage matrix is in an improvement direction” is high. Can be associated. In addition, when there is no change in the amount of normal aggrecan measured in Step 1, “the abnormality (normality) of articular cartilage matrix does not change” or “the abnormality (normality) of articular cartilage matrix does not change”. It is highly possible that there is no possibility ”.
The normal aggrecan amount used as a criterion for detecting abnormalities of the articular cartilage matrix may be a normal aggrecan concentration obtained using a calibration curve prepared for the relationship between the normal aggrecan standard concentration and the detection result of the labeled substance. Further, it may be a ratio to the normal aggrecan amount in the joint fluid of a healthy human (human without articular cartilage matrix abnormality) without using the calibration curve.
[0023]
<3> Measurement kit of the present invention
The measurement kit of the present invention is a normal aggrecan measurement kit including at least the following configuration.
(A) A solid phase on which one or more antibodies selected from the following antibody group are fixed
Antibody group: anti-keratan sulfate antibody, anti-chondroitin sulfate antibody
(B) Protein labeled with a labeling substance and capable of binding to hyaluronic acid
The description of the solid phase of (A) and the protein of (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 includes at least the above (A) and (B), and further, a normal aggrecan standard product of known concentration that serves as a standard for preparing a calibration curve, a detection reagent for a labeled substance, HA and A reagent for labeling a protein having binding ability or a reagent for detecting a protein having binding ability to HA (such as an antibody against the labeled protein) can be added as a constituent. In addition to these components, the blocking substance, the cleaning solution, the sample diluent, the enzyme reaction stop 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.
Measurement of normal aggrecan using the measurement kit of the present invention can be performed according to the measurement method of the present invention <1>.
[0024]
<4> Detection kit of the present invention
The detection kit of the present invention is a detection kit for abnormality of articular cartilage matrix, comprising the measurement kit of the present invention. The description of the configuration of the detection kit of the present invention is the same as that of the measurement kit of the present invention <3>.
The abnormality of the articular cartilage matrix using the detection kit of the present invention can be detected according to the detection method of the present invention <2>.
[0025]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described 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 of 5D4 have been described above.
(1-2) CS56, LY111, MO-225 and MC21C were used as anti-CS antibodies. The characteristics of CS56 and LY111 have been described above. The characteristics of MO-225 and MC21C will be described below.
MO-225:
MO-225 [J. Biol. Chem., 262, 4146-4152 (1987), J. Biol. Chem., 264, 8012-8018 (1989)] uses chicken embryo limb proteoglycan as an antigen and this is used as a mouse. A monoclonal antibody obtained by immunization with the following reactivity:
Recognizing a determinant (D-glucuronic acid-2-sulfate (β1 → 3) -N-acetylgalactosamine-6-sulfate disaccharide structure (D-unit)) on the intact CS chain and repeating the D-unit Those with a continuous structure react more strongly.
MO-225 is sold by Seikagaku Corporation.
MC21C:
MC21C [Dev. Biol., 133, 475-488 (1989),
Differentiation, 43, 37-50 (1990), Int.
Dev. Biol., 34, 191-204 (1990)] is a monoclonal antibody obtained by immunizing a mouse with mature rat bone protein as an antigen, and has the following reactivity:
It reacts with intact CS-C of rats, mice, chickens, sharks, and cows, but 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 [Histochemical J., 21, 455-459 (1989)] is a monoclonal antibody obtained by immunizing a mouse with a proteoglycan prepared from a human Yorksack tumor as an antigen. Have reactivity;
Recognizes the core protein of proteoglycan. In adult tissues, the intima and media of the aorta are stained, and the connective tissue around the blood vessels and muscles reacts weakly, while in the fetus, stroma is strongly stained in all tissues. In cancer tissue, it reacts with all stromal fiber components such as stomach cancer, rectal cancer, breast cancer, uterine cancer, ovarian cancer, etc., and shows a stromal image peculiar to cancer.
2-B-1 is sold by Seikagaku Corporation.
[0026]
(2) Proteins capable of binding to HA
“Biotin-labeled hyaluronic acid binding protein” sold by Seikagaku Corporation was used. This is a product obtained by digesting a core protein extracted from bovine nasal septal cartilage with guanidine hydrochloride after trypsin digestion and then purifying it by affinity chromatography and labeling with N-hydroxysuccinimide biotin.
[0027]
(3) Normal aggrecan
As a standard product of normal aggrecan, bovine cartilage proteoglycan (bovine normal aggrecan) manufactured by Seikagaku Corporation was used.
[0028]
Example 1
Measurement of normal aggrecan by the measurement method of the present invention
(1) The above-mentioned antibody is phosphate buffered saline (pH 7.2 to 7.5, divalent ion-free) [hereinafter sometimes referred to as “PBS (−)”. ], And 50 μL (1 μg / well) of this solution is added to each well of a 96-well immunoplate (manufactured by NUNK; trade name Maxisorp) and stored at 4 ° C. for 16 hours. It was fixed uniformly.
(2) This plate was washed twice with PBS (−), a PBS (−) solution containing 3% bovine serum albumin (BSA; sold by Seikagaku Corporation) as a blocking substance was added, and the plate was allowed to stand at room temperature for 2 hours. I put it.
Next, this plate was washed three times with a washing solution [PBS (−) containing 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.)], and then PBS (−) containing 1% BSA (hereinafter “reaction dilution”). Bovine normal aggrecan having a concentration of 100, 10, 1, 0.1 or 0.01 μg / ml (hereinafter also referred to as “normal aggrecan standard solution”) diluted with The reaction was allowed to proceed for 1 hour at ° C.
After removing the above solution, 50 μl of 5 μg / ml shark shark-derived keratan sulfate diluted with the reaction diluent is added to the well to the plate to which the anti-KS antibody is fixed, and the reaction is applied to the plate to which the anti-CS antibody is fixed. Blocking unreacted antibody by adding 50 μl of 0, 1, 5 or 10 μg / ml chondroitin sulfate D (manufactured by Seikagaku Corporation) diluted in diluent and allowing to stand at 37 ° C. for 60 minutes. did.
The plate was washed 3 times with the above washing solution, biotin-labeled hyaluronic acid-binding protein (sold by Seikagaku Corporation) diluted with reaction diluent was added at a final concentration of 1 μg / ml, and the plate was allowed to stand at 37 ° C. for 60 minutes. To react.
The plate was washed 3 times with the above washing solution, and 50 μl of peroxidase-labeled streptavidin (Jackson, sold by Seikagaku Corporation) diluted 5000 times with the reaction diluent was added, and the plate was allowed to stand at 37 ° C. for 30 minutes to react. I let you. After washing the plate 3 times with the above washing solution, 50 μl of tetramethylbenzidine solution (manufactured by Moss: hereinafter abbreviated as “TMB”) was added as a substrate for peroxidase and reacted at 37 ° C. for 15 minutes to develop color.
After color development, 50 μl of 1N-HC1 was added to the plate to stop the reaction, and the absorbance at a wavelength of 450 nm (control wavelength: 630 nm) (A450 / 630) of the colored solution due to the decomposition of TMB was measured by Well Reader SK601 (Seikagaku Corporation). ). FIG. 1 shows the results of 5D4, FIG. 2 shows the results of CS56, FIG. 3 shows the results of LY111, FIG. 4 shows the results of MO-225, FIG. 5 shows the results of MC21C, and FIG. Each is shown in FIG.
From the results shown in FIGS. 1 to 3, when a solid phase to which 5D4, CS56, and LY111 are fixed is used, a calibration curve is obtained, and it can be quantified in the range of about 0.1 to 10 μg / mL. Recognize. From these results, it was shown that according to the method of the present invention, normal aggrecan can be measured with high sensitivity and good quantitativeness and reproducibility.
On the other hand, as can be seen from FIGS. 4 to 6, when using a solid phase to which MO-225 and MC21C, which are anti-CS antibodies, and 2-B-1, which is an anti-proteoglycan core protein antibody, are used, No line was obtained, indicating that it was not suitable for the measurement of normal aggrecan in the present invention.
As can be seen from the results of FIGS. 1 to 3, the calibration curve for normal aggrecan was not affected by the concentration of chondroitin sulfate D used for blocking.
[0029]
Example 2
hyaluronic acid , Effects of keratan sulfate, hyaluronidase and keratanase
Various concentrations of HA and KS diluted with the reaction diluent were measured by the method of Example 1 (using 5D4 as the antibody), and the specificity of the measurement method of the present invention was examined. Further, according to the method of Example 1, normal aggrecan standard solution and 10 mU / well hyaluronidase SD (Seikagaku Corporation sold) or 5 mU / well Keratanase II (Seikagaku Corporation sold) were added to the well at 37 ° C. The reaction was performed for 60 minutes, and the influence on the measurement method of the present invention was examined.
FIG. 7 shows the results of measuring HA, FIG. 8 shows the results of measuring KS, FIG. 9 shows the results of examining the effects of hyaluronidase SD, and FIG. 10 shows the results of examining the effects of keratanase II.
From these results, the measurement method of the present invention does not react with HA or KS alone, and is inhibited by the addition of hyaluronidase and keratanase, so that normal aggrecan containing KS in the molecule and associated with HA is specific. It was shown that it was measuring. Since the method of the present invention does not require analysis by hyaluronidase treatment, high performance liquid chromatography, or the like, normal aggrecan can be measured simply, quickly and inexpensively.
[0030]
Example 3
Determination of normal aggrecan in arthritic cartilage.
The normal aggrecan present in the left and right femurs and left and right tibia articular cartilages of healthy rabbits (10 cases) and papain-induced arthritic rabbits (10 cases) was quantified by the method described in Example 1. Articular cartilage was lyophilized and then treated with an extract containing guanidine hydrochloride. The results are shown in FIG. From FIG. 11, the average value of normal aggrecan in papain-induced arthritic rabbit cartilage was significantly lower than that in healthy rabbits. Moreover, when the normal aggrecan in the joint fluid of healthy rabbits and papain-induced arthritic rabbits was also measured, similar results were obtained.
[0031]
Example 4
The present measurement kit and the present detection kit
The measurement kit of the present invention and the detection kit of the present invention having the following constitution were prepared.
1. One 96-well immunoplate with CS56 fixed
2. 1 set of normal aggrecan standard solution
3. One biotin-labeled hyaluronic acid-binding protein
4). One peroxidase-labeled streptavidin
5. 1 TMB solution
6). 1 reaction stop solution (1N HCl)
In addition, the above-described measurement kit and the detection kit of the present invention were prepared by replacing the immunoplate of 1 with a 96-well immunoplate to which 5D4 was fixed or a 96-well immunoplate to which LY111 was fixed.
[0032]
【The invention's effect】
The measurement method of the present invention is a method that can measure normal aggrecan with specificity, high sensitivity, good quantification and reproducibility, simply, quickly and inexpensively, and is very practical. Further, the detection method of the present invention is an application of the measurement method of the present invention, and can detect the abnormality of the articular cartilage matrix very easily, and is a highly practical method. In addition, since the measurement kit and the detection kit of the present invention are kits that use the measurement method and the detection method of the present invention, normal aggrecan is specifically and highly sensitive, quantitatively and reproducibly, easily and quickly. It can be measured and provided at low cost.
The present invention is useful not only for grasping the state of cartilage disease, but also as a useful evaluation method for drug development relating to joint diseases.
[Brief description of the drawings]
FIG. 1 shows the measurement results of normal aggrecan when using a plate to which 5D4 is fixed.
FIG. 2 shows the measurement results of normal aggrecan when using a plate to which CS56 is fixed.
FIG. 3 shows the measurement results of normal aggrecan when using a plate with LY111 adhered thereto.
FIG. 4 shows measurement results of normal aggrecan when using a plate to which MO-225 is fixed.
FIG. 5 shows the measurement results of normal aggrecan when using a plate to which MC21C is fixed.
FIG. 6 shows the measurement results of normal aggrecan when using a plate to which 2-B-1 is fixed.
FIG. 7 shows the 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 influence of hyaluronidase in the measurement method of the present invention.
FIG. 10 shows the influence of keratanase in the measurement method of the present invention.
FIG. 11 shows the concentration of normal aggrecan in healthy rabbit cartilage extract and papain-induced arthritis articular cartilage extract.

Claims (8)

A method for measuring normal aggrecan in a specimen, comprising at least the following steps.
Step 1: An analyte is brought into contact with a solid phase to which one or two or more antibodies selected from the group of anti-chondroitin sulfate antibodies reactive with chondroitin sulfate A and having anti-keratan sulfate antibody and chondroitin sulfate A as an antigen are fixed. , A step of forming a complex consisting of “solid-phase-fixed antibody-normal aggrecan-hyaluronic acid”.
Step 2: A protein having binding ability to hyaluronic acid is further brought into contact with the solid phase to form a sandwich complex composed of “solid-phase-fixed antibody-normal aggrecan-hyaluronic acid-protein having binding ability to hyaluronic acid”. Process.
Step 3: A step of detecting the sandwich complex formed in Step 2.   The method according to claim 1, wherein the anti-keratan sulfate antibody is 5D4. 3. The measurement according to claim 1 or 2, wherein the anti-chondroitin sulfate antibody having chondroitin sulfate A as an antigen or reactive to chondroitin sulfate A is one or more antibodies selected from the group consisting of CS56 and LY111. Method.   The method according to any one of claims 1 to 3, wherein the protein having the ability to bind to hyaluronic acid is labeled with a labeling substance.   The method according to any one of claims 1 to 4, wherein the protein having binding ability to hyaluronic acid is a protein derived from cartilage proteoglycan core protein. A method for detecting an abnormality of an articular cartilage matrix, comprising at least the following steps.
Step 1: Normal aggrecan in joint fluid is measured by the measurement method according to claim 1.
Step 2: A step of associating the amount of normal aggrecan in the joint fluid obtained in Step 1 with the abnormality of the articular cartilage matrix. A kit for measuring a normal aggrecan comprising at least the following (A) and (B).
(A) A solid phase on which one or two or more antibodies selected from an antibody group consisting of an anti-keratan sulfate antibody and chondroitin sulfate A as an antigen or an anti-chondroitin sulfate antibody reactive to chondroitin sulfate A are fixed.
(B) A protein labeled with a labeling substance and capable of binding to hyaluronic acid.   The kit for measuring normal aggrecan according to claim 7, which is a kit for detecting abnormality of articular cartilage matrix.

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