JP3768165B2 - Method and kit for measuring anti-carpastatin antibody - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a measurement method and measurement kit for an anti-carpastatin antibody. The present invention also relates to a disease detection method and a detection kit.
[0002]
[Prior art]
Calpain is a kind of calcium-dependent cysteine protease and is considered to be one of the enzymes involved in cartilage destruction of rheumatoid arthritis (hereinafter simply referred to as “RA”). Calpastatin, on the other hand, is a specific endogenous inhibitor of calpain.
[0003]
Since calpain is a kind of protease involved in tissue destruction, autoantibodies (anti-calpastatin antibodies) against calpastatin, which is an inhibitor of such protease, are involved in the development of rheumatic diseases, particularly RA and inflammation. There is a possibility. Therefore, measurement of anti-carpastatin antibody is considered useful for detection of RA, understanding of pathological conditions, and the like.
[0004]
British Journal of Rheumatology, 37, p1164-1171 (1998) and Rheumatology, 40, p1126-1134 (2001) correspond to anti-carpastatin antibody in serum corresponding to 27 amino acids on the C-terminal side of calpastatin (SSKAPKNGGKAKDSAKTTEETSKPKDD) It describes that it was measured by enzyme-linked immunosorbent assay (ELISA) using a solid phase to which a synthetic peptide or purified calpastatin was fixed. However, there is no description or suggestion about the measurement of anti-carpastatin antibodies in synovial fluid.
[0005]
Since calpain is known to increase in synovial cells and synovial fluid of RA, it is possible to detect RA more accurately and detect disease state by measuring anti-calpastatin antibody in synovial fluid. There is sex.
[0006]
[Problems to be solved by the invention]
If a method or kit that can measure anti-calpastatin antibodies in synovial fluid more sensitively, specifically, accurately, with good quantitativeness, and simply can be provided, the anti-calpastatin antibody in synovial fluid is extremely practical. A measurement method is provided, and there is a possibility that highly sensitive detection and understanding of a disease state such as rheumatic diseases will be extremely easy.
[0007]
That is, an object of the present invention is to provide a very practical measurement method and measurement kit for anti-carpastatin antibody in joint fluid, and a disease detection method and detection kit.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a solid phase to which a partial peptide of calpastatin, which is widely used, is fixed when an anti-calpastatin antibody in joint fluid is measured by ELISA. The present invention was completed by using a solid phase to which a full-length polypeptide was fixed instead of using a high-sensitivity, specific and accurate, reproducible and quantitative, and simple measurement. It came to do.
[0009]
That is, the present invention comprises at least the following steps (A) and (B), comprising an anti-carpastatin antibody in joint fluid. Quantitative A method (hereinafter referred to as “the measurement method of the present invention”) is provided.
Step (A): A step of bringing the joint fluid into contact with the solid phase to which calpastatin is fixed.
Step (B): anti-carpastatin antibody bound to calpastatin immobilized on a solid phase amount Detecting.
[0010]
Where anti-carpastatin antibody amount The detection of is preferably performed using an anti-immunoglobulin antibody capable of binding to the antibody. The anti-immunoglobulin antibody is preferably labeled.
[0011]
The present invention also provides an anti-carpastatin antibody in joint fluid. Quantitative And that Quantitative Characterized by associating outcomes with disease Degree A detection method (hereinafter referred to as “the detection method of the present invention”) is provided. Of anti-carpastatin antibody in synovial fluid Quantitative Is preferably performed by the measurement method of the present invention. The disease to be detected is preferably a rheumatic disease, particularly RA.
[0012]
The present invention also provides an anti-carpastatin antibody in joint fluid, comprising at least the following components (A) and (B): Quantitative A kit (hereinafter referred to as “the measurement kit of the present invention”) is provided.
(A) Solid phase to which calpastatin is fixed.
(B) An anti-immunoglobulin antibody capable of binding to an anti-carpastatin antibody.
Here, the anti-immunoglobulin antibody is preferably labeled.
[0013]
Furthermore, the present invention includes at least the components (A) and (B) of the measurement kit of the present invention, Degree A detection kit (hereinafter referred to as “detection kit of the present invention”) is provided. The disease to be detected is preferably a rheumatic disease, especially RA.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described.
First, common matters through the measurement method of the present invention, the detection method of the present invention, the measurement kit of the present invention and the detection kit of the present invention will be described.
[0015]
“Calpastatin” used in the present invention is not a peptide consisting of a part of the amino acid sequence of calpastatin (so-called partial peptide) but a polypeptide having the entire amino acid sequence of calpastatin that exists in nature. In the present specification, when simply expressed as “calpastatin”, it means “a polypeptide having the entire amino acid sequence of naturally occurring calpastatin” and includes a peptide consisting of a part of the amino acid sequence. It is not intended.
[0016]
The method for producing “calpastatin” used in the present invention is not particularly limited as long as “polypeptide having the entire amino acid sequence of naturally occurring calpastatin” can be finally obtained. For example, it may be produced by isolation and purification from a natural product containing calpastatin, may be produced by genetic engineering techniques using DNA encoding calpastatin, etc., or produced by chemical synthesis techniques. May be. Calpastatin used in the present invention may be obtained by any of these production methods. Such a calpastatin can also use what is marketed.
[0017]
Calpastatin is slightly different in amino acid sequence depending on the animal species, but the calpastatin used in the present invention is the same as the animal species from which the anti-calpastatin antibody (joint fluid containing it) to be measured is derived. It is preferable to select an animal species. For example, when measuring anti-calpastatin antibodies in human synovial fluid, human calpastatin is preferably used.
[0018]
The greatest feature of the present invention is that `` a polypeptide having the entire amino acid sequence of calpastatin '' instead of using a peptide consisting of a part of the amino acid sequence as `` carpastatin '', Thereby, the anti-calpastatin antibody in joint fluid can be measured with high sensitivity and good quantitativeness.
[0019]
<1> Measurement method of the present invention
The measurement method of the present invention comprises at least the following steps (A) and (B), the anti-calpastatin antibody in joint fluid: Quantitative Is the method.
Step (A): A step of bringing the joint fluid into contact with the solid phase to which calpastatin is fixed.
Step (B): anti-carpastatin antibody bound to calpastatin immobilized on a solid phase amount Detecting.
[0020]
“Calpastatin-fixed solid phase” used in step (A) means that calpastatin is not released through the usual ELISA processes (blocking, antigen-antibody reaction, washing, enzyme reaction, color reaction, measurement, etc.) Mean solid phase bound to a degree. That is, in this specification, “adhesion” means binding to the extent that it is not released through a process in a normal ELISA method.
[0021]
The “solid phase” that can be used in the step (A) is not limited in its shape and material as long as it is a water-insoluble solid phase to which calpastatin can be fixed.
[0022]
Examples of the shape of the solid phase that can be used in the step (A) include plates (for example, wells of microplates), tubes, beads, membranes, gels, latexes, and the like. Similarly, examples of the solid phase material include polystyrene, polypropylene, nylon, polyacrylamide and the like. Among these, a plate made of polystyrene is preferable.
[0023]
As a method for fixing calpastatin to these solid phases, a general method for preparing an immobilized enzyme such as a physical adsorption method, a covalent bond method, a comprehensive method (immobilized enzyme, 1975, published by Kodansha, No. 9). -See page 75). Among these, the physical adsorption method is preferable because the operation is simple and frequently used.
[0024]
Specific examples of the physical adsorption method include and are preferably the following methods; a calpastatin antibody can be prepared using a buffer solution (for example, a borate buffer solution, a phosphate buffer solution, a phosphate buffered saline solution) having a pH of about 7-9. A method of dissolving in a liquid (PBS), carbonate buffer, etc., contacting with a solid phase, and allowing to stand overnight at 4 ° C. for fixation. After that, the surface of the solid phase may be washed with a washing solution. As the washing solution, a buffer solution (for example, Tris-HCl buffer solution, phosphate buffer solution, PBS or the like) can be used.
[0025]
In addition, after that, it is preferable that the blocking substance is brought into contact with the solid phase to coat the portion where the calpastatin is not fixed. Examples of such blocking substances include serum albumin (BSA), gelatin, casein, skim milk, and the like. These may be used alone or as two or more components. In addition, you may use what is marketed as a blocking substance.
[0026]
Thereafter, the surface of the solid phase may be washed with a washing solution. As the washing solution, a buffer solution (for example, Tris-HCl buffer solution, phosphate buffer solution, PBS or the like) can be used.
As described above, a “solid phase to which calpastatin is fixed” can be produced.
Step (A) comprises a step of bringing the joint fluid into contact with the solid phase thus produced.
[0027]
The “joint fluid” only needs to contain or possibly contain an anti-carpastatin antibody to be measured. Further, the joint fluid does not need to be subjected to a treatment such as isolation / purification. That is, according to the measurement method of the present invention, even when various protein components other than the anti-calpastatin antibody are contained in the joint fluid, the anti-calpastatin antibody to be measured can be specifically measured.
[0028]
When the joint fluid is brought into contact with the solid phase (solid phase to which calpastatin is fixed), the anti-carpastatin antibody in the joint fluid binds to calpastatin fixed to the solid phase.
[0029]
The method of bringing the “solid phase on which calpastatin is fixed” and “joint fluid” into contact with each other is as long as the calpastatin molecule fixed on the solid phase and the anti-calpastatin antibody molecule in the joint fluid are in contact with each other. It is not limited in. Specific examples of the method of bringing both into contact include a method of adding joint fluid to the solid phase, a method of adding the solid phase to the joint fluid, and a method of adding both to a separate container at the same time. It is not limited to this, and those skilled in the art can appropriately determine it according to the shape and material of the solid phase.
[0030]
It is preferable to incubate after contacting both of these to make the antigen-antibody reaction sufficiently. The incubation temperature is not particularly limited as long as it is the temperature at which the antigen-antibody reaction occurs, and room temperature is exemplified. The incubation time is not particularly limited as long as the two react sufficiently, but it can be exemplified by about 0.5 to 2 hours, more preferably about 1 to 1.5 hours.
[0031]
Moreover, it is preferable to wash | clean the surface of a solid phase with a washing | cleaning liquid after reaction. This washing is performed under conditions where calpastatin fixed to the solid phase and anti-calpastatin antibody bound thereto are not released. As the cleaning solution, for example, a buffer solution containing a nonionic surfactant such as a Tween surfactant (for example, Tris-HCl buffer solution, phosphate buffer solution, PBS, etc.) is preferably used.
[0032]
After passing through the above-mentioned process (A), the process proceeds to process (B).
[0033]
In step (B), an anti-carpastatin antibody bound to calpastatin affixed to a solid phase amount It is the process of detecting.
The measurement method of the present invention utilizes an antigen-antibody reaction between calpastatin fixed to a solid phase and anti-carpastatin antibody in joint fluid, and anti-carpastatin antibody bound to calpastatin fixed to the solid phase amount By detecting anti-carpastatin antibody in synovial fluid Quantitative can do.
[0034]
That is, if the amount of anti-carpastatin antibody bound to calpastatin fixed to the solid phase is large, it is determined that the amount of anti-calpastatin antibody in the joint fluid is large. Conversely, if the amount of anti-carpastatin antibody bound to calpastatin fixed to the solid phase is small, it is determined that the amount of anti-calpastatin antibody in the joint fluid is small.
[0035]
For detection of the anti-carpastatin antibody bound to “calpastatin” fixed to the solid phase, for example, an anti-immunoglobulin antibody capable of binding to the anti-calpastatin antibody can be used. The class of immunoglobulin to which this “anti-immunoglobulin antibody” binds is not particularly limited, but IgG is preferred. That is, as the “anti-immunoglobulin antibody”, an “anti-IgG antibody” is preferable.
[0036]
Immunoglobulins have slightly different amino acid sequences depending on the animal species, and there are various anti-immunoglobulin antibodies corresponding to these, but the anti-immunoglobulin antibodies used in the present invention are anti-carpastatin to be measured. It is preferable to select an antibody against immunoglobulin of the same animal species from which the antibody (joint fluid containing it) is derived. For example, when measuring anti-carpastatin antibody in human synovial fluid, it is preferable to use an antibody against human immunoglobulin, more preferably an anti-human IgG antibody.
[0037]
Various anti-immunoglobulin antibodies against various animals can be appropriately prepared by those skilled in the art, and commercially available antibodies can also be used.
This anti-immunoglobulin antibody is preferable because it is labeled with a labeling substance in order to facilitate detection.
[0038]
Labeling substances used for labeling anti-immunoglobulin antibodies include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), fluorescent dyes (luminol, fluorescein isothiocyanate (FITC), etc.), chemiluminescent substances , Biotin, avidin, radioisotope, and the like, but are not particularly limited as long as they are capable of labeling proteins. The labeling method is a known method suitable for the labeling substance, for example, glutaraldehyde method, periodate crosslinking method, maleimide crosslinking method, carbodiimide method, activated ester method, etc. ("Protein Chemistry (below)", Tokyo Chemical Dojin , Published in 1987). When biotin is used as a labeling substance, a method using a hydrazide derivative of biotin (see Avidin-Biotin Chemistry: A Handbook, p57-63, PIERCE CHEMICAL COMPANY, 1994), or when using fluorescein isothiocyanate The method can be appropriately selected from the methods described in JP-B 63-17843.
[0039]
The above-mentioned “substance capable of binding to anti-carpastatin antibody” is brought into contact with a solid phase to which the anti-calpastatin antibody is fixed via calpastatin, thereby binding the substance to the anti-calpastatin antibody. The substance can be fixed to the solid phase via an anti-carpastatin antibody and calpastatin. In this case, the contact method, reaction conditions, washing and the like are the same as described above.
[0040]
When “a substance capable of binding to an anti-carpastatin antibody” is labeled, the anti-calpastatin antibody (solid phase immobilized on the solid phase via calpastatin is detected by detecting the label. Anti-calpastatin antibody bound to calpastatin affixed to the can be detected.
The method for detecting the label can be appropriately determined by those skilled in the art according to the labeling substance used for the label.
[0041]
For example, when an enzyme (such as peroxidase) is used as a labeling substance, an enzyme substrate or a chromogenic substrate is added to this, and a method of detecting the degree of color development of the product by the enzyme reaction by changing the absorbance is adopted. it can. In addition, when a fluorescent substance or a chemiluminescent substance is used as a labeling substance, a method for detecting fluorescence or luminescence of the solution after the reaction can be employed. When a radioisotope is used as a labeling substance, radiation emitted from this isotope may be detected.
[0042]
When biotin is used as the labeling substance, add an enzyme (eg, peroxidase) to which streptavidin is bound to bind biotin and streptavidin, and then add the enzyme substrate or chromogenic substrate. For example, a method of detecting the degree of color development of the product by the enzyme reaction by a change in absorbance can be employed.
[0043]
By detecting the label in this way, the presence or absence of an anti-carpastatin antibody in the joint fluid can be examined. In addition, the amount (concentration) of anti-carpastatin antibody in the joint fluid is also determined using a calibration curve or a calibration curve for the relationship between the anti-calpastatin antibody concentration and the label detection result (for example, absorbance) using an anti-calpastatin antibody standard solution with a known concentration in advance. It is possible to measure by creating a relational expression and using it. That is, in the measurement method of the present invention, “measurement” is a concept including both qualitative measurement of the presence or absence of an anti-carpastatin antibody and quantitative measurement of the amount (concentration) thereof.
[0044]
<2> Detection method of the present invention
The detection method of the present invention comprises anti-carpastatin antibody in joint fluid. Quantitative And that Quantitative Characterized by associating outcomes with disease Degree It is a detection method.
[0045]
The disease to be detected by the detection method of the present invention is not particularly limited as long as the disease causes a change in the amount of anti-carpastatin antibody in the joint fluid.
In addition, anti-carpastatin antibody in joint fluid Quantitative Although the method to perform is not particularly limited, it is preferably performed by the measurement method of the present invention.
[0046]
In addition, the correlation between the amount of anti-calpastatin antibody in the joint fluid and the disease, and the detection of the disease by this can be appropriately performed according to the disease to be detected.
For example, when the disease to be detected is a disease in which the amount of anti-carpastatin antibody in synovial fluid is increased, the joint of an animal in which the amount of anti-calpastatin antibody in synovial fluid is healthy (an animal without the disease) Since the amount of anti-calpastatin antibody in the fluid is increased compared to the amount in the joint fluid of a healthy individual, the amount of anti-calpastatin antibody measured by the measurement method of the present invention is large. It is related to “is the disease” or “highly likely to be the disease”, whereby the disease can be detected.
[0047]
In addition, for example, when the disease to be detected is a disease in which the amount of anti-carpastatin antibody in the joint fluid is decreased, the amount of anti-calpastatin antibody in the joint fluid is normal (a human without the disease). Since the amount of anti-carpastatin antibody in the synovial fluid decreases compared to the amount of anti-calpastatin antibody measured by the measurement method of the present invention or the like compared to the amount in the joint fluid of healthy individuals, It is related to “is the disease” or “highly likely to be the disease”, whereby the disease can be detected.
[0048]
Further, in the case of detecting a disease in which the amount of anti-carpastatin antibody in the joint fluid is increased or decreased as described above, the joint of an individual whose anti-calpastatin antibody amount measured by the measurement method of the present invention is healthy If it is equivalent to the amount of anti-carpastatin antibody in the fluid, it can be related to “not the disease” or “not likely to be the disease”.
[0049]
The detection method of the present invention includes not only the presence or absence of the disease but also the detection of the degree of the disease. For example, when the amount of anti-carpastatin antibody in the joint fluid of an individual is regularly measured by the measurement method of the present invention and the like, and the amount of anti-carpastatin antibody tends to deviate from the level of a healthy individual, “the above disease has progressed. Or “the disease is likely to be progressing”. Further, when the amount of anti-carpastatin antibody measured by the measurement method of the present invention tends to approach the level of a healthy individual, “the above disease is in an improvement direction” or “the above disease is in an improvement direction” Can be related to " Further, when the amount of the anti-carpastatin antibody measured by the measurement method of the present invention does not change within the range of the level of a healthy individual, “there is no change in the health” or “the possibility that there is no change in the health If the amount of anti-carpastatin antibody does not change outside the range of the level of a healthy individual, “the degree of the disease is not changed” or “the degree of the disease is changed”. It is highly possible that there is no possibility ”.
[0050]
The amount of anti-carpastatin antibody that serves as a detection criterion for the above-mentioned diseases is the amount of anti-carpastatin obtained using a calibration curve or a relational expression created for the relationship between the concentration of the anti-calpastatin antibody standard solution and the detection result of the labeled substance. It may be an antibody concentration, or may be a ratio to the amount of anti-carpastatin antibody in the body fluid of a healthy individual (an individual without the above-mentioned disease) without using the calibration curve or the relational expression.
[0051]
For example, in rheumatic diseases, particularly RA, anti-calpastatin antibodies in joint fluid are increased. Therefore, when detecting rheumatic diseases, particularly RA, anti-calpastatin antibody in joint fluid is measured by the measurement method of the present invention, and the amount of this antibody is a healthy individual (human without rheumatic disease (RA)). May be associated with “there is a rheumatic disease (RA)” or “highly likely to have a rheumatic disease (RA)”. it can.
[0052]
In the detection method of the present invention, anti-calpastatin antibody in joint fluid is measured by the measurement method of the present invention, and the amount of anti-calpastatin antibody in joint fluid is associated with rheumatic disease (especially RA), thereby causing rheumatic diseases (especially RA). ) Is preferable.
[0053]
<3> Measurement kit of the present invention
The measurement kit of the present invention comprises at least the following components (A) and (B), and contains anti-carpastatin antibodies in joint fluid. Quantitative It is a kit.
(A) Solid phase to which calpastatin is fixed.
(B) An anti-immunoglobulin antibody capable of binding to an anti-carpastatin antibody.
[0054]
The solid phase (A) and the anti-immunoglobulin antibody (B) are the same as those described in the <1> measurement method of the present invention. Therefore, the anti-immunoglobulin antibody is preferably labeled.
[0055]
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, anti-calpastatin antibody standard product of known concentration that becomes a standard for preparing a calibration curve and a relational expression, a labeling substance Detection reagents, reagents for labeling anti-immunoglobulin antibodies, and the like can be added as constituents. In addition to these components, the blocking substance, the washing solution, a solution for diluting the joint fluid, an enzyme reaction stop solution, and the like may be included.
[0056]
These components can be stored in separate containers and stored as a kit that can be used according to the measurement method of the present invention at the time of use.
[0057]
The measurement of the anti-carpastatin antibody using the measurement kit of the present invention can be performed according to the measurement method of the present invention <1>.
[0058]
<4> Detection kit of the present invention
The detection kit of the present invention comprises at least the components (A) and (B) of the measurement kit of the present invention, Degree It is a detection kit.
[0059]
The components (A) and (B) of the detection kit of the present invention and other points are the same as those described in the above <3> of the measurement kit of the present invention.
[0060]
The disease detected by the detection kit of the present invention is the same as described in <2> above. Therefore, the disease is preferably rheumatic disease, more preferably RA.
Disease detection using the detection kit of the present invention can be performed according to the detection method of the present invention <2>.
[0061]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
[0062]
First, the materials used in this example will be described together.
[0063]
96-well microplate (Iwaki Glass) (code number 3860-096)
Calpastatin (derived from human erythrocytes) (manufactured by Calbiochem; catalog number 208901)
HRPO (horseradish peroxidase) -Afini pure rabbit anti-human IgG (H + L) (manufactured by Jackson Immuno Research Laboratory; code number 309-035-003)
TBS: 20 mM Tris-HCl (pH 7.5) /0.5 M NaCl
TTBS: 0.05% Tween 20 / TBS
OPD: o-phenylenediamine (containing 10 mg in 25 ml of 0.1 M citrate-0.2 M phosphate buffer (pH 5.0)) + H ₂ O ₂ 5 μl
[0064]
Anti-calpastatin antibody detection ELISA (Progen; catalog number PR59191)
This “anti-carpastatin antibody detection ELISA” is commercially available and is a quantification kit for “autoantibodies specific to the C-terminal side of calpastatin” present in serum or plasma. In the present invention, what is fixed to the solid phase is a “polypeptide having the entire amino acid sequence of calpastatin that exists in nature”, whereas in the commercially available kit, “a peptide on the C-terminal side of calpastatin” It is different in some ways. In addition, the subject specimen is also “joint fluid” in the present invention, whereas the commercially available kit is also “serum or plasma”.
[0065]
[Example 1]
<Preparation of solid phase to which calpastatin is fixed>
Calpastatin was dissolved in 0.1 M borate buffer (pH 8.4) to a concentration of 2 μg / ml, added to a 96-well microplate at 50 μl / well, and incubated at 4 ° C. overnight. After washing 3 times with TBS, 100 μl / well of TBS containing 2% BSA / 1% gelatin was added and incubated at 37 ° C. for 1 hour. Next, the well was washed 3 times with TBS to prepare a 96-well microplate to which calpastatin was fixed.
[0066]
<Contact between standard phase and solid phase to which calpastatin is fixed>
To the calpastatin-fixed plate prepared above, various concentrations of human anti-calpastatin antibody (IgG) standard solution (2 ng / ml, 4 ng / ml, 8 ng / ml, 15 ng / ml, 30 ng / ml, 60 ng / ml or 120 ng / ml; diluted with 1% BSA / TBS) was added at 100 μl / well. After incubating at room temperature for 1.5 hours, it was washed 3 times with TTBS.
<Detection of anti-carpastatin antibody fixed to solid phase via calpastatin>
Subsequently, 50 μl / well of HRPO-rabbit anti-human IgG diluted with 1% BSA / TTBS was added to the plate and incubated at room temperature for 1 hour. After washing three times with TTBS, OPD solution was added at 100 μl / well and incubated for 15 minutes at room temperature in the dark. H ₂ SO ₄ Was added at 50 μl / well to stop the color reaction and the absorbance at 492 nm was measured. The relationship between the anti-calpastatin antibody concentration and the absorbance is shown in FIG.
[0067]
Similarly, the absorbances of various concentrations of anti-calpastatin antibody (IgG) standard solution were measured using the above-mentioned commercially available kit. Detailed operation was in accordance with the instructions attached to the kit. The relationship between the anti-calpastatin antibody concentration and the absorbance is shown in FIG.
[0068]
From the comparison between FIG. 1 and FIG. 2, it is clear that the use of a plate to which calpastatin (polypeptide having the entire amino acid sequence of naturally occurring calpastatin) is attached is a commercially available kit (on the C-terminal side of calpastatin). It was found that the measurement sensitivity was higher than when the peptide was used. Further, FIG. 1 shows that the amount of anti-carpastatin antibody can be measured accurately and quantitatively even using a plate to which calpastatin (polypeptide having the entire amino acid sequence of naturally occurring calpastatin) is fixed. . The result of FIG. 1 can be used as a calibration curve as it is.
[0069]
[Example 2]
<Measurement of anti-calpastatin antibody amount in RA patient joint fluid>
The amount of anti-carpastatin antibody in the joint fluid of RA patients (16 people) was measured in the same manner as described above. When contacting the calpastatin fixation plate prepared above, the joint fluid is diluted 2000 times with 1% BSA / TBS. When contacting with a commercially available kit, the joint fluid is diluted 200 times with the same solution. And used respectively. FIG. 3 shows a comparison between the result of measuring the same specimen with the above plate and the result of measuring with a commercially available kit.
[0070]
As is clear from FIG. 3, when the amount of anti-calpastatin antibody in the joint fluid was measured using the calpastatin-fixed plate prepared above, the measurement was performed even though the dilution rate of the specimen was 10 times higher than that of a commercially available kit. The sensitivity was remarkably high. This indicates that, according to the present invention, measurement with higher sensitivity is possible with a smaller amount of joint fluid specimen than a commercially available kit.
[0071]
Moreover, only one sample was positive in the commercially available kit for the cut-off value (30 mU / ml), which is the boundary for determining whether or not it is RA, whereas in the kit prepared above, one sample was excluded. All were positive, indicating that RA can be detected with extremely high sensitivity.
[0072]
Further, it was shown that the anti-calpastatin antibody in the joint fluid can be specifically measured without using the calpastatin-adhering plate prepared above.
[0073]
[Example 3]
<Creation of measurement kit and detection kit of the present invention>
The measurement kit of the present invention and the detection kit of the present invention having the following constitution were prepared.
1. One immunoplate (96-well) with calpastatin fixed to the well
2. One HRPO-rabbit anti-human IgG
3. One color former (OPD)
4). Reaction stop solution (6N H ₂ SO ₄ ) One
5. 1 set of human anti-calpastatin antibody (IgG) standard solution
Standard solution 1 (2ng / ml) 1 bottle
Standard solution 2 (4ng / ml) 1 bottle
Standard solution 3 (8ng / ml) 1 bottle
Standard solution 4 (15ng / ml) 1 bottle
Standard solution 5 (30ng / ml) 1 bottle
One standard solution 6 (60ng / ml)
Standard solution 7 (120ng / ml) 1 bottle
[0074]
【The invention's effect】
The measurement method of the present invention can measure the anti-carpastatin antibody in synovial fluid with higher sensitivity, and therefore requires a small amount of synovial fluid collected from a patient, and is extremely useful. In addition, the measurement method of the present invention is a method that can measure an anti-carpastatin antibody in synovial fluid in a specific, accurate, quantitative and simple manner and is extremely useful. The detection method of the present invention is an application of the measurement method of the present invention, and can detect various diseases such as RA very easily, and is therefore 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, the anti-calpastatin antibody in the joint fluid can be used with high sensitivity, specificity, and accuracy. It can be measured easily with good quantitativeness, and various diseases including RA can be detected very easily.
[0075]
Further, the measurement kit and the detection kit of the present invention are simple in configuration and have few constituent reagents, and therefore can be provided at low cost.
[0076]
The present invention is applied not only to detection of various diseases including RA described in the present specification, but also to grasping a state, determining a treatment policy, confirming a therapeutic effect, and evaluating candidate substances in drug development. Can be very useful.
[0077]
[Brief description of the drawings]
FIG. 1 shows the measurement results (calibration curve) of an anti-carpastatin antibody when a plate on which calpastatin (derived from human erythrocytes) is immobilized is used.
FIG. 2 shows the measurement results (calibration curve) of an anti-carpastatin antibody when a commercially available kit (plate on which a peptide on the C-terminal side of calpastatin is immobilized) is used.
FIG. 3 shows a comparison between the measurement method of the present invention and a method using a commercially available kit for the amount of anti-carpastatin antibody in synovial fluid. In the figure, “RF-SF improved method” indicates the result of the measurement method of the present invention, and “RA-SF Kit” indicates the result of a commercially available kit.

Claims (11)

A method for quantifying an anti-carpastatin antibody in joint fluid, comprising at least the following steps (A) and (B).
Step (A): A step of bringing the joint fluid into contact with the solid phase to which calpastatin is fixed.
Step (B): A step of detecting the amount of anti-carpastatin antibody bound to calpastatin fixed on the solid phase.   2. The method according to claim 1, wherein the amount of the anti-carpastatin antibody is detected using an anti-immunoglobulin antibody having a binding ability to the antibody.   The method according to claim 2, wherein the anti-immunoglobulin antibody is labeled. A method for detecting the degree of disease, characterized in that the anti-calpastatin antibody in joint fluid is quantified by the quantification method according to any one of claims 1 to 3 , and the quantification result is associated with a disease. The detection method according to claim 4 , wherein the disease is a rheumatic disease. The detection method according to claim 5 , wherein the rheumatic disease is rheumatoid arthritis. A kit for quantifying an anti-carpastatin antibody in joint fluid, comprising at least the following components (A) and (B).
(A) Solid phase to which calpastatin is fixed.
(B) An anti-immunoglobulin antibody capable of binding to an anti-carpastatin antibody. The quantification kit according to claim 7 , wherein the anti-immunoglobulin antibody is labeled. A kit for detecting the degree of disease, comprising at least the components (A) and (B) of the quantitative kit according to claim 7 or 8 . The detection kit according to claim 9 , wherein the disease is a rheumatic disease. The detection kit according to claim 10 , wherein the disease is rheumatoid arthritis.

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