JP2013003066A - Method for measuring dmp1 - Google Patents

Abstract

The present invention provides a method for measuring DMP1 that is simpler and more accurate than existing measurement methods, and has good accuracy and reproducibility.
SOLUTION: DMP1 in blood (serum and plasma) or urine specimen is quantified by a sandwich method using a combination of a plurality of specific antibodies against a specific site of DMP1 which is a protein produced by bone cells, and DMP1 is used as an index. A method for non-invasively quantifying bone quality, a method for diagnosing a bone / joint-related disease according to the above method, a method for confirming early reactivity to treatment of a bone / joint-related disease, and a kit for use in the method.
[Selection figure] None

Description

The present invention relates to a method for measuring the concentration of Dentin Matrix Protein 1 (usually abbreviated as “DMP1”) produced by bone cells, and a measurement kit thereof. The measurement method and kit are useful in bone quality evaluation, diagnosis of bone / joint related diseases, and confirmation of early responsiveness to treatment of bone / joint related diseases.

Bone and joint disease causes a decrease in bone strength. When the strength of the bone is reduced, fractures are likely to develop, resulting in a decrease in QOL and bedriddenness, which in turn causes death.

Conventionally, it has been reported that the strength of the bone is defined by the bone mass, but in recent years, it has been reported that the bone quality affects the bone strength as another index (Non-patent Document 1). In addition, indexes for evaluating the bone quality include the degree of bone turnover, the degree of bone mineralization, and the number of microfractures. It must be collected.

Alternatively, bone-related cells are also recognized as another index related to the evaluation of the bone quality, and bone cells are attracting attention among the bone-related cells. In particular, the relationship between the number of bone cells and the number of microfractures has been confirmed, and the confirmation method is also based on an invasive examination (Non-patent Document 2).

On the other hand, a method for grasping the number and bone quality of bone cells using a protein or the like specifically produced by bone cells as an index has been presented in a prior patent. As one of the proteins, Dentin M
atrix Protein 1 (DMP1) is mentioned (Patent Document 1). DMP
One gene is a known gene, and the sequence of DMP1 has been reported from 10 kinds of animals including humans. DMP1 is an extracellular matrix protein having 400 to 550 amino acids and is an acidic protein, and the amino acid sequence and gene sequence thereof are disclosed in Non-Patent Documents 3 to 5 by Toyozawa et al.

In addition, as a non-invasive measurement method of DMP1, TaKaRa Code M176 (anti-DMP1 peptide antibody, manufactured by Takara Bio Inc .: see Non-Patent Document 6) antibody is used, and (1) Western blot analysis (2 (1) ELISA method, (3) radioimmunoassay method, (4) method for measuring sugar chain bound to protein, etc. (Patent Document 1)
Is presented. However, the antibody presented in this document is an antibody against rat DMP1, and no results of measurement using actual human biomaterials have been reported.
Therefore, it is not clear whether the antibody can actually be used for measurement of human biomaterials, and whether the actual condition of bone quality, the ability to evaluate bone-joint-related diseases and the effectiveness of therapeutic agents can be determined. It was. Furthermore, Patent Document 1 also describes that the ELISA method is desirable in terms of protein measurement accuracy. However, the TaKaRa Code M176 (hereinafter referred to as “
Also referred to as “M176 antibody”. The ELISA method using only one kind of antibody such as) seems to have a problem in measurement because of its theoretically lower sensitivity and specificity than the sandwich method.

However, an immunological measurement method using an antibody against a peptide that maintains the immunogenicity of DMP1 is also described in the prior document (Patent Document 2). Among them, as an immunogenic amino acid sequence for obtaining an antibody against DMP1, “DAYHNKPIGDQDDND” (
SEQ ID NO: 6) has been identified, and by using a purified anti-DMP1 antibody obtained by sensitizing New Zealand white rabbits with this peptide and immunostaining with the purified anti-DMP1 antibody, DMP1 in a pathological tissue section A method to confirm a positive reaction is shown. However, although immunostaining can indicate the presence of DMP1 during tissue disruption, it is impossible to quantify the concentration of DMP1 in serum, plasma, urine, etc. collected non-invasively from biological materials. is there.

JP 2007-178356 A JP 2004-292321 A

"Osteoporosis Prevention and Treatment Guidelines 2006 Edition" Osteoporosis Prevention and Treatment Guidelines Committee, Life Sciences Publishing (2006) Bone, 2002; 30: 201-206 J. et al. Mol. Evol. 1999; 48: 160-167. Gene, 1999; 234: 307-314. J. et al. Mol. Evol. 2000; 50: 31-38. BIO VIEW 2003; 43: 18-20 (Published by Takara Bio Inc.)

Therefore, there is a need for a method for measuring DMP1 that can provide more accurate and high detection sensitivity. That is, as described above, Patent Document 1 presents a method for grasping the number and quality of bone cells using a protein or the like specifically produced by bone cells as an index, and DMP1 is one of the proteins. Although it is mentioned, it is clear that the anti-rat DMP1 peptide antibody (M176 antibody) exemplified only as an antibody for measuring DMP1 in this document cannot measure human DMP1, as shown in the reference examples described later. It became.

Furthermore, the amino acid sequence “DAYHNKPIGDQDDND” (SEQ ID NO: 6) used as an immunogen for obtaining an antibody against DMP1 in Patent Document 2 is similar to the amino acid sequence of SEQ ID NO: 5 in the present application. The inventor prepared an antibody having immunological specificity for the peptide consisting of the amino acid sequence of SEQ ID NO: 5 and attempted to measure the DMP1 concentration by sandwich ELISA using the antibody, but the sensitivity was further increased. There was room for improvement.

As a result of various studies in view of the above problems, the present inventors have found that when an immunologically specific antibody against a specific sequence site of human DMP1 is used, the measurement sensitivity is significantly higher than when other antibody combinations are used. As a result, the present invention has been completed. According to the invention, D
The presence of MP1 can be accurately and sensitively identified non-invasively, and its concentration can be measured. Furthermore, according to the present invention, an excellent DMP1 measurement kit is also provided. It has also been found that the kit can be used in, for example, a method for evaluating bone quality, a method for diagnosing a bone / joint-related disease, and a method for confirming early responsiveness to treatment of a bone / joint-related disease. That is, the present invention includes the following aspects.

(1) A method for measuring DMP1 or a fragment thereof in a sample, comprising an antibody having immunological specificity for a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, and an amino acid represented by SEQ ID NO: 2 The method according to claim 1, wherein DMP1 or a fragment thereof in the sample is measured using a combination with an antibody having immunological specificity for the peptide comprising the sequence.
(2) The method according to (1) above, wherein the measurement method is a sandwich ELISA method.
(3) A solid phase on which an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is immobilized, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 The method according to (1) or (2) above, wherein a labeled antibody labeled with an antibody having immunological specificity is used.
(4) The method according to (3) above, wherein the label is horseradish peroxidase (HRP).
(5) An antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 At least one of which is a polyclonal antibody (1) to (4)
The method in any one of.
(6) An antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 At least one of which is a monoclonal antibody (1) to (4)
The method in any one of.
(7) The sample is selected from the group consisting of urine, blood, serum and plasma (1)
Thru | or the method in any one of (6).

(8) An antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 A kit for measuring DMP1 or a fragment thereof.
(9) Antibody and sequence having immunological specificity for the peptide consisting of the amino acid sequence represented by column number 1 for use in the measurement method described in any of (1) to (7) above A kit for measuring DMP1 or a fragment thereof, comprising an antibody having immunological specificity for a peptide consisting of the amino acid sequence represented by No. 2.
(10) The above, which is used for diagnosing whether or not the patient has osteoporosis, osteoarthritis or rheumatoid arthritis by measuring DMP1 or a fragment thereof in a patient-derived sample (
The measurement kit according to 8) or (9).
(11) The degree of detection of each average DMP1 or a fragment thereof in a normal person and a patient diagnosed with osteoporosis, osteoarthritis, and rheumatoid arthritis is measured in advance, and the degree of detection and target Including comparing the degree of detection of the patient
The measurement kit according to 10).
(12) D in a sample from a patient suspected of having osteoporosis, osteoarthritis or rheumatoid arthritis
The measurement kit according to (8) or (9), which is used for predicting a therapeutic effect on the disease when a bone formation promoter is administered to the patient by measuring MP1 or a fragment thereof.
(13) The degree of detection of each average DMP1 or a fragment thereof in a healthy person and a patient diagnosed with osteoporosis, osteoarthritis, and rheumatoid arthritis is measured in advance, and the degree of detection and the target Including comparing the degree of detection of the patient
The measurement kit according to 12).
(14) The measurement kit according to (12) or (13), wherein the osteogenesis promoter is PTH or a calcium sensing receptor antagonist.
(15) Further, an antibody having immunological specificity for a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, or two or more thereof
The measurement kit according to any one of (8) to (14).

(16) For samples of patients suspected of having osteoporosis, osteoarthritis or rheumatoid arthritis,
An antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 And detecting a measurement index of DMP1 or a fragment thereof in the sample, or an index that changes in response thereto, and diagnosing the measurement index in advance as a healthy person, osteoporosis, osteoarthritis, or rheumatoid arthritis Whether or not the patient has osteoporosis, osteoarthritis or rheumatoid arthritis by confirming the average degree of detection in each patient and comparing the degree of detection with the degree of detection of the subject patient How to diagnose.
(17) The method according to (16) above, wherein the measurement method according to any one of (1) to (7) is used.
(18) A method for predicting a therapeutic effect on a disease when an osteogenesis promoter is administered to a patient suspected of having osteoporosis, osteoarthritis or rheumatoid arthritis, wherein the sequence of SEQ ID NO: 1 is applied to the sample of the patient. An antibody having immunological specificity for a peptide consisting of the amino acid sequence represented by the following, and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, Either DMP1 in the sample or a fragment thereof, or any of the indicators that change corresponding to them, and the detection level is compared with the average level of detection in a healthy person or the patient who has been confirmed separately, Said method.
(19) The method according to (18) above, wherein the measurement method according to any one of (1) to (7) is used.
(20) The method according to the above 18) or (19), wherein the osteogenesis promoter is PTH or a calcium sensing receptor antagonist.

Since the method for measuring DMP1 or a fragment thereof of the present invention enables accurate measurement in spite of non-invasive measurement, it is sufficient to collect any sample of serum, plasma, urine, for example. Thus, the burden on the patient is reduced. Furthermore, by using the method and kit for measuring DMP1 or a fragment thereof of the present invention, it becomes possible to accurately evaluate the number of human bone cells, the number of bone cavities, and bone quality. Diagnosis of joint-related diseases and determination of the efficacy of a therapeutic agent from an early stage are possible, and thus the significance of the present invention is extremely great. In addition, even in healthy individuals, it is expected that it will be possible to evaluate individual physical ages, not calendar ages, by analyzing characteristic aging changes in measured values of DMP1, so far As a physical age assessment method, it is expected to have great utility value in preventing metabolic syndrome and locomotive syndrome and maintaining the healthy life expectancy of the people.

It is the figure which showed each antigen peptide sequence of DMP1. It is a standard curve of DMP1 sandwich ELISA using each DMP1 antibody as a primary and secondary antibody. The result of having plotted DMP1 density | concentration in the serum sample of a healthy adult male according to age is shown. The result of having plotted the DMP1 density | concentration in the serum sample of a healthy adult woman according to age is shown. The average value of serum DMP1 concentration of healthy adult males and rheumatoid arthritis (RA) male patients is shown. The average value of the serum DMP1 concentration of healthy adult women and rheumatoid arthritis (RA) women patients is shown.

(DMP1)
In the present specification, DMP1 means human DMP1 unless otherwise specified. Den
The tin Matrix Protein 1 (DMP1) gene is a known gene,
The amino acid sequence and gene sequence are described in “J. Mol. Evol. 199” by Toyosawa et al.
9; 48: 160-167; Gene 1999; 234: 307-314; Mol
. Evol. 2000; 50: 31-38 "(Non-Patent Documents 3 to 5).
Thus, for example, any peptide fragment of DMP1 protein that can be used as an immunogen to immunize a test animal is based on the sequence described in the above literature, eg, a peptide synthesizer (
It can be prepared by a chemical synthesis method using a peptide synthesizer 430A type, manufactured by PerkinElmer Japan, etc.

(Immunological specific antibody against a specific sequence site of DMP1)
The antibody used in a preferred embodiment of the present invention is an amino acid sequence of each SEQ ID NO (SEQ ID NO: 1 of 22 human DMP1; SGDDTFGDDDSGPGPKDRQEGG, SEQ ID NO: 2
Is an antibody having immunological specificity for a peptide consisting of: LNEDRVDSKPEGGDSTQ and SEQ ID NO: 4: 19; ENSNSRDTGLSQPRRDSKG
-4 antibody may be abbreviated).

In the present specification, “an antibody having immunological specificity for the peptide having the amino acid sequence represented by SEQ ID NO: 1” is included in the peptide having the amino acid sequence represented by SEQ ID NO: 1. By specifically recognizing any epitope
It means an antibody that binds to a protein or any fragment containing the peptide moiety, but does not substantially bind to other proteins and other fragments not containing the peptide. Therefore, in the present invention, the antibody having immunological specificity for the peptide having the amino acid sequence represented by SEQ ID NO: 1 is any of those present in the peptide having the amino acid sequence represented by SEQ ID NO: 1. Also included are monoclonal antibodies specific for these epitopes and specific polyclonal antibodies raised in the body of experimental animals inoculated with the peptide having the amino acid sequence represented by SEQ ID NO: 1 as an immunogen. The same applies to antibodies having immunological specificity for other peptides having the amino acid sequence represented by SEQ ID NO.

In a further preferred embodiment of the present invention, the above DMP1-1 antibody, DMP1-2 antibody, D
It is preferable to use any two of the three types of MP1-4 antibodies. In another embodiment, the amino acid sequence of the following SEQ ID NO: SEQ ID NO: 3 FRKSRISEDEDRS
Antibodies having immunological specificity for peptides consisting of EL, SEQ ID NO: 5; VDAYHNKPIGDQDNDC (may be abbreviated as DMP1-3 antibody and DMP1-5 antibody, respectively) can be used in the same manner or in contrast. .

A method for producing an immunologically specific antibody against a peptide having a known amino acid sequence is known. Specifically, an immunogen containing an adjuvant, if desired, for example, a peptide containing any one of the amino acid sequences of SEQ ID NOS: 1 to 5 is subcutaneously administered to an immunized animal, and it is determined at appropriate intervals (for example, one week). The whole blood is collected after the last immunization by repeating the number of times (for example, 5 times), and antiserum is obtained by separating the whole blood. Such a method is, for example, CUREN
T PROTOCOLS IN IMMUNOLOGY, Chapter 2.4, John Wil
ey & Sons, Inc. , New York et al. Subsequently, the polyclonal antibody is purified from the antiserum by using a peptide having any one of the amino acid sequences of SEQ ID NOS: 1 to 5 used for animal immunization as a chromatography resin, such as CNBr activated Sepharose or HiTrap NHS- activated (both Amersha
m Pharmacia), and the antibody is adsorbed on the resin by specifically adsorbing the antibody in the antiserum by applying the antiserum to the solidified resin and applying the antiserum to the immobilized resin. Can be achieved by elution using an appropriate buffer, chaotropic ions, or the like, but is not limited thereto. Other methods for producing and processing polyclonal antisera include, for example, Mayer and Walker (1987): IMMUNOCHEMICAL M
ETHOD SNCELLAND MOLECULAR BIOLOGY (Academ
ic Press. London). When immunizing an immunized animal, if it is desired to increase the immunogenicity of the immunogen, for example, a peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 5 may be converted to bovine thyroglobulin or keyhole. What is necessary is just to combine with carriers, such as limpet hemocyanin (KLH), and to use as an antigen.

When the antibody of the present invention is obtained as a monoclonal antibody, a laboratory animal immunized with an immunogen prepared in the same manner as described above, preferably a rodent such as a mouse, a rat or a hamster, using a technique well known to those skilled in the art. Fusing parental cells for cell fusion, such as spleen cells of animals and myeloma cell lines, and selecting and cloning a suitable one from the obtained hybridomas,
Subsequently, the fused cells are cultured in vitro or in vivo, and a monoclonal antibody having a higher specificity than this culture mixture is collected. In addition, as described above, a permanently proliferating antibody-producing cell line can be prepared by cell fusion, but in addition, the permanent proliferating antibody-producing cell line can also be used for B lymphocytes using oncogenic DNA. Direct transformation or Epstein-Barr
It can also be prepared by other methods such as transfection with viruses. For example, J. et al. Virol. , 60: 1153, Schreier. M.M. Et al. (19
80), Virology 162: 167, Hammerling et al. (1981), B
ritish Medical J. , 295: 946, Kennett et al. (1980).
Please refer to.

The antibody of the present invention also includes an antigen-binding fragment of the antibody obtained by enzymatic digestion of the above antibody. Examples of such fragments include Fab fragments, Fa
b 'fragment, F (ab') ₂ fragment, F (v) fragment, H chain monomer or dimer, L chain monomer or dimer, dimer composed of one H chain and one L chain, and the like. The fragment can be obtained, for example, by digesting a complete antibody with a protease such as pepsin or papain, or by treating with a reducing agent as necessary after digestion. The H chain and L chain monomers can also be obtained by treating a complete antibody with a reducing agent such as dithiothreitol and then separating the purified chain. Furthermore, human disease diagnosis and
These antibodies can also be humanized in order to be usable for therapy.

(Method for measuring DMP1 or a fragment thereof in a sample)
In the detection and / or quantification of DMP1 or a fragment thereof in a biological sample collected from a test subject, preferably a sample collected non-invasively, It can be used advantageously. Non-invasive collection in the present invention includes collection of biological fluid samples such as urine, blood, serum or plasma.

As will be described later, the detection and quantification results of DMP1 or a fragment thereof in the sample that can be obtained by using the method and kit of the present invention are the bone quality (degree of bone calcification,
It can be an extremely important index in the evaluation of the degree of microfracture etc.), diagnosis of bone / joint related diseases, or confirmation of early response to treatment of bone / joint related diseases. Useful in.

Detection and quantification of DMP1 or a fragment thereof of the present invention can be performed according to a protocol based on, for example, a competitive analysis method, a direct reaction type analysis method, or a sandwich type analysis method. These protocols typically use solid supports or can utilize immunoprecipitation methods. Taking a sandwich type analysis method, which is one of the representative techniques for the detection, as an example, any labeled antibody of the present invention is prepared. For example, the antibody can be labeled with a radioactive substance, colored particles such as gold colloid, a fluorescent or chemiluminescent label, or an enzyme. Any method known to those skilled in the art can be employed as a method for labeling an antibody. Biochem. vol. 11, pages 395-399 (19
79), J. et al. Biochem. vol. 14, 41-57 (1982), Immuno.
fluorescence and related technologies, Else
vier / North Holland Biomedical Press, 215-
The method described on page 225 (1978) can be used.

In the sandwich type analysis method, any antibody of the present invention, preferably an antibody different from the labeled antibody described above, is immobilized on a solid support. The solid phase can be achieved, for example, by dissolving the antibody in a carbonate buffer (about pH 8.6), adding the solution to the well of the microplate, and incubating for a predetermined time. In short, any of the antibodies of the present invention is used for coating a solid support such as the bottom of a well to provide a capture side antibody, and any of the antibodies of the present invention (preferably an antibody different from the capture side antibody) ) Is labeled with a radioactive substance, colored particles or an enzyme to provide a detection-side antibody. A biological sample from the test subject is added to the well having the capture side antibody and incubated for a predetermined time, and then the sample is removed from the well. After the well is thoroughly washed with a suitable buffer or the like, the detection-side antibody is added to the well. After predetermined incubation, the inside of the well is washed to detect the formation of a capture side antibody-measurement object-detection side antibody complex.

Detection depends on the nature of the labeling substance labeled on the detection-side antibody. If radioactive labeling, the radiation dose is colored, if colored particle labeling, the color development amount or absorbance, and if it is enzyme labeling (ELISA method), Further, an appropriate substrate is added to the well, and the absorbance after a predetermined incubation is detected. In the example of the ELISA method, the enzyme used is not particularly limited, and for example, an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase is advantageously used. When labeling with horseradish peroxidase, 3,3 ', 5,5 as the substrate of the enzyme
'-Tetramethylbenzidine or the like can be used. When alkaline phosphatase is used, p-nitrophenyl phosphate is used as a substrate.

To describe the preferred embodiment of the present invention more specifically, the DMP1-1 antibody of the present invention, DMP1
-2 antibodies or DMP1-4 antibodies, or combinations thereof, are advantageously used. For example, one antibody against the same DMP1 epitope, multiple antibodies against different DMP1 epitopes, and combinations thereof may be used.

In the ELISA method which is one of the preferred embodiments of the present invention, the result of detection and / or quantification depends on the binding between the enzyme and the antigen or antibody, and the activity of the bound enzyme is used as a label for the detection / quantification. . For example, the antibody of the present invention is immobilized on a solid phase (such as a microplate or a plastic cup), incubated with a sample dilution such as serum, and then washed.
An enzyme-labeled antibody is added and incubated, and the solid phase is washed again. A suitable enzyme for labeling is, for example, horseradish peroxidase. The activity of the enzyme bound to the solid phase is converted into an antigen concentration by adding a substrate specific to the enzyme exemplified above and measuring the production of the product or the utilization rate of the substrate by a colorimetric method.

The solid phase is preferably an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 of the present invention or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4. An antibody having immunological specificity is immobilized on each plate, and the immobilized first antibody (
Prepare as capture side antibody). On the other hand, in the labeling, an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention is labeled with HRP and prepared as a labeled second antibody (detection side antibody). . A sample suspected of the presence of DMP1 is brought into contact with the solid-phased first antibody, washed, and then contacted with a labeled second antibody.
Measure the concentration.

(DMP1 or a fragment measurement kit thereof)
A kit having reagents labeled for immunodiagnosis can be obtained by packaging appropriate materials in appropriate containers. This suitable material may include an antibody (DMP1-1 antibody) having immunological specificity for a peptide consisting of at least the amino acid sequence represented by SEQ ID NO: 1 of the present invention. Alternatively, the appropriate material may include an antibody (DMP1-2 antibody) having immunological specificity for a peptide consisting of at least the amino acid sequence represented by SEQ ID NO: 2 of the present invention. Alternatively, the appropriate material includes an antibody having immunological specificity (DMP1-1 antibody) against a peptide consisting of at least the amino acid sequence represented by SEQ ID NO: 1 of the present invention and SEQ ID NO: 2 of the present invention. An antibody (DMP1-2 antibody or DMP1-4 antibody) having immunological specificity for a peptide consisting of the amino acid sequence represented by 4 can be included in combination. When a plurality of antibodies are included in combination, it is preferable that each antibody is put in a separate container. In addition, the above-described antibody (or one antibody when a plurality of antibodies are included) may be labeled.

Furthermore, when appropriate, as an appropriate material for the measurement kit, an antibody having immunological specificity for standard DMP1 or a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 or 5 (DMP1-3 antibody Or DMP1-5 antibody). DMP1-3 antibody or DM
The P1-5 antibody may be used, for example, for comparison of detection results. A particularly preferred embodiment of the kit of the present invention is directed to an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. A combination of antibodies having immunological specificity may be included.

The kit of the present invention further comprises the remaining reagents and materials necessary to perform the desired assay,
In addition, an appropriate set of assay instructions may be included. The kit for the ELISA method may contain a dilution buffer, a substrate specific for the labeling enzyme, and the like as necessary remaining reagents.

(Use of measurement results of DMP1 or a fragment thereof)
1. Use for Diagnosis of Bone / Joint Related Diseases (Including Preliminary Judgment) According to the present invention, DMP1 produced by bone cells present in a sample, particularly in a biological sample collected from a test subject, can be detected and / or Or, it is quantified, and the result of detection / quantification is, for example, evaluation of bone quality in which bone cells are involved (degree of bone calcification, degree of micro fracture, etc.), diagnosis of bone / joint related disease, or bone / joint related disease It can be used for confirming the responsiveness from the early stage to the treatment.

Non-limiting examples of bone-joint-related diseases diagnosed by the present invention include osteoporosis, osteoarthritis, rheumatoid arthritis, tumor-induced osteomalacia, which are diseases in which the balance of bone resorption / formation is lost. Other metabolic bone and joint diseases are included.

2. Prediction of effects of osteogenesis promoter administration to patients with suspected bone / joint-related diseases DMP1 is a substance produced from bone cells, and since PTH receptors exist in bone cells, production of DMP1 by administration of PTH The amount can change. Therefore, when an osteogenesis promoter is administered to a patient suspected of having a bone / joint-related disease, such as osteoporosis, osteoarthritis or rheumatoid arthritis, the measurement method of the present invention is used. DMP1
Or detecting or quantifying the fragment, or detecting the amount of change in an index (for example, bone density or bone metabolism marker) that changes in accordance with the fragment, and the therapeutic effect index of osteoporosis, osteoarthritis or rheumatoid arthritis By contrast, the ability to judge the therapeutic effect of the osteogenesis promoter can be confirmed.

Here, the osteogenesis promoter is a general term for substances that act on osteoblasts to promote the osteogenesis function, and include parathyroid hormone (PTH) and calcium sensing receptor antagonists such as ACS.
Med. Chem. Lett. , 2011; 2: TT-238-242.
5 and Bone, 2010; 46: 534-542, and the like. Examples thereof are known to those skilled in the art to act on osteoblasts and promote osteogenic function. As long as it is a substance, it is not limited to the exemplified substance.

The PTH of the present invention includes natural PTH, PTH produced by genetic engineering techniques, and chemically synthesized PTH, preferably human PTH consisting of 84 amino acids (human PTH (1- 84)). The term PTH in the present invention includes biologically active derivatives thereof. That is, the derivative may be a physiologically active PTH fragment or a modified form thereof. For example, the PTH partial peptide, PTH itself, or a partial amino acid of the partial peptide partially substituted with another amino acid, PTH A natural type PT, such as a peptide in which a part of the constituent amino acids of the peptide itself or a partial peptide thereof is deleted, and a peptide in which one or more amino acids are added to PTH itself or the partial peptide
It may be a peptide having all or some of the biological activities of H. The PTH
As more specific examples of the derivatives, for example, human PTH (1-34), human PTH (1-3)
1), human PTH (1-36), human PTH (1-64), human PTH (35-84),
Human PTH (1-14), bovine PTH (1-34), etc. are mentioned.

In particular, PTH (1-34) is a 34-amino acid 34 amino acid from the N-terminus of parathyroid hormone.
Corresponding to a parathyroid hormone fragment consisting of a single amino acid,
It is known that the biological activity of TH is reproduced by this PTH (1-34) (Biochemical Dictionary, Tokyo Kagaku Dojin, 1984). Clinically, it is a diagnostic agent for parathyroid function and a therapeutic agent for osteoporosis. It is used as Accordingly, preferred examples of PTH in the present invention include human PTH (1-84), human PTH (1-34), human PTH (1-37), human PTH.
TH (1-38), such as human PTH (1-34) -NH ₂ and the like, more preferably include human PTH (1-84) or human PTH (1-34) it is.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

(Acquisition of specific antibody)
1. Production of DMP1 Human DMP1 was produced and purified based on recombinant cell technology using the cDNA obtained by extracting RNA from a portion of bone tissue extracted by surgery, synthesizing cDNA with reverse transcriptase. . Specifically, cDNA with the His tag sequence added except for the stop codon of human DMP1 cDNA was inserted into pGEX-6p-1 (Pharmacia). GST and His-tag fusion human DMP1 protein was expressed in E. coli based on a conventional method. GST and His-tag fusion human DMP1 protein was purified by a method according to the manufacturer's manual using glutathione sepharose 4B (Pharmacia) and Ni-NTA agarose (Qiagen).

2. Preparation of antigen Peptides consisting of amino acid sequences of SEQ ID NOs: 1, 2, 3, 4 and 5
-1 was requested to synthesize to American Peptide Co. based on the sequence (however, when preparing the conjugate with thyroglobulin described below, the N-terminus of SEQ ID NOs: 1, 3 and 4 and the C-terminus of SEQ ID NO: 2) A cysteine residue was added.).
A conjugate of the obtained peptide and bovine thyroglobulin was prepared by the EMCS method as follows according to a conventional method. Each peptide was dissolved in 1 ml of distilled water. On the other hand, bovine thyroglobulin 5 mg / ml dissolved in phosphate buffer and EMCS dissolved in dimethylformamide were mixed so as to have the above molar equivalents, respectively, and thyroglobulin-EMC was mixed.
An S complex solution was prepared. By adding this complex solution to the peptide solution in an amount corresponding to the molar amount, a conjugate of each peptide crosslinked with EMCS and bovine thyroglobulin was prepared. The obtained conjugate was used as an antigen in the following experiments.

3. Preparation of antibody Three JW rabbits were immunized with the prepared antigen. Immunity is about every 10 weeks.
This was carried out by administering an antigen equivalent to 0 μg. The antigen was mixed with Freund's complete adjuvant only for the first immunization, and with Freund's incomplete adjuvant from the second time. After 8 immunizations, whole blood was collected and serum was separated. The obtained rabbit antiserum was mixed with a thiol Sepharose gel immobilized with the above synthetic peptide as a specific antigen at 4 ° C. overnight to be reacted.
After the gel was packed in the column, unreacted components were washed and removed with PBS. While monitoring the protein elution position at 280 nm, the antibody was eluted from the column with 0.2 M citrate buffer. Tris buffer was added to the eluted antibody solution to neutralize it. The obtained antibodies DMP1-1, DMP1-2, DMP1-3, DMP1-4 and DMP1-5 were used as purified antibodies.

4). Confirmation of antibody specificity The antigen specificity of each of the obtained purified antibodies was confirmed by the following method. DMP1 was left in a 96-well plate at a concentration of 1 μg / mL (50 ng / well) at 4 ° C. overnight to prepare a plate to which blocking solution (BSA) was added and a plate to which only blocking solution was added without adding DMP1. Each plate was washed, purified antibody diluted twice from 10 μg / ml was added and allowed to react for 0.5 hours, and then washed with PB containing 0.05% Tween20. Furthermore HRP
A labeled anti-rabbit secondary antibody was added to each well for reaction, and then washed with PB containing 0.05% Tween20. Substrate (OPD) was added to each well to develop color, the absorbance was measured at 490 nm, and the absorbance of the DMP1 adsorption well and the non-adsorption well was compared. As a result, it was confirmed that any antibody specifically bound to the DMP1 antigen.

5. Sandwich ELISA
Among the acquired specific antibodies, DMP1-2, DMP1-3, DMP1-4 and DMP
1-5 was prepared to 20 μg / ml with 0.1 M carbonate buffer, and 0.1 ml was dispensed onto a 96-well ELISA plate, and allowed to stand at 4 ° C. overnight to prepare a solid-phase antibody. Plate 2 with PBS
After washing twice, 0.2 ml of 1% BSA-containing PBS was added and allowed to stand at 4 ° C. for 16 hours to block nonspecific adsorption. After washing twice with washing solution again, 1% BSA, 0.05% Tw
A 320 pM DMP1 solution dissolved in PBS containing een20 was mixed with 1% BSA, 0.05%
The mixture was diluted from 160 pM to 2.5 pM with PBS containing Tween 20, 0.1 ml was added, and the mixture was reacted at 37 ° C. for 1 hour. After washing with washing solution 7 times, 1% BSA, 0.05% Twee
Horseradish peroxidase (HRP) labeled DMP1-1 dissolved in PBS containing n20
0.1 ml of antibody was dispensed and reacted at 4 ° C. for 30 minutes. After washing 9 times with washing liquid, TMB
0.1 ml of the substrate solution (color developing solution) was added, and the enzyme reaction was performed in the dark at room temperature for 30 minutes. Stop solution (
1N H ₂ SO ₄ ) was added to stop the reaction, and the absorbance at 450 nm was measured (see FIG. 2).
.

Since linearity was observed between the absorbance and the DMP1 concentration in any combination, the correlation between the two was confirmed. On the other hand, from the absorbance value, the measurement sensitivity is DMP1-1 and DMP.
MP1-2 antibody combination is the highest, hereinafter DMP1-1 and DMP1-4 antibody combination,
DMP1-1 and DMP1-3 antibody combination order, DMP1-1 and DMP1-5
The antibody combination was the least sensitive.

6). Additional recovery rate Next, DMP1 with different concentrations in serum, plasma (EDTA treatment, Heparin treatment), urine
Using the sample to which DMP1 was added, the DMP1 concentration was measured by the above sandwich ELISA,
The recovery rate was calculated from the comparison with the theoretical value (see Table 1). Recovery rates are DMP1-1 and DMP1
-2 antibody combination was the highest. The combination of DMP1-1 and DMP1-5 antibodies has the worst recovery rate, and the combination of DMP1-1 and DMP1-3 antibodies, DMP1-1 and DMP1-
The combination of 4 antibodies was in the middle.

From the above results, it is suggested that the combination of DMP1-1 and DMP1-5 is more strongly affected by serum, plasma and urine components than the other combinations, and the quantitativeness is inferior.

Next, using serum, plasma (EDTA treatment or Heparin treatment), and urine obtained from a healthy person as samples, the absorbance of each combination of DMP1 was measured (see Table 2). From the absorbance, D
The combination of MP1-1 and DMP1-2 antibodies has the highest sensitivity, hereinafter DMP1-1 and DM
P1-4 antibody combination, DMP1-1 and DMP1-3 antibody combination in order, DMP
The combination of 1-1 and DMP1-5 antibodies had the lowest absorbance reactivity. DMP1
In the combination of -1 and DMP1-5, no linearity of the change in absorbance according to the dilution rate was observed in all the collected samples.

From the above results, DMP1-1 antibody and DMP1-2 antibody, DMP1-3 antibody or DMP
DMP1 was measured by the combination with the 1-4 antibody, and it was confirmed that the combination of the DMP1-1 and DMP1-2 antibodies can be measured with particularly high sensitivity. In addition, DMP1-1 antibody and DMP
The combination of 1-2 antibodies was not affected by serum, plasma and urine components, and in this respect as well, the combination of antibodies was considered to be the most suitable combination for measuring DMP1 in a specimen.

7). Reference Example -Measurement with anti-rat DMP1 antibody-
An equivalent product of the anti-rat DMP1 antibody (M176 antibody) described in Patent Document 1 was prepared, and human DMP1 was measured.
As a method, anti-rat DMP1 and anti-human DMP1 1-2 and 1-4 antibodies (rabbit IgG 1 μg / well / 100 μL) were immobilized on a plate (solid phase plate), and rat or human DMP1 0.156 as a standard product. 10 ng / mL was added, and biotinylated anti-rat DMP1-1 antibody (Takara commercially available Code. M716 equivalent, rabbit IgG) was used as a labeled antibody, and the presence or absence of color development was confirmed. The experimental conditions were as follows.
1) Add 100 μl / well of standard product to solid phase antibody plate (37 ° C., 30 min)
2) Adjust biotinylated antibody to 1 μg / well and add 100 μl / well (37
℃ 30min)
3) Addition of Avidin-HRP 100 μl / well (37 ° C., 30 min)
4) Addition of TMB (chromogenic substrate) 100 μl / well, light shielding (room temperature 30 min)
5) Absorbance (A450) measurement As a result, as shown in Table 3 below, when anti-rat DMP1 antibody was used for the solid phase plate and anti-rat DMP1-1 antibody was used for the labeled antibody, the color development in the rat DMP1 standard was confirmed. However, color development with human DMP1 standard was not confirmed, and sandwich ELISA was not established. In addition, even when an anti-human DMP1 antibody is used for the solid phase plate, the labeled antibody is treated with anti-rat DM.
When P1-1 antibody is used, color development in human DMP1 standard is not confirmed, and sandwich EL
The ISA was not successful. As shown in the experimental results in “4. Confirmation of antibody specificity”, when the solid-phase plate is an anti-human DMP1 antibody and the labeled antibody is an anti-human DMP1-1 antibody, a human DMP1 standard product is used. Thus, it was confirmed that human DMP1 could not be detected with rat DMP1 antibody.

8). Confirmation of age-related changes in healthy individuals The DMP1 concentration in serum samples of healthy adult men was measured by sandwich ELISA using the combination of DMP1-1 and DMP1-2 antibodies of the present invention, and the results plotted according to age are shown in FIG. It was. The DMP1 concentration in healthy men decreased from 20 years old to 40 years old, and thereafter the reduced state persisted. This aging change is shown in D.C. Vashishth et al., Bone, V
ol. 26, no. 4, 2000: 375-380, and S.H. Qiu et al., Bone, Vo
l. 31, no. 22002: 313-318, which is similar to the age-related changes in the number of bone cells and the density of bone cavities in which bone cells are present, confirmed in invasively collected bone,
It was confirmed by one measurement that these can be measured approximately.

Next, the plot according to age of the DMP1 density | concentration in a healthy adult woman measured by the sandwich ELISA method by the combination of DMP1-1 and DMP1-2 antibody was shown in FIG. DM
The P1 concentration decreased from the age of 20 to 40 as in the case of healthy adult males, but increased over time after the age of 45. This was thought to reflect the rapid increase in bone resorption after menopause, a phenomenon peculiar to women. This aging change is observed in M.P. Iki et al. Osteoporos
Int. , 15, 2004: 981-991, which is consistent with the results of age-related changes in markers released following bone destruction (bone resorption), but the rate of change was greater for DMP1. The reason for this is that DMP1 accumulated in the bone is released with bone resorption and the number and quality of the bone cells themselves are changed to increase the blood concentration of DMP1 higher. .

In the above, the increased state of DMP1 with aging, confirmed in healthy adult women, is due to fluctuations in bone matrix protein degradation products (markers released with bone destruction (bone resorption)) due to increased bone resorption There is a possibility that the existence site of the bone cell has an influence as a factor that greatly varies in comparison.
That is, many of the bone cells are present in cortical bone, not cancellous bone. Moreover, 90% or more of the bones of the whole body are cortical bone, and the quantitative ratio of cancellous bone is small. When bone turnover increases after menopause, bone matrix protein degradation products increase in blood and urine due to increased bone resorption in both cancellous and cortical bone. There is no difference between cancellous bone and cortical bone in the presence of bone matrix protein, but bone cells are present in cortical bone at a very high density. As the amount of whole body bone, the ratio of cortical bone is large, which suggests that it strongly reflects the increase caused by destruction of many bone cells accompanying resorption of cortical bone.

Therefore, the clinical usefulness of the measurement of DMP1 is expected to be a diagnostic marker that captures changes in bone resorption more sensitively than conventional markers and reflects bone characteristics associated with aging that have not been known so far. it can. Furthermore, it becomes possible to evaluate individual body ages, not the calendar age, through bones, and can be used for preventive medicine that maintains the healthy life expectancy of the people.

9. Confirmation of the diseased state of bone / joint-related diseases DMP1 concentration in serum of rheumatoid arthritis patients was measured by sandwich ELISA using a combination of DMP1-1 and DMP1-2 antibodies, and DM in serum samples of healthy men and healthy women
The results of comparison with the P1 concentration are shown in FIGS. Serum DM in patients with rheumatoid arthritis
The P1 concentration was significantly lower in both men and women than in healthy individuals (P <0.001).

10. Diagnosis of bone / joint-related disease disease DMP1-1 and DMP1-2 with rheumatoid arthritis patients as cases and healthy subjects as non-cases
Sensitivity / specificity analysis was performed using the DMP1 concentration obtained by the sandwich ELISA method using a combination of antibodies (see Table 4). For both men and women, the DUC1 AUC is 0.
Since it exceeded 9, and both sensitivity and specificity exceeded 0.8 at each cut-off value, it was confirmed that the diagnostic ability of rheumatoid arthritis by DMP1 was extremely high.

From the above, it was possible to determine whether or not the patient is rheumatoid arthritis by measuring the patient sample with the measurement method of the present invention. If it carries out similarly, those skilled in the art can easily understand that it can be judged whether it is osteoporosis or osteoarthritis.

11. Prediction of effects of therapeutic agents for bone and joint-related diseases DMP1 is a substance produced from bone cells, and since PTH receptors exist in bone cells, there is a possibility that the production amount of DMP1 may be changed by administration of PTH is there. An antibody that recognizes the amino acid sequence of SEQ ID NO: 1 when a bone formation promoter such as PTH and / or a calcium sensing receptor antagonist is administered to a patient suspected of having osteoporosis, osteoarthritis or rheumatoid arthritis; And using an antibody that recognizes the amino acid sequence of SEQ ID NO: 2 to detect the amount of change in DMP1 and / or a fragment thereof, or an index that changes correspondingly in the sample, and osteoporosis, osteoarthritis, or rheumatoid arthritis By comparing with the therapeutic effect index, the ability to judge the therapeutic effect of the osteogenesis promoter can be confirmed.
By conducting the above experiments, it is confirmed that in each patient with osteoporosis, osteoarthritis or rheumatoid arthritis, the degree of therapeutic effect on each disease when an osteogenesis promoter is administered is predicted. Conceivable.
In the measurement method and determination of the present invention, the method of directly calculating the concentration of DMP1 is exemplified above, but it is determined by some other index (for example, absorbance, etc.) corresponding to DMP1 and / or a fragment thereof. However, it goes without saying that it can be similarly implemented.

Since the non-invasive human bone quality can be accurately evaluated by using the method and kit for measuring DMP1 or a fragment thereof of the present invention, the present invention can be used in the medical device industry field and the like. .

Claims (15)

A method for measuring DMP1 or a fragment thereof in a sample, comprising an antibody having immunological specificity for a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence represented by SEQ ID NO: 2 The method as described above, wherein DMP1 or a fragment thereof in the sample is measured using a combination with an antibody having immunological specificity for the peptide.   The method according to claim 1, wherein the measurement method is a sandwich ELISA method. A solid phase on which an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is immobilized, and an immunological test against the peptide consisting of the amino acid sequence represented by SEQ ID NO: The method according to claim 1 or 2, wherein a labeled antibody obtained by labeling an antibody having specificity is used.   4. The method of claim 3, wherein the label is horseradish peroxidase. At least one of an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 The method according to any one of claims 1 to 4, wherein is a polyclonal antibody. At least one of an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 The method according to any one of claims 1 to 4, wherein is a monoclonal antibody. The method according to claim 1, wherein the sample is selected from the group consisting of urine, blood, serum and plasma. An antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and an antibody having immunological specificity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. A kit for measuring DMP1 or a fragment thereof. An antibody having immunological specificity for a peptide consisting of the amino acid sequence represented by column No. 1 and / or represented by SEQ ID No. 2 for use in the measurement method according to any one of claims 1 to 7. A kit for measuring DMP1 or a fragment thereof, comprising an antibody having immunological specificity for a peptide comprising the amino acid sequence. By measuring DMP1 or a fragment thereof in a patient-derived sample, the patient has osteoporosis,
The measurement kit according to claim 8 or 9, which is used for diagnosing whether or not the disease is osteoarthritis or rheumatoid arthritis. The degree of detection of each average DMP1 or a fragment thereof in a healthy person and a patient diagnosed with osteoporosis, osteoarthritis and rheumatoid arthritis is measured in advance, and the degree of detection and the target patient The measurement kit according to claim 10, comprising comparing the detection levels of. In order to predict the therapeutic effect on the disease when an osteogenesis promoter is administered to the patient by measuring DMP1 or a fragment thereof in a sample derived from a patient suspected of osteoporosis, osteoarthritis or rheumatoid arthritis The measurement kit according to claim 8 or 9, which is used. The prediction is performed by measuring the average degree of detection of each average DMP1 or a fragment thereof in healthy persons and patients diagnosed with osteoporosis, osteoarthritis and rheumatoid arthritis, and the degree of detection and the target patient. The measurement kit according to claim 12, comprising comparing the degree of detection. The measurement kit according to claim 12 or 13, wherein the osteogenesis promoter is PTH or a calcium sensing receptor antagonist. Furthermore, the antibody which has immunological specificity with respect to the peptide which consists of an amino acid sequence represented by sequence number 3, sequence number 4, or sequence number 5, or two or more of them, The any one of Claims 8 thru | or 14 A measurement kit according to claim 1.

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