WO2002079256A1 - Antibody and utilization thereof - Google Patents

Abstract

An antibody binding to a complex of osteoclastogenesis inhibitory factor (OCIF) with a soluble OCIF-binding molecule (sOBM) (OCIF/sOBM complex) occurring in bodily fluids; a hybridoma producing this antibody; a process for producing the above antibody with the use of this hybridoma; and preventives or remedies for bone metabolic errors containing this antibody as the active ingredient. A method of diagnosing bone metabolic errors (in particular, rheumatoid arthritis) by quantifying the OCIF/sOMB complex; and a kit for diagnosing bone metabolic errors. The above-described antibody is useful in diagnosing, preventing and treating bone metabolic errors (in particular, rheumatoid arthritis) or as an analytical reagent for laboratory use.

Description

 Antibodies and their use

[Technical field]

 The present invention relates to an antibody that binds to a complex of an osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM).

 Further, the present invention provides a hybridoma producing the antibody, a method for producing the antibody, a preventive / therapeutic agent for bone metabolism disorders using the antibody as an active ingredient, or a diagnostic agent for bone metabolism disorders using the antibody. The present invention relates to a diagnostic method and a diagnostic kit.

[Background technology]

 Bone metabolism depends on a balance between the activity of osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption (Chambers, TJ, et al., Vitam. Horm., 46, 41-86). (199 D) o Bone metabolism disorders are thought to be caused by an imbalance between bone formation and bone resorption, as diseases associated with bone metabolism disorders such as rheumatoid arthritis, osteoarthritis, osteoporosis, Known are hypercalcemia, bone Paget's disease, and renal osteotrophic disease Rheumatoid arthritis is an intractable inflammatory disease with the synovium as the main lesion. If it progresses, it causes cartilage 'bone destruction, leading to joint function deterioration.In addition, various extra-articular symptoms may spread inflammatory diseases to systemic organs, resulting in the patient's QOL ( quality of life).

The criteria for early rheumatoid arthritis are being prepared because it is necessary to start treatment for early rheumatoid arthritis and to suppress the progression of bone dystonia. The diagnostic criteria are (1) Morning stiffness lasting for more than 15 minutes for more than one week, (2) Swelling of three or more joint areas lasting for more than one week, (3) Wrist joint, metacarpophalangeal joint (MCP) , Proximal phalangeal joint (PIP), ankle or metatarsophalangeal joint (MTP) swelling lasting more than one week, (4) symmetric swelling lasting more than one week, (5) detection of rheumatoid factor, (6 ) X-ray changes of the hand or foot, soft tissue fusiform swelling and bone atrophy, or bone erosion p43 (1988)), and rheumatoid arthritis is diagnosed if 4 out of 6 items apply. However, setting a sensitive standard for such early diagnosis will inevitably sacrifice specificity. For this reason, specific disease markers for early diagnosis of rheumatoid arthritis are currently being sought.

As mentioned above, the cells responsible for bone metabolism are osteoblasts and osteoclasts. These cells are known to interact closely, and this phenomenon is called force-linking. In other words, for the differentiation and maturation of osteoclasts, various cytokines secreted by osteoblast-like stromal cells, such as interleukin (IL) _1, IL-6, IL-11, macrophage colony stimulating factor (M- CSF), tumor necrosis factor (TNF c, transformer growth factor-β (TGF- β) such as is this and force ^s SaiwaiushitoraTsuge acting promoted or inhibited Rereru (Raisz ·· Disorder of Bone and Mineral Metabolism, 287-311, 1992; Suda et al.,: Principle of Bone Biology, 87-102, 1996; Suda et al., ··· Endocrine Reviews, 4, 226-270, 1995; Lacey et al., · ' (Endocr inology, 136, 2367-2376, 1995) ₀ Osteoclast-like stromal cells adhere to immature osteoclast precursor cells and osteoclasts to differentiate, mature and mature osteoclasts, respectively. It is known that it plays an important role in functions such as bone resorption. As a factor involved in osteocyte formation, a molecule called osteoclast differentiation factor (ODF) expressed on the membrane of osteoblast-like stromal cells was assumed (Suda et al.,: Endocrine Rev. 13, 66-80, 1992; Suda et al.,: Bone, 17, 87S-91S, 1995).

 Tsuda and colleagues report that bone marrow in the culture of human fetal lung fibroblasts IMR-90 (ATCC CCL-186)? ^ We discovered an osteoclastogenesis inhibitory factor (0CIF) and succeeded in confirming the in vivo effect of improving bone metabolism by recombinant 0CIF (W096 / 26217). And it was shown that 0CIF promotes osteogenesis by specifically inhibiting osteoclast differentiation and maturation. 0CIF is sometimes called osteoprotegerin (W097 / 23614). In addition, an osteoblast-like stromal cell line, ST-2 cDNA library

(0CIF binding molecule; 0BM) was successfully cloned. It became clear that this 0BM was the virtual 0DF. Around the same time as the discovery of 0BM, the same protein as 0BM was published as TRANCE (W099 / 29865) or RAKL (W098 / 28426). This 0BM is a type II membrane-bound protein Yes, along with soluble OBM (sOBM), whose transmembrane region has been deleted, is a factor that supports and promotes osteoclast differentiation and maturation in the osteoclastogenesis system in vitro. (W098 / 46644). 0BM is present in osteoblasts, activated T cells, etc., and 0CIF suppresses osteoclast formation by binding to 0BM and blocking its biological activity.

 Yano et al. Confirmed the presence of 0CIF in human serum and found that it strongly correlated with aging and bone metabolic disorders (Yano et al., Journal of Bone and Mineral Research, 14 (4) , 518-527 (1999)). It is expected that sOBM will be produced with the progression of bone metabolism disorder, and this will bind to 0CIF present in serum to form a complex.However, a measurement system for the complex of 0CIF and sOBM. Not developed.

[Disclosure of the invention ί

 The present inventors have conducted intensive searches in view of such a situation, and as a result, have found a monoclonal antibody having extremely high affinity for a complex of OCIF and sOBM (OCIF / sOBM complex). Furthermore, an enzyme immunoassay (enzyme immunoassay; EIA) method capable of measuring the OCIF / sOBM complex using these antibodies has been established. In addition, as a result of measuring the amount of OCIF / sOBM complex in plasma of healthy subjects and rheumatoid factor-positive patients using this enzyme immunoassay, more OCIF / sOBM complexes were detected in rheumatoid factor-positive patients.

 Accordingly, the present invention provides an antibody that binds to the OCIF / sOBM complex, a polyclonal antibody that binds to the OCIF / sOBM complex, a monoclonal antibody that binds to the OCIF / sOBM complex, a humanized monoclonal antibody that binds to the OCIF / sOBM complex, A method for producing these antibodies, which comprises collecting these antibodies from a culture of cells producing these antibodies; a pharmaceutical composition containing these antibodies; Prophylactic and therapeutic agents for bone metabolism disorders (rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone jet disease, renal osteodystrophy, etc.), and bone metabolism disorders containing these antibodies It is an object of the present invention to provide a disease diagnosis kit, a diagnostic method thereof, an OCIF / sOBM complex measurement kit, a measurement method thereof, and the like.

 The present invention has been made to solve the above problems.

That is, the present invention (1) an antibody that binds to a complex of an osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM);

 (2) the antibody according to (1), which binds to osteoclast formation inhibitory factor (0CIF);

(3) the antibody of (1) or (2), which binds to a soluble 0CIF binding molecule (sOBM);

 (4) The body according to any one of (1) to (3), which is a polyclonal antibody.

 (5) the antibody according to any one of (1) to (3), which is a monoclonal antibody;

 (6) the antibody according to (5), which is a humanized antibody;

 (7) the antibody according to (5), which is produced by Hybridoma 01-30 (FERM BP-7872);

 (8) The body according to the above (5), which is produced by hybridoma Doma H-0BM1 (FERM BP-6264).

 (9) The antibody according to (5), which is produced by hybridoma # 207 (FERM BP-7953).

 (10) Hybrid Dorma 01-30 (FERM BP-7872),

 (11) Hybrid Dorma H-0BM1 (FERM BP-6264),

 (12) Hybrid Dorma # 207 (FERM BP-7953),

 (13) A cell producing the antibody according to any one of (1) to (9) is cultured, and then the antibody according to any one of (1) to (9) is cultured from the culture. The method for producing an antibody according to any one of (1) to (9),

(14) A pharmaceutical composition comprising the antibody according to any one of (1) to (9),

(15) a preventive or therapeutic agent for bone metabolism disorder, comprising the antibody according to any one of (1) to (9) as an active ingredient;

 (16) The bone loss tf disorder is one selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone pageet disease, and renal osteodystrophy. The preventive or therapeutic agent for bone metabolism disorder according to (15),

(17) a method for diagnosing bone metabolism disorder, comprising the following steps [1] and [2]: [1] Step of measuring the amount of the complex of 0CIF and sOBM contained in a sample of a subject or a healthy subject:

 [2] When the amount of the complex contained in the sample of the subject is larger than the amount of the complex contained in the sample of a healthy subject, Determining that the subject is suffering from

 (18) The diagnostic method according to (17), wherein at least one antibody according to any one of (1) to (9) is used.

 (19) The diagnostic method according to (17) or (18), wherein at least two antibodies according to any one of (1) to (9) are used.

 (20) Antibodies produced by hybridoma 01-30 (FERM BP-7872) and antibodies produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) Characterized by using the diagnostic method according to any one of (17) to (19),

 (21) An antibody produced by Hypri-Doma 01-30 (FERM BP-7872) is used as an immobilized antibody, and Hypri-Doma H-0BM1 (FERM BP-6264) or Hybridoma # 207 (FERM BP-7872) is used. The diagnostic method according to any one of (17) to (20), wherein the antibody produced by -7953) is used as a labeled antibody.

(22) The diagnostic method according to any one of (17) to (21), wherein the sample is plasma or synovial fluid,

 (23) The diagnostic method according to any one of (17) to (22), wherein the bone metabolic disorder is rheumatoid arthritis.

 (24) A diagnostic kit for bone metabolism disorder, comprising at least one antibody according to any one of (1) to (9).

 (25) The diagnostic kit according to (24), which comprises at least two antibodies according to any one of (1) to (9).

 (26) Antibody produced by hybridoma 01-30 (FERM BP-7787) and antibody produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) The diagnostic kit according to (24) or (25),

(27) An antibody produced by hybridoma 01-30 (FERM BP-7872) is contained as an antibody for immobilization, and hybridoma H-0BM1 (FERM BP-6264) or hybrid is used. The diagnostic kit according to any one of (24) to (26) above, which comprises an antibody produced by Redoma # 207 (FERM BP-7953) as a labeling antibody.

 (28) An osteoclast inhibitor (0CIF) and a soluble 0CIF binding molecule, comprising using at least one of the antibodies according to any one of (1) to (9).

(sOMB) complex measurement method,

 (29) The measurement method according to (28), wherein at least two antibodies according to any one of (1) to (9) are used.

 (30) Antibodies produced by hybridoma 01-30 (FERM BP-7872), and hybridomas produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) The method according to (28) or (29), wherein an antibody is used,

'' (31) An antibody produced by hybridoma 01-30 (FERM BP-7872) was used as an immobilized antibody, and hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7872) was used. BP - ⁷ the antibody produced by 953), characterized in that you use as a labeled antibody, the measurement method according to any one of (28) to (30),

(32) Measurement of a complex of an osteoclast inhibitor (0CIF) and a soluble 0CIF-binding molecule (sOMB), comprising at least one antibody according to any one of the above (1) to (9). Kit,

 (33) The measurement kit according to (32), which comprises at least two antibodies according to any one of (1) to (9).

 (34) Antibodies produced by hybridoma 01-30 (FERM BP-7872) and hybridomas produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) The kit for measurement according to (32) or (33), further comprising an antibody.

 (35) Antibodies produced by Hypri-Doma 01-30 (FERM BP-7872) are contained as antibodies for immobilization, and Hypri-Doma H-0BM1 (FERM BP-6264) or Hybridoma # 20a (FERM) The measurement kit according to any one of the above (32) to (34), which comprises an antibody produced by BP-7953) as a labeling antibody.

 Etc.

The antibody that binds to the OCIF / sOBM complex provided by the present invention is useful for prevention, treatment, and / or diagnosis of bone metabolism disorders. In the present invention, bone metabolism disorders include primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis), endocrine osteoporosis (hyperthyroidism, parathyroidism, Cushing's syndrome and acromegaly) ), Osteoporosis associated with hypogonadism (hypopituitarism, Klinefelter syndrome and Turner syndrome), hereditary and congenital forms of osteoporosis (osteogenesis imperfecta, homocystinuria, menkes disease, Di syndrome), osteopenia due to reduced gravitational load or fixation or immobilization of limbs, ぺ single jet disease, osteomyelitis, infectious lesions due to bone loss, solid tumors (breast cancer, lung cancer, kidney cancer, prostate cancer, etc.) Hypercalcemia, hematological malignancies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, hyperthyroidism or kidney Hypercalcemia due to dysfunction, osteopenia due to steroid administration, and other drugs (methodrexet and immunosuppressants such as cyclosporin A, heparin and antiepileptic drugs) Osteopenia, osteopenia due to renal insufficiency, surgery, gastrointestinal disorders (small bowel disorders, large bowel disorders, chronic hepatitis, gastrectomy, primary biliary cirrhosis and liver cirrhosis), osteopenia associated with rheumatoid arthritis Osteopenia due to various types of rheumatism, bone rupture and joint destruction due to various types of rheumatism such as rheumatoid arthritis, mutilance type rheumatism, osteoarthritis, periodontal bone loss, bone metastasis of cancer (osteolytic metastasis ), Traumatic injuries, Gaucher disease, sickle cell anemia, bone dystrophy or bone cell death associated with systemic erythematosus erythema or nontraumatic injuries, osteodystrophy such as renal osteodystrophy, Force rephosphata Ze hypertriglyceridemia, osteopenia associated with diabetes, osteopenia accompanying nutritional disorder or eating disorders, and other osteopenia and the like.

The antibody provided by the present invention is not particularly limited as long as it binds to the OCIF / sOBM complex, and may be either a polyclonal antibody or a monoclonal antibody. Such antibodies, High Priestess dormer 01-30 provided by the present invention (FERM BP - 7872) by ^the: Ri site monoclonal antibodies produced specifically binds (epito pe) to specifically bind to monoclonal antibodies Specific to the site (epitope) that specifically binds to the monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953). Monoclonal antibodies and the like that bind thereto can be exemplified. As a preferred example, hybridoma 01-30

(FERM BP-7872), monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953). it can. Examples of animals immunized when the antibody of the present invention is obtained include humans, mammals other than humans, and birds. Suitable animals are different from the species from which the antigen is derived. In the present invention, the term “antigen” includes the meaning of an immunogen (i-thigh unogen).

 Antigens used for obtaining the antibodies of the present invention include 0CIF derived from humans or non-human mammals, analogs thereof, mutants thereof, derivatives thereof, and the like (above, TO96 / 26197, WO097 Hereinafter, all of them are simply referred to as “0CIF.”;), Human or non-human mammal-derived 0BM, its analogs, its mutants, its derivatives, etc.

(W098 / 46644, W099 / 29865, W098 / 28426, W098 / 46751); hereinafter, all of them are simply referred to as “0BMj.”, SOBM (W098 / 4664, W099 / 29865). Publication No., W098 / 28426, W098 / 46751), an OCIF / sOBM complex derived from a human or a non-human mammal, etc. When 0CIF is used as an immunogen, it is preferably used. A mixture of 0CIF monomer and 0CIF dimer is used.

The OCIF / sOBM complex is generally used for the isolation and purification of proteins from biological samples (tissue, blood, etc.) or cells (cultured cells, cell lines, etc.) collected from humans or non-human mammals. (2) 0CIF derived from a human or non-human mammal previously obtained and isolated and purified as necessary, and human or non-human mammals. It can be obtained by dissolving animal-derived sOBM in a solvent, mixing and then keeping the mixture warm. 0CIF or sOBM may be recombinant.Recombinant 0CIF or sOBM should be collected from a culture of prokaryotic cells or animal cells such as E. coli into which nucleotides encoding 0CIF or sOBM have been introduced. Can be. The species derived from 0CIF and sOBM in (2) is not particularly limited, but the species derived from 0CIF and the species derived from sOBM are preferably the same. The solvent for dissolving 0CIF or sOBM may be any solvent that is usually used for dissolving a protein, and examples thereof include a phosphate buffer. The solvent may contain sodium salt, a surfactant and the like. The pH range of the solvent is 5 to 10, preferably 6 to 8. The range of the mixing ratio of 0CIF and sOBM is 0CIF: sOBM = 1: 0.1 to 1: 100, preferably 0CIF: sOBM = 1: 0.2 to 1: 5. The range of the heat retention temperature is 0 to 40 ° C, preferably 0 to 37 ° C, more preferably 4 to 25 ° C. The range of this time depends on the reaction temperature, the concentration of 0CIF and sOBM, the mixing ratio thereof, and the like, but is usually 1 hour to 1 week, preferably 6 hours to 2 days. After warming, anti The desired OCIF / sOBM complex can be obtained by electrophoresis (such as PAGE under non-denaturing conditions), EIA using a combination of anti-0CIF antibody and anti-sOBM antibody or anti-0BM antibody, etc. We can confirm that we got body. The OCIF / sOBM complex thus obtained and confirmed is purified, if necessary, and then purified or selected from an antigen for immunizing an animal when obtaining the antibody of the present invention, and an antibody of the present invention. The method can be used as a standard for measuring the amount of the OCIF / sOBM complex in the test sample.

 The polyclonal of the present invention can be obtained by collecting blood from an animal previously immunized with an antigen to obtain a serum fraction, and then obtaining it by affinity chromatography using an OCIF / sOBM complex.

The monoclonal antibody of the present invention can be obtained by the following method. That is, the above-mentioned antigen is diluted with a solvent (for example, a physiological saline solution), and administered together with an immunological adjuvant (for example, Freund's complete adjuvant) intraperitoneally or intravenously, if necessary. Immunization is generally performed 3 to 4 times at intervals of 1 to 2 weeks. Alternatively, an in vitro sensitization method can also be used. After intravenously administering an antigen diluted with saline or the like to the animal, spleen cells prepared from the spleen extracted on the third day are fused with a bone marrow tumor cell line (myeloma) to produce a hybridoma by a conventional method. I do. Examples of mouse-derived myeloma include P3X63, Ag8.653 and Sp2 / 0-Agl4. Cell fusion between splenocytes and myeloma is generally performed by a known method, for example, the method of Koehler and Milstein (Koehler, G. and Milstein, C. Nature, 256, 495-497, 1975). The sensitized splenocytes and myeloma are mixed in the usual ratio of the number of cells, and fusion is performed by adding polyethylene glycol to a medium without fetal calf serum (FCS), followed by culturing in HAT selection medium with FCS. And select the fused cells (Hypridoma). Using the hybridoma culture supernatant, a hybridoma producing the target antibody that specifically recognizes the antigen is selected by a commonly used antibody detection method such as the EIA method. By cloning the resulting cells by the limiting dilution method, it is possible to establish stable hybridomas (Harlow, E. & Lane, D. , Antibodies, Cold Spring Harbor Lab. (1988)) 0

Antibodies can be purified from the culture supernatant obtained by culturing the hybridoma according to a conventional method, or from ascites obtained by inoculating intraperitoneally into an animal. Antibodies contained in the culture supernatant or ascites are analyzed by salting out, ion exchange and gel chromatography, protein It can be purified by a commonly used method such as affinity chromatography using A or G (Harlow, E. & Lane, D., Antibodies, Cold Spring Harbor Lab. (1988)). From the monoclonal antibodies obtained in this way, (1) After selecting monoclonal antibodies that bind to 0CIF, 0BM or sOBM, and then selecting antibodies that bind to the OCIF / sOBM complex from among them. Or (2) By selecting an antibody that directly binds to the OCIF / sOBM complex, a hybridoma producing the desired antibody can be obtained. From the obtained hybridoma culture, an antibody that binds to the OCIF / sOBM complex is obtained by using affinity chromatography using the OCIF / sOBM complex in addition to the above-described purification method. be able to.

 The antibody of the present invention thus obtained can be used for OCIF / sOBM immunoassay or assay. Examples of such measurement or assay include Western blotting, immunoprecipitation, EIA, radioimmunoassay (RIA) and the like. In addition, such measurements or assays include samples from humans or non-human mammals (blood, plasma, serum, tissue, synovial fluid, urine, lymph, etc.), cells (cultured cells, cell lines, etc.), The culture supernatant, their extracts, their partially purified fractions, and the like can be used as samples, and suitable samples are plasma or synovial fluid.

 The present invention provides a method for diagnosing a bone metabolism disorder in a human or a non-human mammal. The diagnostic method is not particularly limited as long as it includes a step of measuring the amount or concentration of the OCIF / sOBM complex, and includes, for example, the following steps [1] and [2];

 [1] Step of measuring the amount of OCIF / sOBM complex contained in a sample from a subject or a healthy subject:

 [2] When the amount of the complex contained in the sample of the subject is larger than the amount of the complex contained in the sample of a healthy subject, the subject has a bone metabolic disorder Determining that the subject is suffering from the disease.

In a preferred diagnostic method, in the above step [2], the amount of the complex contained in the sample of the subject is twice as large as the amount of the complex contained in the sample of a healthy subject. If the number is large, it is determined that the subject is suffering from bone metabolism disorder, or the amount of the OCIF / sOBM complex contained in the sample of the subject or a healthy subject is determined three times each. Measuring The average value of the OCIF / sOBM complex contained in the sample of the subject is twice the standard deviation of the average value of the OCIF / sOBM complex contained in the sample of the healthy subject. If the value is equal to or greater than the value obtained by adding the calculated values, it is determined that the subject is suffering from bone loss and haze disorder. In a more preferred diagnostic method, the antibody provided by the present invention is used when measuring the amount of the OCIF / sOBM complex in the above step [1]. As a measurement method using such an antibody, various EIAs such as ELISA, sandwich EIA and the like can be exemplified. Preferably, sandwich EIA (Harlow, E. & Lane, D., Antibodies , Cold Spring Harbor Lab. (1988)). In the case of sandwich EIA, at least two antibodies of the present invention are selected as a primary antibody and a secondary antibody, the primary antibody is immobilized on an insoluble carrier or the like, and the secondary antibody is used as a labeled antibody for OCIF / sOBM detection. The preferred antibodies of the present invention are monoclonal antibodies produced by hybridoma 01-30 (FERM BP-7872) and hybridoma H-0BM1 (FE RM BP-6264) or hybridoma # 207 (FERM BP -When combining the monoclonal antibody (# 207) produced by 7953), it is preferable to use the former as the primary antibody and the latter as the secondary antibody.

 Further, in the diagnostic method of the present invention, the phrase “j is subject having bone metabolism disorder” includes, in addition to the fact that the subject already has bone metabolism disorder, In the present invention, the subject and the healthy subject in the present invention are mammals other than humans or humans.

 The above step [1] in the diagnostic method of the present invention is included in the present invention as a method for measuring an OCIF / sOBM complex.

 The present invention provides a kit for diagnosing a bone metabolism disorder in a human or a non-human mammal. The kit is not particularly limited as long as it contains at least one antibody of the present invention, but preferably contains at least two antibodies of the present invention when used for measurement by sandwich EIA. One of them is an antibody immobilized on an insoluble carrier, and the other is a labeled antibody. In addition, the kit of the present invention appropriately contains a lysing agent, a diluent for a sample, a diluent for a labeled antibody, an OCIF / sOBM complex standard, a detergent, a substrate for a labeled enzyme, a reaction stopping solution for a labeled enzyme, and the like. Is also good.

As the insoluble carrier, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacryl nitrile, latex, polymers such as magnetic fine particles in which latex is coated with metal, and combinations thereof can be specified. Wear. The shape of the insoluble carrier can be various shapes such as tray shape, spherical shape, container shape, test tube, porous filter, and microtiter plate. As the labeling substance of the labeled antibody, it is advantageous to use enzymes, fluorescent substances, luminescent substances, radioactive substances and the like. Enzymes include peroxidase (hereinafter referred to as “P0D”), alkaline phosphatase,] 3-galactosidase, glucose oxidase, and the like. Fluorescent substances such as fluorescein isothiosinate, and Fi co pyridinium protein, as the light-emitting substance, Isorushinoru, etc. Cie genin, and as the radioactive material as possible out to the like ^{125 1, 131 1, 14 c} ,. Further, the present invention is not limited to those exemplified above, and is not particularly limited as long as it can be used for an immunological assay for biotin and the like.

When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure its activity. When using the POD as enzymes, the 0 ₂ as substrate, 2 a color former, 2 '- Ajinoji - (3-E chill benz thiazoline sulfonic acid) Anmoniumu salt (ABTS), 5-Aminosarichiru acid. -Phenylenediamine, 3,3 ', 5,5'-tetramethylbenzine, etc., as substrate when using enzyme phosphatase as enzyme. When using -D-galactosidase as the enzyme, fluorescein-di- (β-D-galatatoviranoside), 4 -nitrophenylphosphate, 4-methylumbelliferyl phosphate, etc. For example, -methylumberiferi / le / 3-D-galactopyranoside can be used.

 Any lysing agent may be used as long as it is commonly used for immunological measurement.For example, the pH range of phosphate buffer, Tris-HCl buffer, acetate buffer, etc. ranges from 6.0 to 8.0. Are shown as preferred examples. Further, as the detergent, those commonly used for immunological measurement are also used as they are. Examples include physiological saline, phosphate buffer containing saline, Tris buffer containing saline, and mixtures thereof. These detergents may further contain a nonionic surfactant such as Triton X-100, Tween20 or Brij35, and an ion surfactant such as sodium dodecyl sulfate or CHAPS.

 Moreover, the kit for diagnosing bone metabolism disorders provided by the present invention is also encompassed in the present invention as a kit for measuring an OCIF / sOBM complex.

The present invention provides a pharmaceutical composition containing the antibody of the present invention, and an agent for preventing or treating bone metabolism disorders. When the monoclonal antibody is contained in the pharmaceutical composition of the present invention and administered to a human, and the monoclonal antibody is derived from a mammal other than a human, the monoclonal antibody is preferably humanized.

 For example, when a mouse-derived monoclonal antibody is administered to a human, a human antimouse antibody response (hereinafter referred to as “HAMA”) occurs (Schloff et al., Cancer Res., 45, 879-). 85 (1985)). When a mouse-derived monoclonal antibody is to be administered to humans for a long time or continuously, the monoclonal antibody is humanized.

In general, an antibody is composed of two immunoglobulin heavy chains each having a molecular weight of about 50,000 (hereinafter referred to as “heavy chains”) and two immunoglobulin light chains each having a molecular weight of about 23,000 (hereinafter referred to as “light chains”). It is configured. The heavy chain and the light chain each have a region having a different amino acid sequence called a variable region at about 110 residues from the amino terminal. Furthermore, among these variable regions, the frequency of amino acid sequence mutation is particularly high, and the region is called a hypervariable region, which is located near the 30th, 50th, and 95th positions from the amino terminus, respectively. In an antibody molecule, these hypervariable regions are assembled and folded so as to form a surface structure to which an antigen can bind. As described above, since the hypervariable region (variable region) is involved in the formation of the antigen binding site, the hypervariable region is referred to as a complementarity determining region (CDR). The part sandwiched between the two CDRs is called a framework (frame work region: hereinafter referred to as “FR”). Kabat et al. Collected a large number of primary sequences of the variable regions of the heavy and light chains, and based on the conservation of the sequences; created a table in which each primary sequence was classified into CDRs and frameworks (Kabato et al. SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, EA Kabatt et al.). In addition, each framework was classified into a plurality of subgroups having a common amino acid sequence. In addition, a corresponding framework was found to exist between human and mouse. The humanized antibody of the present invention includes a chimeric antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (Chem.), In which a variable region in a monoclonal antibody derived from a non-human animal is conjugated to a human-derived constant region. 1984)), and antibodies in which a site other than the CDR is replaced with a human antibody amino acid sequence (CDR-grafted antibody: PT Jones et al., Nature 321, 522 (1986)). Has less immunogenic CDR-grafted anti- (Riechmann, L., et al., Nature 332, 323-327 (1988); Isaacs, JD. Et al., Lancet 340, 748-752 (1992)).

 In order to prepare a CDR-grafted antibody, it is necessary to set the amino acid sequence of the variable region such that the entire CDR sequence and a part of the amino acid residues of the FR sequence are grafted to a human antibody. This design follows the following method.

 Generally, an antibody derived from a non-human mammal having a CDR to be transplanted is defined as a “donor”, and a human antibody to which the CDR is transplanted is defined as an “acceptor”. The present invention also complies with this definition. .

 When designing for humanization, guidelines for selecting subgroups of receptor are:

 (1) Using the natural combination of known immunoglobulin heavy and light chains of human antibodies with natural amino acid sequences in their entirety,

 (2) The combination is preserved as a subgroup to which the heavy and light chains belong, but the heavy and light chains are derived from different human antibodies and have high identity to the amino acid sequences of the donor heavy and light chains Either a sequence or a consensus sequence is selected. In the present invention, the above guidelines can be followed.

 (3) It is also possible to adopt a method of selecting the heavy chain and light chain FRs having the highest identity with the donor FR from the human antibody primary sequence library without considering the subgroup combination. is there. By these selection methods, the identity of the amino acid in the FR moiety between the donor and the acceptor can be at least 70% or more. By employing this method, the number of amino acid residues to be transplanted from the donor can be reduced, and the induction of the HAMA response can be reduced.

In addition, the operation of predicting the tertiary structure from the primary sequence of an antibody molecule has limited prediction accuracy, and the role of an amino acid residue that rarely appears in the subgroup to which the donor belongs cannot be sufficiently specified. According to the method of Queen et al. (See Japanese Patent Application Laid-Open No. 4-502408), it is difficult to determine which amino acid residue of a donor or an acceptor should be selected at such a position. According to the selection method of (3), the chance of making such a judgment can be significantly reduced. When an antibody that binds to the OCIF / sOBM complex is used for the prevention or treatment of various bone metabolic disorders, it can be administered in various forms, but the type of disease, the degree of the disease, the age of the patient, the sex of the patient, etc. Can be appropriately selected according to the conditions. For example, tablets, capsules, powders, granules, syrups are orally administered, injections are administered intravenously, alone or mixed with normal replenishers such as glucose and amino acids, or intramuscularly administered alone It is administered subcutaneously, intradermally, intraperitoneally, and suppositories are rectally. These preparations can be used in the pharmaceutical preparation field, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspensions, coatings, etc., according to the usual methods. It can be formulated using an auxiliary agent.

 For molding into a tablet form, a wide variety of carriers known in the art can be used. Examples of such carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and caicic acid; water, ethanol, propanol, simple syrup, dextrose, Binders such as starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; etc .; —dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene Disintegrators for sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose; disintegrators for sucrose, stearin, cocoa butter, hydrogenated oil, etc .; quaternary ammonium base, sodium lauryl sulfate Absorbents such as glycerin and starch; Adsorbents such as starch, lactose, kaolin, bentonite, and colloidal keic acid; Lubricants such as purified talc, stearate, borate powder, polyethylene glycol, etc. Can be mentioned. The tablets may be tablets coated with ordinary skin as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

 For molding into a pill form, a wide variety of carriers known in the art can be used. Examples of such carriers include excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, and ethanol; laminaran, agar, and the like. And the like.

For shaping in the form of suppositories, a wide variety of carriers known in the art can be used. Such carriers include, for example, polyethylene glycol, cocoa butter, Higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like can be mentioned.

 When prepared as an injection, the liquid preparation and the suspension are preferably sterilized and isotonic with blood. In order to form these solutions, emulsions and suspensions, any diluent known in the art can be widely used, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, Polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be mentioned. In this case, a sufficient amount of salt, glucose, glycerin, etc. may be included in the pharmaceutical preparation to maintain isotonicity with blood, and ordinary solubilizing agents, buffers, soothing agents, etc. May be added.

 In addition, if necessary, coloring agents, preservatives, flavors, flavors, sweeteners, and other medicines may be included.

 Although the amount of the antibody that binds to the OCIF / sOBM complex contained in these pharmaceutical preparations is not particularly limited, it is usually 0.1 to 70% by weight, preferably 1 to 30% by weight. %.

 The dosage of the antibody that binds to the OCIF / sOBM complex depends on the symptoms, age, body weight, dosage form, dosage form, etc., but is generally 500 to l, 000 mg / day and 10 to 10 days / day for adults. The preferred range is 50 to 500 mg.

The frequency of administration of a drug containing an antibody that binds to the OCIF / sOBM complex as an active ingredient depends on the dosage form, dosage form, etc., but is once every few days, once a day, or several times a day. The present invention also provides a hybridoma that produces the monoclonal antibody of the present invention. Such a hybridoma is not limited as long as it is a cell that produces an antibody that binds to OCIF / sOBM. The site where the monoclonal antibody produced by hybridoma 01-30 (FERM BP-7872) binds ( hybridoma that produces a monoclonal antibody that specifically binds to an epitope), or a monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) And hybridomas that produce a monoclonal antibody that specifically binds to an epitope (epitope), such as hybridoma 01-30 (FERM BP-7872) and hybridoma H-0BM1. (FERM BP-6264), hybridoma # 207 (FERM BP-7953) and the like. [Brief description of drawings]

 FIG. 1 shows the results when the OCIF / sOBM complex of the present invention (Example 3) was subjected to electrophoresis (Native PAGE) under non-denaturing conditions.

 Description of symbols>

 Lane 1: OCIF / sOBM complex

 Lane 2: Human 0 CIF monomer

 Lane 3: human sOBM

 Fig. 2 shows the results of measurement of the OCIF / sOBM complex by sandwich EIA using the anti-sOBM monoclonal antibody of the present invention (Example 4) as a solid-phased antibody and a perch anti-OCIF polyclonal antibody as a labeled antibody. .

 Fig. 3 shows the results of OCIF / sOBM complex measurement by Sandwich EIA constructed using anti-sOBM monoclonal antibody # 207 as the immobilized antibody and anti-OCIF monoclonal antibody as the labeled antibody in this effort (Example 4). Is shown.

 FIG. 4 shows the measurement of OCIF / sOBM complex by sandwich EIA in which anti-sOBM monoclonal antibody # 207 and anti-OCIF monoclonal antibody 01-30 of the present invention (Example 4) were respectively immobilized or labeled. The results are shown.

 FIG. 5 shows a calibration curve prepared using the OCIF / sOBM complex standard prepared in Example 3 of the present invention (Example 5).

 FIG. 6 shows the results obtained by using a sandwich EIA constructed with anti-sOBM monoclonal antibody # 207 of the present invention (Example 6) as a labeled antibody and anti-OCIF monoclonal antibody 01-30 as a solid-phased antibody in serum or rheumatism of a healthy subject. 4 shows the results of measuring the amount of OCIF / sOBM complex in plasma of factor-positive patients.

[Best Mode for Carrying Out the Invention]

 Next, the present invention will be described in more detail with reference to examples. However, these are merely examples, and the present invention is not limited thereto. Example 1 Preparation of anti-OCIF monoclonal antibody

Anti-OCIF monoclonal antibody was prepared according to the method of Yano et al. (J. Bone & Mineral Res., 14, 518-527 (1999)). That is, BALB / c mice were immunized with a mixture of human 0CIF monomer and dimer purified according to the method of Tomoyasu et al. (Biochem. Biophys. Res. Co Rat, 245, 382-387 (1998)). Next, the spleen was collected from the mouse. A hybridoma was prepared by cell fusion of the obtained spleen cells and mouse myeoma cells (P3 X63. Ag8.63), and 0CIF was converted to 0CIF using EIA immobilized with 0CIF. Cells producing antibodies that specifically bind were selected. The operation of cloning the hybridoma in which production of an antibody that specifically binds to 0CIF was recognized by the limiting dilution method was performed 3 to 5 times, and a clone having a high antibody production was selected. The obtained clone was administered intraperitoneally to BALB / c mice to which pristane (Aldrich Chemical Co., Ltd.) had been administered at a concentration of 1 to 10 × 10 ⁶ cells / animal. Ascites was collected. From this ascites, affinity chromatography was performed using Affigel Protein A Sepharose. (Piorad) according to the protocol attached to the kit to purify 40 types of antibodies. When the obtained purified antibody was subjected to SDS-PAGE, a uniform band was detected at a position having a molecular weight of about 150,000. Example 2 Preparation of anti-sOBM monoclonal antibody

 The anti-sOBM monoclonal antibody was prepared basically according to the method of Yano et al. (J. Bone & Mineral Res., 14, 518-527 (1999)).

That is, BALB mice were immunized with human sOBM purified according to the method described in W098 / 46644, and then spleens were collected from the mice. A hybridoma was prepared by fusing the obtained spleen cells and mouse myeloma cells (P3X63.Ag8.653), and sOBM-specific EIA was performed by immobilizing sOBM from these cells. Cells producing antibodies that bind to were selected. Cloning of hybridomas, in which production of an antibody specifically binding to sOBM was observed, by limiting dilution was performed 3 to 5 times, and clones with high antibody production were selected. The obtained production strain was administered intraperitoneally to BALB / c mice to which pristane (Aldrich Chemical Co., Ltd.) had been administered at a concentration of 1 to 10 × 10 ⁶ cells / animal. Was collected. From this ascites fluid, affinity chromatography was performed using a protein A column (Pharmacia) according to the protocol attached to the kit to purify 40 types of antibodies. When the obtained purified antibody was subjected to SDS-PAGE, a uniform band was detected at a position of about 150,000 in molecular weight. Example 3 Preparation of Standard OCIF / sOBM Composite

To 10 mM phosphate buffer (pH 7.4) containing 0.1% Tween20 -0.15 M NaCl (PBST), use the method of Tomoyasu et al. (Biochem. Biophys. Pres. Commun., 245, 382-387 (199 8 )), And human sOBM obtained according to the method described in W098 / 46644 is dissolved at 40 g / ml, respectively, so that the human 0CIF monomer obtained according to After incubation at 4 ° C for 18 hours, a human 0CIF / human sOBM complex was prepared (100 / ig / ml as a complex). The formation of the complex was examined by electrophoresis (Native PAGE) under non-denaturing conditions by partially modifying the method of Davis et al. (Ann. NY Acad. Sci. 121, 404 (1964)). That is, the above-mentioned human 0CIF / human sOBM complex and human 0CIF monomer and human sOBM were used in an equal amount of sample buffer (0.0625M Tris-HCl (pH 6.8), 15% Glycerol, 0.001% BPB), and 10 μΐ of the sample mixture was analyzed by Native PAGE. The electrophoresis gel used was Multigel 4/20 (Daiichi Kagaku), the electrophoresis buffer was 0.025M Tris-0.192M Glycine (pH 8.4), and the gel was stained with Silverstein KANTO (Kanto Chemical). Fig. 1 shows the results. As a result, human 0CIF monomer (lane 2) and human sOBM (lane 3) showed single bands at different positions on Native PAGE. In the complex (lane 1) prepared by mixing them, a band was detected at a position different from that of human 0CIF monomer or human sOBM. Further, in lane 1, no protein was detected at the position corresponding to human 0CIF monomer or human sOBM. The above results indicated that human 0CIF monomer and human sOBM reacted under these conditions to form a complex. ^J Example 4 Measurement of OCIF / sOBM complex by EIA

 (1) Selection of anti-sOBM monoclonal antibody

 Sandwich EIA was constructed by using 40 kinds of anti-sOBM monoclonal antibodies obtained in Example 2 as solid-phased antibodies, and using a rabbit ego anti-0CIF polyclonal antibody described in W096 / 26217 as a labeled antibody.

標識 Labeling of a heron anti-0CIF polyclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). Dissolve each of the 40 anti-sOBM monoclonal antibodies in 0.1 M sodium bicarbonate solution (pH 9.6) to a concentration of 10 μg / ml, and add 100 μl aliquots to each well of a 96-well Elymno plate (Nunc). In addition, the mixture was allowed to stand at 4 ° C for immobilization. Discard each well and add 25% (V / V) Solution (manufactured by Dainippon Pharmaceutical Co., Ltd.)

 did. Thereafter, the plate was washed with PBST.

 Next, each of the OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% block ace, added to each well in a volume of ΙΟΟμΙ, and allowed to react at room temperature for 2 hours. Two hours later, the plate was washed with PBST, and POD-labeled 'Egret anti-0CIF polyclonal antibody, diluted 1000-fold with PBST containing 25% Block Ace, was added to each well, and allowed to react at room temperature for 2 hours. After washing the plate with PBST, add 100 1 enzyme-substrate solution (TMB, ScyTec) to each well to develop color, then add 停止 μΙ of reaction stop solution (ScyTec) to each well to perform enzyme reaction. Stopped. The absorbance at 450 nm of each well was measured using a microplate reader (Imnoleader I NJ2000: Intermed Japan). As a result, of the 40 anti-sOBM monoclonal antibodies, 8 were confirmed to bind to the OCIF / sOBM complex. As shown in FIG. 2, # 207 had the highest reactivity with the OCIF / sOBM complex.

 In addition, on November 5, 1997, Hypri-Doma, which produces # 207, was established on November 5, 1997 in Tsukuba East, Ibaraki, Japan. Tsukuba East Co., Ltd. 1-1-1 was deposited internationally as H-0BM1 with the 6th Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology (AIST) under the accession number FERM BP-6264. Hypri-Dorma, which produces # 207, was established on February 19, 2001 at 1-1-3 Tsukuba-Higashi, Ibaraki, Japan, by the Ministry of International Trade and Industry, Ministry of International Trade and Industry. Tsukuba East Co., Ltd. 1-1-1 was deposited with the National Institute of Advanced Industrial Science and Technology (AIST) at # 6, the # 6 National Institute of Advanced Industrial Science and Technology (AIST), and was assigned the accession number FERM P-18210. Transferred to the International Depositary on 11 March and assigned Accession No. FERM BP-7953. ,

 (2) Selection of anti-0CIF monoclonal antibody

A sandwich EIA was constructed using the anti-sOBM monoclonal antibody # 207 obtained in (1) above as a solid-phased antibody and the 40 anti-OCIF monoclonal antibodies obtained in Example 1 as labeled antibodies. Labeling of the anti-OCIF monoclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). 0. 1M bicarbonate Natoriumu solution to anti-sOBM monoclonal antibody (ί½0 ⁷⁾ becomes 10 μ g / ml (pH9. 6) To溶角牟, each 100 μ ϊ 96 © El I Takeno plates (Nunc ) Then, the mixture was allowed to stand still at 4 ° C. to solidify. The solution in each well was discarded, 300 μl of 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added, and the mixture was left standing at room temperature for 2 hours for blocking. Thereafter, the plate was washed with PBST.

 Next, each of the OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% Block Ace, and 100 μl of each was added to each well, followed by reaction at room temperature for 2 hours. After the reaction, wash the plate with PBST, add 100 1 of each of 40 POD-labeled anti-OCIF monoclonal antibodies diluted 1000-fold with PBST containing 25% Block Ace to each well, and react at room temperature for 2 hours. I let it. After washing the plate with PBST, add ΙΟΟμΙ of enzyme substrate solution (TMB, ScyTec) to each well to develop color, and add 100μl of reaction stop solution (ScyTec) to each well. The reaction was stopped. The absorbance at 450 nm of each well was measured with a microplate reader. As a result, it was confirmed that out of the 40 types of anti-0CIF monoclonal antibodies, 6 types bind to the OCIF / sOBM complex. As shown in FIG. 3, it was revealed that 01-30 had the highest reactivity with the OCIF / sOBM complex. On April 14, 1999, the Hypri-Doma was established by the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry of 1-1-3 Tsukuba, Ibaraki, Japan (currently 1-1-1 East, Tsukuba, Ibaraki, Japan). Deposit No. 01-30 with the 6th Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology (AIST), assigned accession number FERM P-17366, and transferred to the International Depositary on February 1, 2002. Accession No. FERM BP-7872.

 (3) Selection of immobilization and labeling

The anti-human sOM monoclonal antibody # 207 obtained in (1) above or the anti-0CIF monoclonal antibody 01-30 obtained in (2) above was added to a 0.1 M sodium bicarbonate solution at 10 μg / ml. (pH 9.6), 100 μl of each solution was added to each plate of 96 Ueno Reim Nobrate (Nunc), and the mixture was allowed to stand at 4 ° C. for solidification. The solution in each well was discarded, 25% Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) (300 μΐ) was added, the mixture was allowed to stand at room temperature for 2 hours, and blocking was performed. Thereafter, the plate was washed with PBST. Next, 5 ng / ml of the OCIF / sOBM complex obtained in Example 3 was dissolved in PBST containing 40% block ace, diluted, added to each well in 100 μl portions, and reacted at room temperature for 2 hours. . Two hours later, the plate was washed with PBST, and when # 207 was immobilized, POD-labeled 01-30 was used.When 01-30 was immobilized, POD-labeled # 207 was used for 25 times each. Dilute with PBST containing Block Ace and add ΙΟΟμΙ to each well. The reaction was allowed to proceed at room temperature for 2 hours. After washing the plate with PBST, add 100 1 enzyme substrate solution (TMB, ScyTec) to each well to develop color, then add 100 μl of stop solution (ScyTec) to each well to perform enzyme reaction. Stopped. The absorbance at 450 nm of each well was measured with a microplate reader. The results are shown in FIG. As a result, the OCIF / sOBM complex can be detected with higher sensitivity when 01-30 is used as the immobilized antibody (primary antibody) and # 207 is used as the labeled antibody (secondary antibody) compared to the other combination. Was confirmed. Example 5 Preparation of calibration curve

 Using the OCIF / sOBM complex standard prepared in Example 3 in the concentration range of 0 to 100 ng / ml, the method of Example 4 (3), that is, using 01-30 as the immobilized antibody, and # The measurement was performed by sandwich EIA using 207 as a labeled antibody, and a calibration curve was prepared. Figure 5 shows a typical result. This EIA method confirmed that the absorbance increased with increasing complex concentration, and this calibration curve revealed that the OCIF / sOBM complex could be quantified in the concentration range of 1 to 25 ng / ml. Example 6 Measurement of the amount of OCIF / sOBM complex contained in a sample

 The amount of the OCIF / sOBM complex in the serum of healthy subjects or the plasma of rheumatoid factor-positive patients was measured by sandwich EIA using 01-30 as the immobilized antibody and # 207 as the labeled antibody.

Dissolve 01-30 to a concentration of 10 μg / ml in 0.1 M sodium bicarbonate solution (pH 9.6), add ΙΟΟμΙ to each well of a 96-well immunoplate (Nunc), and add The 01-30 antibody was immobilized by allowing the mixture to stand at ° C. The solution in each well was discarded, 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.) 300 ^ 1 was added, and the mixture was allowed to stand at room temperature for 2 hours to block. Thereafter, the plate was washed with PBST. Normal serum (BioWhitaker) and rheumatoid factor-positive patient plasma (International Enzymes, Inc.) were each used at 40 ° /. It was dissolved in PBST containing PROC Ace, diluted 2-fold, added to each well in triplicate at 100 / zl, and reacted at room temperature for 2 hours. After the reaction, the plate was washed with PBST, POD-labeled # 207 was diluted with PBST containing 25% Block Ace, and 100 was added to each well, followed by reaction at room temperature for 2 hours. After the reaction, the plate is washed with PBST, and ΙΟΟμ 酵素 of enzyme substrate solution (TMB, ScyTec) is added to each well. After color development, the enzyme reaction was stopped by adding ΙΟΟμΙ of a reaction stop solution (ScyTec) to each well. The absorbance at 450 nm of each gel was measured with a microplate reader, and the average and standard deviation were calculated for the measured values. A Student's t-test was performed on the calculated values.

 The results are shown in FIG. The average amount of OCIF OBM complex in plasma of rheumatoid factor-positive patients is larger than twice the average amount of OCIF / sOBM complex in serum of healthy subjects, and It was larger than the average value of the amount of the OCIF / sOBM complex plus twice its standard deviation. The amount of OCIF / sOBM complex contained in the plasma of rheumatoid factor-positive patients was statistically significantly higher than that in the serum of healthy subjects. Example 7

 (1) Manufacture of OCIF / sOBM complex measurement kit (for 80 samples)

 (1) Antibody produced by Hypri-Doma 01-30 was immobilized by the method of Example 4 and blocked in advance with Block Ace.

(2) P207-labeled # 207 antibody by the method of Example 4: 10 μl

 (3) OCIF / sOBM complex standard: 1, 2.5, 5, 10, 25 ng / ml 5 concentrations 400 μ1 each

(4) Sample dilution (PBS solution containing 0.01% Tween20 and 40% Block Ace): 10 ml

 (5) Labeled antibody diluent (PBS solution containing 0.01% Tween20 and 25% Block Ace): 10 ml

 (6) 96-L plate washing solution (PBS solution containing 0.1% Tween20): 1 liter

(7) Substrate solution for measuring the activity of the labeled enzyme (here, TMB solution): 10 ml

(8) TMB stopping reagent: 10ml

 (2) Measurement using kit

To each well of the plate (1), add the sample diluted with the diluent (4), normal pooled serum and OCIF / sOBM complex standard (3) in triplicate 100 // 1 each. After leaving at room temperature for about 2 hours, wash each well of the plate 5 to 6 times with the washing solution (300 ^ 1). An automatic plate washer may be used for this washing operation. To each well of the washed plate, add 100 μΐ of a solution obtained by diluting P0D-labeled # 207 antibody (2) 500-fold with diluent (5), and let stand at room temperature for about 2 hours. Wash plate (6) with washing solution Wash each well 5-6 times. You can use an automatic plate washer for this washing operation. Add enzyme substrate solution (7) 1¾31 to each well and leave at room temperature for 20 ~ 30 minutes. Stop the enzyme reaction by adding ΙΟΟμΙ of Reaction Stop Solution (8) to each well. Measure the absorbance of each well at 450 nm using a microplate reader. A calibration curve of the OCIF / sOBM complex is prepared from the absorbance at 450 nm of each well to which the OCIF / sOBM complex standard (3) has been added, and the OCIF / sOBM complex concentration of each sample is calculated from the calibration curve.

[Possibility of industrial use]

 According to the present invention, an antibody that binds to the OCIF / sOBM complex and a hybridoma that produces the antibody can be obtained.

 The antibody of the present invention is useful for prevention, treatment or diagnosis of bone metabolism disorders. Further, the present invention provides a method for diagnosing bone metabolism disorder by quantifying the amount of the OCIF / sOBM complex. Further, the present invention provides a kit for diagnosing bone metabolism disorder containing the antibody. Further, the antibody of the present invention can also be used as a research analysis reagent.

[Reference to deposited biological material]

 (1) G. The name and address of the depositary institution that deposited the biological material

 Name: National Institute of Advanced Industrial Science and Technology

 Patent Organism Depositary

 Address: Tsukuba East 1-chome, Ibaraki Prefecture, Japan 1 Chuo Exit No. 6

 November 5, 1997 (Original deposit date)

 February 20, 1998 (Transfer date to deposit based on the Budapest Treaty) Deposit number assigned to deposit by HA institution

 FERM BP-6264

(2) b. Name and address of the depositary institution that deposited the biological material

 Name: National Institute of Advanced Industrial Science and Technology

 Patent Organism Depositary

Address: Tsukuba East 1-chome, Ibaraki Prefecture, Japan 1 Chuo No. 6 Date of deposit with the institution

 April 14, 1999 (date of original deposit)

 February 1, 2002 (Transfer date to the deposit based on the Budapest Treaty) Deposit number assigned to deposit by HA institution

 FERM BP-7872 B. Name and address of the depositary institution that deposited the biological material

 Name: National Institute of Advanced Industrial Science and Technology

 Patent Organism Depositary

 Address: Tsukuba East 1-chome, Ibaraki, Japan 1 Chuo Exit No. 6 Date of deposit with the institution

 February 19, 2001 (Original deposit date)

 March 2002 H (Transfer date to deposit based on the Budapest Treaty) Deposit number assigned to deposit by NOV.

 FERM BP— 7953

Claims

The scope of the claims

1. An antibody that binds to a complex of osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM).

 2. The antibody according to claim 1, which binds to osteoclast formation inhibitory factor (0CIF).

 3. The antibody according to claim 1 or 2, which binds to a soluble 0CIF binding molecule (sOBM).

 4. The antibody according to any one of claims 1 to 3, which is a polyclonal antibody.

 5. The antibody according to any one of claims 1 to 3, which is a monoclonal antibody.

 6. The antibody according to claim 5, which is a humanized antibody.

 7. The antibody according to claim 5, which is produced by hybridoma 01-30 (FERM BP-7787).

 8. The antibody according to claim 5, which is produced by hybridoma H-0BM1 (FERM BP-6264).

 9. The antibody according to claim 5, which is produced by hybridoma # 207 (FERM BP-7953).

 10. Hybrid Dorma 01-30 (FERM BP-7872).

 1 1. Hybrid Dorma H-0BM1 (FERM BP-6264).

 1 2. Hybrid Dorma # 207 (FERM BP-7953).

 13. A cell producing the antibody according to any one of claims 1 to 9 is cultured, and then the culture is cultured for any one of claims 1 to 9. The method for producing an antibody according to any one of claims 1 to 9, wherein the antibody according to any one of claims 1 to 9 is collected.

 1 4. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9.

15. An agent for preventing or treating bone metabolism disorders, comprising the antibody according to any one of claims 1 to 9 as an active ingredient.

1 6. The bone metabolism disorder is one selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone pageet disease and renal osteodystrophy. Item 16. The preventive or therapeutic agent for bone metabolism disorder according to Item 15 above.

1 7. A method for diagnosing a bone metabolism disorder comprising the following steps [1] and [2]:

 [1] Step of measuring the amount of a complex of 0CIF and sOBM contained in a sample of a subject or a healthy subject:

 [2] When the amount of the complex contained in the sample of the subject is larger than the amount of the complex contained in the sample of a healthy subject, the subject has a bone metabolic disorder A step of determining that the subject is affected.

 18. The diagnostic method according to claim 17, wherein at least one antibody according to any one of claims 1 to 9 is used.

 19. The diagnosis according to claim 17 or 18, wherein at least two antibodies according to any one of claims 1 to 9 are used. Method.

 20. Antibodies produced by hybridoma 01-30 (FERM BP-7872) and antibodies produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953) Claims 17 to 17

19. The diagnostic method according to any one of paragraphs 9 to 19.

. 2 1 Use as High Priestess dormer 01 ³ 0 immobilized antibody antibodies produced by (FERM BP-7872), and hybridoma H - 0BM1 (FERM BP - 6264 ) or Hype re dormer # 207 (FERM BP -The diagnostic method according to any one of claims 17 to 20, wherein the antibody produced by -7953) is used as a labeled antibody. ·

22. The diagnostic method according to any one of claims 17 to 21, wherein the sample is plasma or synovial fluid.

 23. The diagnostic method according to any one of claims 17 to 22, wherein the bone metabolism disorder is rheumatoid arthritis.

 24. A diagnostic kit for bone metabolism disorder, comprising at least one antibody according to any one of claims 1 to 9.

25. The diagnostic kit according to claim 24, comprising at least two antibodies according to any one of claims 1 to 9.

2 6. Antibodies produced by hybridoma 01-30 (FERM BP-7872) and antibodies produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-795 3) The diagnostic kit according to claim 24 or claim 25, comprising:

 27. Antibodies produced by Hydpridoma 01-30 (FERM BP-7872) are contained as antibodies for immobilization, and hybridoma H-0BM1 (FERM BP-6264) or Hydpridoma # 207 (FERM BP-7872) 27. The diagnostic kit according to any one of claims 24 to 26, comprising an antibody produced by BP-7953) as a labeling antibody.

28. An osteoclast inhibitor (0CIF) and a soluble 0CIF binding molecule (sOMB), characterized in that at least one antibody according to any one of claims 1 to 9 is used. ) The method for measuring a complex.

 29. The method according to claim 28, wherein at least two antibodies according to any one of claims 1 to 9 are used.

 30. Antibodies produced by hybridoma 01-30 (FERM BP-7872) and antibodies produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FEKM BP-795 3) The measurement method according to claim 28 or 29, wherein the measurement method is used.

 3 1. The antibody produced by hybridoma 01-30 (FERM BP-7872) is used as the immobilized antibody, and hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-78) is used. -7953), wherein the antibody produced by the method according to any one of claims 28 to 30 is used as a labeled antibody.

 32. Measurement of a complex of osteoclast inhibitor (0CIF) and soluble 0CIF binding molecule (sOMB), comprising at least one antibody according to any one of claims 1 to 9 kit.

 33. The measurement kit according to claim 32, comprising at least two antibodies according to any one of claims 1 to 9.

3 4. Antibodies produced by Hydpridoma 01-30 (FERM BP-7787) and antibodies produced by Hybridoma H-0BM1 (FERM BP-6264) or Hybridoma # 207 (FERM BP-7953) The measurement kit according to claim 32 or 33, comprising:

3 5. The antibody produced by Hybridoma 01-30 (FERM BP-7872) is contained as an antibody for immobilization, and hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM The measurement kit according to any one of claims 32 to 34, comprising an antibody produced by BP-7953) as a labeling antibody.