Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-05-24
2004-02-17
Smith, Lynette R. F. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S007100, C435S007200, C435S007710, C435S007230, C435S007920, C435S007940, C435S069700, C435S069100, C435S181000, C530S350000, C530S388240, C530S388260, C530S388150, C530S300000, C530S387300, C530S388200, C530S388500, C530S389100, C530S387100, C530S809000, C514S012200, C514S024000, C424S133100, C424S141100, C424S172100, C424S184100, C800S014000
Reexamination Certificate
active
06693175
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of diagnosing metabolic bone diseases, especially osteoporosis and arthral diseases. In addition, the present invention relates to monoclonal antibodies used in the diagnosis and kits for diagnosis using the monoclonal antibodies. The present invention is useful as a method of diagnosing metabolic bone diseases, especially osteoporosis and arthral diseases, or as assay reagents for research use thereof.
BACKGROUND ART
Bone metabolism depends on integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. In a healthy adult, the balance of bone formation and bone resorption is kept and the bone mass is maintained constant. Metabolic bone diseases are thought to develop by losing this balance. As metabolic bone diseases, osteoporosis, hypercalcemia, Pajet's disease, renal osteodystrophy, rheumatoid arthritis and osteoarthritis etc. are known. Osteoporosis is exemplified as a typical metabolic bone disease. Osteoporosis is thought to be a disease accompanied with decrease in bone mass and shows clinical symptoms, such as bone fracture or bone pain (lumbago and/or dorsalgia) caused by decrease in bone mass. Decrease in bone mass is induced by various causes such as aging after growing period, bone metastasis, or hyperthyroidism. As a method of diagnosing osteoporosis, bone mineral mass and/or bone density are determined by an apparatus to measure physical bone mass, such as X-ray diffraction (MD method), DPA (Dual photon absorptiometry), DEXA (Dual energy X-ray absorptiometry), CXD (Computed X-ray Densitometry) and low-frequency supersonic waves. The criterion of osteoporosis using these diagnostic methods is always rectified depending on technical revolution.
The risk of bone fracture in future might be surely predicted by decrease in bone mineral mass and/or bone density. However, decrease in bone mineral mass and/or bone density is not a sole risk factor of bone fracture and risk of bone fracture is thought to increase by phenomena accompanying with aging such as decrease in elasticity of collagen fiber, qualitative deterioration of bone structure, lowered muscular strength. At present risk factor except lowered muscular strength can not be measured non-invasively and non-invasive measurement is an important object to be solved in future. Further, decrease in bone mineral mass and/or bone density is just a result of losing the balance of bone metabolism and neither a cause of the disease or a diagnostic parameter thereof.
As supplement covering these defects of measurement of bone density, measurement of serum level and/or urinary excretion of factors regulating bone metabolism (parathyroid hormone (PTH), active form of vitamin D
3
and calcitonin etc.), various kinds of factor released from bone tissue accompanying with bone remodeling (bone alkaline phosphates, acid phosphatase, pyridinoline, deoxypyridinoline, type-I procollagen peptide, osteocalcin etc.) are tried to use to diagnose the disease. These factors would reflect of bone metabolic state at the time of measurement and are expected as an early parameter of bone loss and the extent thereof. However, as for these markers of bone metabolism, there are still problems, for example, they do not express local bone metabolic change, they can be affected by diet or circadian rhythm, so that changes in the level of these above factors do not necessarily reflect specific changes in bone metabolism. From these situations, development of highly specific and precise measurement of a novel marker involved in bone metabolism is expected for establishment of methods of suitable diagnosis, prevention and treatment of various kinds of metabolic bone diseases such as osteoporosis.
The present inventors found that osteoclastgenesis inhibitory factor (OCIF) was present in a culture medium of human fetal lung fibroblasts, IMR-90 (ATCC CCL186) and succeeded in isolation thereof. In addition, the inventors also succeeded in cloning of cDNA encoding this protein and confirmed the usefulness thereof as an agent for improving bone metabolism by evaluating pharmacological effect of recombinant OCIF (rOCIF) in vitro and in vivo (WO 96/26217). Further, the present inventors confirmed that administration of rOCIF significantly improved bone density and bone strength in various kinds of animal model of metabolic bone disease and that administration of a large amount of rOCIF also significantly increased bone mass and bone volume in a normal animal without was not any side-effect in the examination of various organs other than osseous tissue, hematology and clinical biochemistry and hemolytic cell. From the results of in vivo experiment, it was found that OCIF is a highly tissue-specific cytokine having an action only on osseous tissue. In addition, the present inventors confirmed that, in an animal cell, OCIF was secreted as homodimer form of OCIF with a molecular weight of about 120 kDa and the homodimer type of OCIF was converted into monomer form of OCIF with a molecular weight of about 60 kDa by protease processing. And as it was confirmed that both types of OCIF were present in a culture medium of human cell line (Tsuda et al. :Biochem. Biophys. Res. Commun. 234, 137-142)(1997)), it is expected that both types of OCIF are present in humor of mammal including human being.
Accordingly, for elucidating whether or not OCIF can be a novel marker of bone metabolism, it is necessary to study precisely the correlation between various kinds of metabolic bone diseases and the level of each type of OCIF or total concentration of both types of OCIF in one of patients with metabolic bone diseases. Therefore, an antibody recognizing equally both types of OCIF and an antibody recognizing only homodimer are required for the above purpose. Any anti-OCIF monoclonal antibody having such features has not been obtained yet.
DISCLOSURE OF THE INVENTION
Considering these situations, the present inventors eagerly studied and found monoclonal antibodies with markedly high affinity (dissociation constant thereof was less than 10
−9
M) recognizing equally both of monomer- and homodimer-types of OCIF and monoclonal antibodies recognizing specifically homodimer type of OCIF. Further, the inventors constructed a highly sensitive enzyme immunoassay kit (sandwich ELISA) using these antibodies. As the results of measurement of serum concentration of OCIF in young adults, the aged, patients with osteoporosis, hyperthyroidism and various kinds of disease including cancer using the sandwich ELISA, a high inverse-correlation was found between serum concentration of OCIF and bone density. As the results of measurement of the concentration of OCIF in synovial fluid of patients with arthrosis such as rheumatoid arthritis, osteoarthritis, trauma and gouty seizure etc., OCIF concentration in synovial fluid of a patient with progressed joint destruction was found to be significantly low.
OCIF was found to be useful as a novel diagnostic marker of metabolic bone disease, because the determination of OCIF in serum and synovial fluid by the present sandwich ELISA makes it to precisely predict the dynamics of bone density and the progress of joint disruption, respectively, and thereby predict the decrease in bone mass and joint disruption at early stage of these bone decreases. Accordingly, an object of the present invention is to provide a method of diagnosing metabolic bone disease, especially osteoporosis, and joint destruction caused by rheumatism, characterized by determination of the concentration of human osteoclastgenesis inhibitory factor and monoclonal antibodies used therein and a kit for measurement of OCIF using the antibodies.
The present invention relates to a method of diagnosing metabolic bone disease by determination of the concentration of osteoclastgenesis inhibitory factor (OCIF) in sample humor.
The diagnosis of the present invention is especially useful for diagnosis of osteoporosis and arthrosis. As humor, serum or synovial fluid can be used. Diagnosis of osteoporosis can b
Goto Masaaki
Higashio Kanji
Kobayashi Fumie
Tsuda Eisuke
Washida Naohiro
Arnold & Porter
Portner Ginny Allen
Sankyo Co. Ltd.
Smith Lynette R. F.
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