Method for the diagnosis of metabolic bone diseases

 

The invention relates to medicine, in particular for the diagnosis of metabolic bone diseases. The method provides high specificity and accuracy of level measurement a new token, which allows produces more accurate diagnosis of metabolic bone diseases. Conduct the study of the biological fluid, when taken in the biological fluid to determine the concentration of Monomeric and homodimeric types of factor that inhibits osteoclastogenesis (OCIF), using monoclonal antibodies, equally recognize Monomeric and homodimeric types of OCIF having a dissociation constant less than 10-9M antigen or selectively recognize dimeric type OCIF having a dissociation constant less than 210-7M antigen. For the quantitative determination of the concentration of OCIF also stated monoclonal antibody, which is able to equally recognize Monomeric and homodimeric types of OCIF, and a monoclonal antibody capable of selectively recognize dimeric type OCIF, as well as the set containing the above antibodies. 4 C. and 11 C.p. f-crystals, 1 tab., 6 Il.

Technical field the Present invention relates to a method of diagnosis is the finding relates to monoclonal antibodies, used in the diagnosis and diagnostic kits using monoclonal antibodies. The present invention can be used as a method for the diagnosis of metabolic bone diseases, especially osteoporosis and diseases of the joints, or as reagents for the quantitative determination of their use in scientific research.

Prior art Bone metabolism depends on the total activity of osteoblasts, which form bone, and osteoclasts that cause bone resorption. In a healthy adult, held the balance of bone formation and bone resorption and constant bone mass. It is believed that the metabolic bone diseases develop when the loss of this balance. As metabolic bone disease known osteoporosis, hypercalcemia, Paget's disease, renal osteodystrophy, rheumatoid arthritis, osteoarthritis etc., Osteoporosis is a typical example of metabolic bone disease. It is believed that osteoporosis is a disease accompanied by a decrease in bone mass and manifests with symptoms such as bone fractures or bone pain (lumbago and/or back pain), calling the ed after a period of growth, metastases in bone or hyperthyroidism. As a way to diagnose osteoporosis bone mineral mass and/or bone density is determined by using a device for measuring physical bone mass, such as x-ray diffraction method (MD), DPA (dual photon absorptiometry), DEXA (dual energy x-ray absorptiometry), CXD (computed x-ray densitometry) and low-frequency ultrasonic waves. Depending on the technical level the criteria of osteoporosis when using these diagnostic methods are constantly also corrected.

The risk of bone fracture in the future can be forecasted reliably by reducing bone mineral mass and/or bone density. However, the decrease in bone mineral mass and/or bone density is not the only risk factor for fracture, and it is considered that the risk of bone fracture increases under the influence of the phenomena that accompany aging, such as decreased elasticity of the collagen fibers, the qualitative deterioration of bone structure, reduced muscle strength. Currently, with the exception of reduced muscle strength, risk factors cannot be determined non-invasive and non-invasive measurement of medieval Inn/or bone density is the result of loss of balance and bone metabolism and is not the cause of the disease and its diagnostic indicator.

In addition, overlapping these defects bone density measurement for the diagnosis of the disease was tested application level measurement of serum and/or excretion of urine factors regulating bone metabolism (parathyroid hormone (PTH), active form of vitamin D3, calcitonin and so on ), different types of factors released from bone related bone reconstruction (bone Alp, acid phosphatase, pyridinoline, deoxypyridinoline, the peptide of procollagen type 1, osteocalcin and so on). These factors should reflect the state of bone metabolism during measurement and, as expected, are an early indicator of osteoporosis and its degree. However, with regard to these markers of bone metabolism, there are still problems, for example they do not Express the local changes of bone metabolism, they can influence the diet or circadian rhythm so that changes in the level of the factors mentioned above do not necessarily reflect specific changes in bone metabolism. Based on these situations, it is expected to develop highly specific and accurate measurements of the level of the new marker that is involved in bone metabolism, to establish ways p is osteoporosis.

The authors of the present invention have found that in culture medium lung fibroblasts fetal human IMR-90 (ATSS CCL186) was attended by a factor inhibiting osteoclastogenesis (OCTF), and successfully isolated. In addition, the inventors have also successfully carried out the cloning cdnc that encodes this protein, and confirmed the possibility of its use as a means of improving bone metabolism by assessing the pharmacological effect of recombinant OCIF (rOCIF) in vitro and in vivo (WO 96/26217). In addition, the authors of the present invention in various experimental models of metabolic bone diseases confirmed that the introduction of rOCTF significantly improved bone density and bone strength, and that the introduction of a large number rOCIF also significantly increased bone mass and bone volume in healthy animals without any side effects in the study of various organs except bone, gematologicheskih and clinical biochemical parameters and hemolytic cells. Based on the results of in vivo experiments, it was found that OCIF is a cytokine with high tissue specificity, which act only on the bone tissue. In addition, the authors nastoyasheva approximately 120 kDa, and homodimeric type OCIF under the influence of protease turned into a Monomeric form OCIF with a molecular weight of approximately 60 kDa. And as was confirmed by the presence of both types of OCIF in culture medium line of human cells (Tsuda et ai.: Biochem. Biophys. Res. Commun. 234, 137-142) (1997), it is expected that in the biological fluids of mammals, including humans, are present in both type OCIF.

Accordingly, to determine whether OCIF to be a new marker of bone metabolism or not, it is necessary to accurately study the correlation between different types of metabolic bone diseases and the level of each type of OCIF or total concentration of both types of OCIF one of the patients with metabolic bone diseases. Therefore, for the above purposes are required antibody equally recognize both types of OCIF, and an antibody that recognizes only glycosilated. To date still not received any monoclonal antibody against OCIF having such characteristics.

Disclosure of the invention when Considering these situations, the authors present invention thoroughly researched and discovered monoclonal antibodies with very high affinity (dissociation constant was less than 10-9M), equally discriminating and Monomeric, and g is lsua these antibodies, the inventors have created a highly sensitive kit for quantitative enzyme-linked immunosorbent assay (sandwich ELISA). In the measurement of serum concentration of OCIF using sandwich ELISA in adults, young people, older people, patients with osteoporosis, hyperthyroidism and various diseases, including cancer, was found to have a high inverse correlation between the concentration of OCIF in the serum and bone density. In the result of measurement of the concentration of OCIF in the synovial fluid of patients with arthritis, such as rheumatoid arthritis, osteoarthritis, injury, and gout, and so on, it was found that the concentration of OCIF in the synovial fluid of patients with progressive destruction of the joints was significantly low.

It was found that OCIF can be used as a new diagnostic marker of metabolic bone diseases, because the definition of OCIF level in serum and synovial fluid using this sandwich ELISA provides the ability to accurately predict respectively the dynamics of changes in bone density and progression of the destruction of the joints and, thus, predict the decrease of bone mass and destruction is the provision of a method for the diagnosis of metabolic bone diseases in particular osteoporosis and destruction of the joints caused by the rheumatic process, characterized by determining the concentration of the human factor, enzyme inhibition osteoclastogenesis, and used here monoclonal antibodies, and set to measure OCIF antibody.

The present invention relates to a method for the diagnosis of metabolic bone diseases by determining the concentration of a factor that inhibits osteoclastogenesis (OCIF) in the sample of biological fluid.

Diagnostics of the present invention may particularly be used for the diagnosis of osteoporosis and osteoarthritis. As a biological fluid can be used serum or synovial fluid. Diagnosis of osteoporosis may be performed by determining the concentration of OCIF in the serum, and diagnosis of osteoarthritis can be done by determining the concentration of OCIF in synovial fluid. Diagnostics of the present invention may particularly be used to diagnose osteoporosis. As a biological fluid can be used serum or synovial fluid.

In addition, the present invention relates to monoclonal antibodies used dinkova recognize and Monomeric and dimeric types of OCIF, and the antibody selectively recognizes only dimeric type OCIF. In addition, monoclonal antibodies include antibodies with high affinity, which recognizes a different epitope with a dissociation constant less than 210-7M antigen.

In addition, the present invention relates to a kit for determining the content of OCIF, including these monoclonal antibodies. The diagnostic method of the present invention can be carried out using a sampling of biological fluids, such as blood (serum), synovial fluid, of the object of diagnosis and OCIF concentration measurement using the kit to determine the level OCIF using the above monoclonal antibodies.

Monoclonal antibodies can be obtained using the method described below. That is, as an antigen for immunization, which is necessary for obtaining monoclonal antibodies against OCIF, can also be used of human OCIF IMR-90 isolated from culture medium of pulmonary fibroblasts of the human fetus in accordance with the method described in WO 96/26217. Can also be used recombinant human OCIF. Rekombinantnymi and ekspressirovali him in a cage animal, such as cell SNO, cell KSS and Namalwa cell, etc. or insect cell, followed by purification. In accordance with the method Tsuda et al. (Biochem. Biophys. Res. Common. 234, 137-142 (1997)) Monomeric and dimeric types of OCIF can be purified using chromatography with reversed phase. Further, both types of OCIF can be purified using a combination of SP-sepharose, the sulfated tellurophene and column chromatography with resource S instead chromatography with reversed phase. To obtain hybridoma spleen cells obtained from a mammal immunized with an antigen, or lymphocytes immunized in vitro, can merge with the line of myeloma cells. Using as antigens highly purified Monomeric and homodimeric types of OCIF and culture medium above hybridoma may be established cell line using screening hybridoma producing the antibody, equally recognize both types of OCIF or an antibody specific recognizes only homodimeric type OCIF with subsequent cloning of hybridoma. Further, with cultivation installed and sustainable hybridoma can be obtained of the target antibodies.

Although immunization of a mammal to obtain a hybrid of the organization OCIF as antigen may divorce in physiological saline to the appropriate concentration, and its solution may be injected intravenously or intraperitoneally, if necessary together with him, the animal may be full of beta-blockers, in General, 3-4 times at intervals of 1-2 weeks. To facilitate obtaining the target monoclonal antibodies and as much as possible to increase the titer of OCIF in the blood after receiving having a high affinity monoclonal antibodies against OCIF (dissociation constant less than 210-7M) can be immunized 3 times with intervals of 1 week. and then another 4 times with intervals of 1 week. can be used for immunization with the antigen with incomplete adjuvant's adjuvant. 3 days after the last immunization immunized as described above are slaughtered and remove the spleen. As the immunized cells may be used splenocytes. As the myeloma cells can for example be used a cell line derived from mice for hybridization with the immunized cells P3/63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, FO, P363 Ag8, 653 and S194. Hereinafter referred to as a cell line derived from rat, can be used, for example, R-210.

For the production of human antibodies human lymphocytes can immunize acetow person, transformed by Epstein-Barr. The fusion of immunized cells with cell myeloma line can be carried out in accordance with a customary method, for example by the method of Koehler and Milstein et al. (Koehier et al., Nature, 256, 495-497, 1975), but can also be used electric pulse method. Immunized lymphocytes and cell line myeloma can be mixed with normal speed by the number of cells, and for carrying out mergers cells in commonly used culture medium can be added to the polyethylene glycol (not including serum fetal calf FCS), and fused cells (hybridoma) can be selected by culturing in HAT selective medium containing FCS.

Selection of hybridomas producing monoclonal antibody, equally discriminating and Monomeric, and homodimeric types of OCIF, and the antibody selectively recognizes homodimeric type OCIF can be carried out in accordance with the method of detecting antibodies, such as the enzyme linked immunosorbent assay (ELI3A), quantification of plaques, the Ouchterlony method or way agglutination. Methods ELISA using purified Monomeric and homodimeric types of OCIF can very easily and accurately identify the investigational antibody. It was difficult to obtain an antibody with the solid phase ELI3A. That is, when using conventional solid phase, cultural environment hybridoma (50-100 μl) was placed in a 96-cell immune tablets coated with antigen (Nunc) for the initial reaction, and then to carry out the secondary reaction is added to the antibody against mouse IgG, labeled enzyme label such as peroxidase (POD). Then, to complete the enzymatic reaction in each cell in the immune tablet is added a solution of enzyme substrate (50-100 µl), and in each cell is determined by the optical absorption. Cultural environment hybridoma having a high optical density, can be considered not only producing a large number of antibodies with low affinity, but also producing antibody with high affinity, even if the productivity of antibodies is low. It is impossible to determine what happens in reality.

Therefore, in the present invention for detection hybridoma producing antibody with high affinity, was carried out improvement of the conventional solid-phase ELISA, as described below. That is, in each of the 96 cells of the immune tablets antigen coated, add human or bovine serum followed by the addition in each cell Nebolsin 80-90% serum. In such conditions can be excluded hybridoma producing antibodies with low affinity for the antigen, even if antibody is high. Thus, the modified solid-phase ELISA provided an opportunity for selective screening using hybridomas producing antibodies with high affinity to the antigen. Using advanced solid-phase ELISA can be selected hybridoma producing antibodies that are equally recognize and Monomeric, and homodimeric types of OCIF as antigens, and other hybridoma producing antibodies that recognize specific homodimeric type OCIF, and using cloning 3-5 times by the method of limited cultivation can be installed hybridoma producing each antibody. So established hybridoma can subculturalists using commonly used method of cultivation and if you want to persist with freezing. Hybridoma can be cultured using a conventional method, and the antibody can be released from the culture medium. Further, the antibody can be emitted from ascitic fluid obtained from a mammal, which hybridoma implanted intraperitoneally. Antibody in cultural salting out, ion-exchange or gel permeation chromatography, chromatography on affinity to protein a or G.

The resulting antibody is an antibody equally discriminating and Monomeric, and homodimeric types of OCIF, and the antibody selectively recognizes OCIF homodimeric type. Each antibody can be used to measure the amount of OCIF (monomer type OCIF + homodimeric type OCIF) and number homodimeric type OCIF. These antibodies can metalsa radioactive isotope or an enzyme and used in the radioimmunoassay (RIA) or enzyme-linked immunosorbent analysis (ELISA) to determine the amount of OCIF (the number of Monomeric type OCIF + homodimeric type OCIF) or number only homodimeric type OCIF. In particular, the antibody of the present invention, selectively recognize homodimeric type OCIF may clarify that in Monomeric and homodimeric types of OCIF, there are different epitope (epitope), and recognize an epitope present only in homodimers type OCIF, and absent in the monomer type OCIF.

The amount of OCIF and the number homodimeric type OCIF can be determined using antibodies obtained by the present invention and are equally discriminating and monomer, and EDINOGO enzyme using antibodies equally discriminating and Monomeric, and homodimeric types of OCIF, and antibodies that selectively recognize only homodimeric type OCIF respectively with a radioactive isotope or enzyme as secondary antibodies.

Further, if the solid-phase antibody preferably define only the number homodimeric type OCIF, can also be used antibody 0I-26 selectively recognize homodimeric type OCIF as described in example 6 (table. 1), as labeled antibodies - antibodies 0I-19 or 0I-4, equally discriminating and Monomeric, and homodimeric types of OCIF. Using these systems, quantitative analysis, you can determine the amount of OCIF or only the number homodimeric type OCIF in biological fluids such as blood, urine and synovial fluid, etc. or in the environment of cell culture.

The kit of the present invention includes (i) any of the primary antibody and a secondary antibody which is an antibody 0I-19 or antibody 0I-26 and (ii) another antibody which is an antibody 0I-4, and the usual combination of reagents used in conventional sandwich method. That is set for immunological analysis includes (1) a primary antibody immobilized strata and the reagent, stopping the reaction, to determine the enzyme activity in the case of enzyme labeling. As the insoluble carrier can for example be used polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorinated resin, transverse cross-linked dextran, a polysaccharide, a latex, the latex polymer containing magnetic particles coated with metal, and so on, paper, glass, metal, agarose and the combination of the above media. As a form insoluble carrier may be used tray, sphere, fiber, plate, container, cell, tube, porous filter. Further, as the materials labels used to obtain the labeled antibody may preferably be used, enzymes, fluorescent substances, luminescent substances and radioactive substances. As enzymes can be used peroxidase, alkaline phosphatase,-D-galactosidase, glucose oxidase, malatdegidrogenaza, glucose-6-phosphatedehydrogenase, invertase. As the fluorescent substances may be used fluorescein isothiocyanate and phycobiliprotein. As the fluorescent substances can be used isoleucinol, lucigenin. And as radioactive are examples only, and can be used any substance used in the immunological analysis.

When the material of the label is an enzyme, to determine the enzyme activity can be used a substrate and, if necessary, the color developer. If the enzyme is peroxidase as a substrate used2About2and as the color developer can be used ammonium salt of 2,2'-asindi[3-ethylbenzothiazoline sulfonic acid] (ABTS), 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantipyrine, 3,3',5,5'-tetramethylbenzidine, homoseuality acid and tyramine.

And when the enzyme is used alkaline phosphatase, the substrate can be used on-nitrophenylphosphate and 4-methylumbelliferone. When the enzyme is used-D-galactosidase, the substrate may be used fluorescein-di-(-D galactopyranoside), 4-methylumbelliferyl-D-galactopyranoside.

As the solubilizer, disclosed in (3) above kit for immunological analysis, can be used any solubilizer, usually used for immunological analysis, nab is which can be considered as suitable examples. Further, as a means for washing disclosed in (4), can be used by any tool that is usually used for immunological analysis. For example, as an example, physiological saline, phosphate buffer solution, buffer solution of Tris-Hcl and mixed solution. Further, in the above means for washing can be added non-ionic surfactant, such as Triton X-100, Tween 20 or Brij 35, or may be added ionic surfactant such as sodium dodecyl sulphate (SDS) or CHAPS.

A brief description of the drawings Fig. 1 shows a standard curve of ELISA using antibodies 0I-19 and antibodies 0I-4 in example 7.

[The symbols]: homodimeric type OCIF: Monomeric type OCIF In Fig. 2 shows the calibration of the ELISA using antibodies 0I-26 and antibodies 0I-4 in example 7.

[The symbols]: homodimeric type OCIF: Monomeric type OCIF
In Fig.3 shows the concentration of OCIF in the blood in patients with osteoporosis and in healthy individuals in example 8.

In Fig. 4 shows the correlation between the concentration of pyridinoline in the urine and the concentration of OCIF in the blood in example 8.

In Fig. 5 shows the corre the curtain concentration of OCIF in the synovial fluid of patients with artiele in example 9.

[The symbols]
RA - rheumatoid arthritis; OA - osteoarthritis; Tr - trauma; P - gout.

The preferred embodiment of the invention
The present invention will be described in more detail with illustrations of examples. However, this is just examples, and the range of claims of the present invention is not limited to these examples.

Example 1 Purification of Monomeric type OCIF or homodimeric type OCIF as antigen
Producing IF cells SNO described in WO 96/26217, seeded in medium EX-CELL 301 (JRH Bioscience) at a density of 1 cells105cells/ml and cultured at 37oC for 7 days with the use of the vessel for cell culture container holding 2 litres). In the resulting culture medium was added CHAPS (3-[(3-cholamidopropyl)-dimethylammonio-] -1-propanesulfonate, Sigma) to a concentration of 0.1%. After adjusting the pH of the environment; to 6.0 with acetic acid medium is filtered through a filter with pore size of 0.22 μm (MiniDisc, Millir). The culture medium is applied on a column of SP sepharose
for high-performance chromatography (2,610 cm, Pharmacia), equilibrated with 50 mm buffer solution of bis-Tris-HCl containing 0.1% CHAPS. After washing the column with the same buffer and the eluate separated into fractions of 8 ml each. In accordance with the method described in WO 96/26217 determine the OCIF activity in each fraction so that they get a fraction of OCIF. After 10-fold dilution fraction OCIF 50 mm buffer solution of bis-Tris-Hcl with pH 6.0, containing 0.1% CHAPS, it put on sulphatoethylsulphonyl column (2,610 cm, Seikagaku-koqyo), equilibrated with 50 mm buffer solution of bis-Tris-Hcl with a pH of 6.0. The column was washed with 50 mm buffer solution of bis-Tris-Hcl at pH 6.0, containing 0.1% CHAPS, with subsequent manifestation for 100 min with a linear gradient from 0 to 1.5 M NaCI at a flow rate of 4 ml/min, and separated into fractions of 8 ml each. As described above, determine the OCIF activity in each fraction.

A portion of each fraction is subjected to electrophoresis in polyacrylamide gel with SDS in non conditions and collect fractions having the activity of OCIF and a molecular weight of 60 kDa, and a pool of OCIF call 1 faction. In addition, non conditions collected fractions having the activity of OCIF and a molecular weight of 120 kDa, and a pool of OCIF referred to as a 2 faction. After 10-fold dilution, respectively, 1 and fraction 2 fraction of 50 mm buffer solution in Tris-HCl with pH 7.0, containing 0.1% CHAPS, every faction is applied to the column RESOURCE S (0,643 cm, Pharmacia), the leader of sodium phosphate with a pH of 7.0, containing 0,01% Polysorbate, column show for 15 min with a linear gradient from 0 to 0.6 M NaCl at a flow rate of 1 ml/min and separated into fractions of 0.5 ml each. As described above, determine the activity of OCIF in each of the 1 and 2 fractions, collecting fractions having the activity of OCIF, so get Monomeric type of OCIF 1 faction and homodimeric type OCIF of the 2 factions.

Example 2
Immunization of mice and obtaining hybridoma
Monomeric and homodimeric types of OCIF, purified as described in example 1, dissolved in physiological saline to concentrations of 100 µg/ml of each mixture containing the same number of both types of OCIF and prepared as described above, add the same volume of complete adjuvant's adjuvant. The mixed solutions are well emulsifiers and for immunization of mice of Balb/c enter them 3 times with intervals of 1 week. intraperitoneally (200 μl/ mouse). Then in a mixture containing 25 μg/ml each of both types of OCIF, add the same amount of incomplete adjuvant's adjuvant for the preparation of the emulsion, 200 μl of which introduce each of the above mice of Balb/c 4 times with intervals of 1 week.

After 1 week. after the 4th booster immunization the mice Way/with intravenously administered 100 μl smeshannoj the t spleen, isolated spleen cells and produce fusion with cells of the mouse myeloma P363-AG8. 653 (ATSS CRL-1580) in accordance with the usual method (Koehler, G. and Milstein, C., Nature, 256, 495 (1975)). After you merge the cells of the cell suspension for 10 days cultivated in the medium HAT containing gipoksantin, aminopterin and thymidine. After the appearance of hybridoma instead of myeloma cells continue cultivation of cell cultures by replacement of the culture medium on Wednesday HT, excluding from protection HAT aminopterin.

Example 3
Selection and cloning of hybridoma
10 days after the merge cells appears hybridoma. Conduct screening to identify hybridoma producing antibody with high affinity, are equally discriminating and Monomeric, and homodimeric types of OCIF, and hybridoma producing an antibody that selectively recognizes homodimeric type OCIF. That is, Monomeric and homodimeric types of OCIF was dissolved in 0.1 M sodium bicarbonate solution (pH 9,6) to concentrations of 5 μg/ml, and 50 μl of a solution of each antigen is added to each well in 96-cell immune plates (Nunc), and the tablets overnight stored at 4oWith to cover each cell of each antigen. The solution of the antigen in each cell widow cell add 40 μl of fetal bovine serum.

Then in each cell add 10 ál of medium containing hybridoma, and then the tablets for 2 h and incubated in the conditions of 80% of serum concentration at room temperature. After incubation tablets washed S-R, in each of the wells add 50 μl of the solution of antibodies against mouse IgG (KPL), labeled with peroxidase, with a 5000-fold dilution with physiological saline solution containing 25% BlockAce, and tablets incubated at room temperature for 2 hours After washing tablets S-P in each cell for the manifestation of color add 50 ál of a solution of enzyme substrate (TMB, ScyTek). Then the enzymatic reaction turn by adding 50 µl of reagent to stop the reaction (ScyTek). Conduct screening for hybridomas producing antibodies, using the definition of the optical absorption of each cell at 450 nm using the apparatus for a microplate reader (immunoreader NJ2000, Nihon-intermed). Conduct screening, identifying, respectively, a hybrid having a high optical absorption, producing the antibody, equally discriminating and Monomeric and dimeric type OCIF, and hybrid having a high optical absorption of producing an antibody that selectively recognizes only the Yu re-cloning 3-5 times each hybridoma using the method of limiting dilution. Among the hybridomas producing antibodies, choose hybridoma with high productivity of the studied antibodies.

So, get hybridoma 0I-19 and 0I-4, which respectively produce antibodies 0I-19 and 0I-4, equally discriminating and Monomeric, and homodimeric types of OCIF. In addition, get hybridoma 0I-26, producing the antibody 0I-26 selectively recognize homodimeric type OCIF. These hybridoma were deposited in National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology, and 0I-4, 0I-19 and 0I-26 have been assigned Deposit numbers respectively FERM BP-6419, FERM BP-6420 and FERM BP-6421.

Example 4
Production and purification of monoclonal antibodies
Cultivate accordingly hybridoma producing with high affinity antibody, equally discriminating and Monomeric, and homodimeric types of OCIF, and hybridoma producing the antibody selectively recognizes homodimeric type OCIF obtained in example 3, and 1106cells hybridoma 1 mouse injected intraperitoneally to mice of Balb/c, which was introduced Pristina (Aldrich). After 2 weeks. after the introduction collect accumulated ascitic fluid containing monoclonal antibody of the present invention. The purified antibody obtained by x is oksanalove antibodies
The dissociation constant of the monoclonal antibody is determined in accordance with the method Betrand Friguet (Journal of Immunolgical Methods, 77, 305-319, 1986). That is, the purified antibody obtained in example 4, diluted in 0.2 M Tris - HCl with pH 7.4 (first buffer), containing 40% BlockAce (Snow Brand Milk Products) and 0.1% Polysorbate 20, to a concentration of 5 ng/ml To the above solution add the same amount of 6.25 ng/ml - 10 μg/ml of purified Monomeric type recombinant OCIF or homodimeric type recombinant OCIF obtained in example 1 and diluted in the first buffer, and the mixture is kept for 15 h at 4oWith so OCIF is contacted with a monoclonal antibody. After 15 h determine the dissociation constant of the monoclonal antibody against Monomeric type OCIF or homodimeric type OCIF by measuring the amount of antibodies that are not associated with OCIF, using solid-phase ELISA, which immobilized Monomeric type rOCIF or homodimeric type rOCIF (10 μg/ml, 100 µl/1 cell).

Example 6
Quantitative determination of the class and subclass of monoclonal antibodies
Quantitative determination of the class and subclass of the monoclonal antibodies of the present invention is carried out with the use of the kit for the quantitative determination of the class and subclass ykazannoj in the instructions to the set. The results obtained in examples 5 and 6, shown in the table. 1.

Based on these data, it was found that antibodies 0I-4 and 0I-19 antibodies are equally discriminating and Monomeric, and homodimeric types of OCIF and antibody 0I-26, as detected is an antibody that selectively recognizes only homodimeric type OCIF. Further, all antibodies belong to IgGl and, as found, are antibodies with extremely high affinity with a dissociation constant less than 210-7M for monomer and homodimer types of OCIF.

Example 7
The definition of OCIF by ELISA
Sandwich ELISA create using 3 types of antibodies, obtained as described above, i.e. antibodies 0I-4, 0I-26 and 0I-19, as the immobilized antibody and labeled antibody. Tagging of antibodies is carried out with a set of peroxidase, activated maleimido (Pias). Antibody 0I-19 as the primary antibody in the case of ELISA, which determines the number and Monomeric, and homodimeric types of OCIF or antibody 0I-26 as the primary antibody in the case of ELISA, selectively determining the number homodimeric type OCIF, dissolved in 0.1 M sodium bicarbonate solution (pH 9,6) to concentrations of 10 ág/ml, 1 is the first cell of the tablets stored over night at 4oC. Solution of each cell and remove to lock in each cell add 300 ál of 50% BlockAce (Snow Brand Milk Products) at room temperature for 2 hours After blocking tablets are washed with saline phosphate buffer (PBS-P) containing 0.1% Polysorbate 20. Monomeric and homodimeric types of OCIF dissolved in a 0.2 M solution of Tris-Hcl (pH 7.4) containing 40% BiockAce (Snow Brand Milk Produces) and 0.1% Polysorbate 20 (primary buffer), and then diluted to obtain solutions of each type OCIF with different concentrations.

In each of the wells add 100 ál solutions of Monomeric type or homodimeric type OCIF with different concentrations for the reaction at room temperature for 2 hours Over 2 hours tablets washed with PBS-P and in each cell in tablets add labeled POD antibody 0I-4, diluted in 0.1 M solution of Tris-Hcl (pH 7.4) containing 25% BiockAce and 0.1% Polysorbate, as antibodies, are equally discriminating and Monomeric, and homodimeric types of OCIF, and tablets left for 2 h at room temperature. Tablets washed with PBS-P and in each of the wells add 100 ál of a solution of enzyme substrate (TSR, ScyTek). After developing, to stop the enzymatic reaction in each well add 100 ál, ostanovlyu the each cell at 450 nm. The results in the case of antibodies 0I-19, the same recognition and Monomeric, and homodimeric types of OCIF as the primary antibody, shown in Fig.1, and the results in the case of antibodies 0I-26 selectively recognize homodimeric type OCIF as the primary antibody, shown in Fig.2.

As a result, as shown in Fig.1, it was found that the sensitivity of the sandwich ELISA is approximately 25 PCG/ml and it is able to detect extremely small amount of OCIF as immobilized antibodies used antibody 0I-19, the same recognition and Monomeric, and homodimeric types of OCIF, and as antibodies, labeled POD antibody was used 0I-4, equally recognize both types of OCIF. Next, as shown in Fig.2, found that the sensitivity of detection sandwich ELISA is 50 PCG/ml and with high sensitivity capable of selectively identify homodimeric type OCIF when used as immobilized antibodies antibody 0I-26 selectively recognize homodimeric type OCIF, and as antibodies, labeled POD antibody 0I-4, equally discriminating and Monomeric, and homodimeric types of OCIF.

Example 8
The concentration of OCIF in the serum of krenov with osteoporosis (based on criteria established Japanese Society for Bone and Mineral Research) determined with the use of partially improved system ELISA OCIF (monomer type OCIF + homodimeric type OCIF), described in example 7. That is, for determining the concentration of OCIF (monomer type OCIF + homodimeric type OCIF) antibody 0I-19, equally recognize both types of OCIF, Immobiliser 96-cell tablet as described in example 7, and in each cell add 50 ál of the first buffer (0.2 M solution of Tris-HCl, pH 7.4, containing 40% BlockAce and 0.1% Polysorbate 20) containing 20 μg/ml purified mouse IgG. Then in each cell add 50 ál of human serum diluted 4 times the first buffer, and tablets left for 2 h at room temperature. After 6 times washing tablets PBS-P in each plate add 100 ál of the solution labeled POD antibody 0I-4, equally recognize both types of OCIF, diluted 3000 times the secondary buffer (0.1 M solution of Tris-HCl, pH 7.4, containing 25% BlockAce and 0.1% Polysorbate 20) containing 10 μg/ml purified mouse IgG, and tablets left for 2 h at room temperature. Tablets 6 times washed with PBS-P and in each of the wells add 100 ál of a solution of enzyme substrate (TMB, ScyTek). To continue the enzymatic reaction tablets leave at room I reaction (ScyTek). Using the apparatus for reading microplates determine the optical density of each cell at 450 nm. As for canids buffer containing a known amount of OCIF, perform the same procedure as described above, and construct a standard curve OCIF, as shown in Fig.1. Serum concentration of OCIF is determined on the basis of the optical density of the serum sample.

The results of determination of serum concentration of OCIF (monomer type OCIF + homodimeric type OCIF) in healthy people and in patients with osteoporosis are shown in Fig.3. Statistical analysis of significant differences of the results in Fig. 3-5 carried out using an unpaired t student test. The results revealed no significant difference in serum concentration of OCIF (monomer type OCIF + homodimeric type OCIF) in healthy people and in patients with osteoporosis. Serum concentration of OCIF in patients with osteoporosis is higher than in healthy people. Accordingly, it was found that OCIF is a new marker for the diagnosis of osteoporosis, because by defining serum concentration of OCIF can be traced to the pathogenesis of osteoporosis.

In addition, in Fig.4 and 5 show the relationship respectively between serum concentration of OCIF (m is favorote and concentration deoxypyridinoline in urine from healthy volunteers and in patients with osteoporosis. The concentration of pyridinoline 42 pcmall/mmol CR (number of pyridinoline in pcmesh 1 mol creatinine) and the concentration of deoxypyridinoline 6.2 pcmall/μmol CR represent the upper limit of the normal level of the inhabitants of Japan.

Data obtained in patients with a higher concentration of pyridinoline and/or deoxypyridinoline serum concentration of OCIF was significantly higher. Pyridinoline and deoxypyridinoline represent transverse cross-linked collagen molecule is produced in the bone substrate after activation of collagen in bone substrate and released by the destruction of bone due to bone resorptive. It is believed that these molecules are highly specific marker of bone resorption and is widely used for the evaluation of the clinical facility. As was recognized correlation between the concentration of OCIF and these two markers, it was found that the concentration of OCIF in the serum can be used as a marker of bone metabolism.

Example 9
The concentration of OCIF in the synovial fluid of patients with articular hernia
Samples of synovial fluid taken from patients with rheumatoid arthritis (RA 43 cases), osteoarthritis (the which in the differential diagnosis was diagnosed with synovial hernia and who agreed to participate in the study on the basis of informed consent. The OCIF concentration in synovial fluid is determined with the use of partially improved system ELISA OCIF (monomer type OCIF + homodimeric type OCIF), described in example 8, that is, after a 16-fold dilution synovial fluid in the first buffer, 50 μl of which is added to each well of 96-cell immunoblasts with immobilized antibody 0I-19. Other than described above, were the same as in example 8.

The results of the study the concentration of OCIF (monomer type OCIF + homodimeric type OCIF) in synovial fluid of patients with articular disc shown in Fig. 6. For statistical analysis of the data shown in Fig.6, studies have been conducted Well-Wallis and Mann-Whitney. Based on these results, the concentration of OCIF in the synovial fluid of patients with rheumatoid arthritis was statistically significantly lower than in patients with gout (p= 0,0023). Further, although the lowest concentration of OCIF in the synovial fluid of patients with osteoarthritis (6 cases) was $ 4.79 ng/ml, the number of patients with rheumatoid arthritis, in which the concentration of OCIF in synovial fluid was below 4.0 ng/ml, was 15 of 43 patients.

Based on these results, it was assumed that not the m showing a lower concentration of OCIF in synovial fluid. It was found that OCIF-ELISA can be used for the diagnosis and study of the pathogenesis of rheumatoid arthritis (RA), osteoarthritis (OA), injuries (Tr) and gout (N).

Example 10
The correlation between the concentration of OCIF in the fluid of the knee joint of patients with rheumatoid arthritis (RA) pathogenesis
To study the correlation between the concentration of OCIF in synovial fluid and progression of destructive processes in the joint was conducted a retrospective cohort study in 2 patients with rheumatoid arthritis.

Case 1, male 66 years
At the age of 50 years in 1982, he had subjective pain in many joints. In 1983 he visited the hospital, where on the basis of the criterion of classification of the American College of Rheumatology was diagnosed with RA. Then, since the application antirheumatoid funds (mercaptan) he caused a remission of the disease, the use of antirheumatoid funds was discontinued. In 1990, due to relapse RA application antirheumatoid funds was resumed. March 18, 1992, was observed swelling of his right knee, and with the help of puncture was taken synovial fluid. Concentration of 43 patients with RA was 6 ng/ml). The level of C-reactive protein in plasma at this time amounted to 5.4 mg%, which clearly testified to the existence of inflammation. With-reactivity protein did not become negative until 1997 remained at the level of about 3 mg%. From 1983 to 1997, he held x-ray of the knee joint, and the main symptom was osteoarthritis. In the joints of the fingers and wrist bone erosion was not observed.

Case 2 a woman 60 years
At age 49 in 1987 she had a subjective pain in many joints. August 17, 1993, she came to the hospital, where on the basis of the criterion of classification of the American College of Rheumatology was diagnosed with PA. At this time, she had been a swelling of the right knee joint, and with the help of puncture was taken synovial fluid. The OCIF concentration in synovial fluid was 3.0 ng/ml, which was low, and the level of C-reactive protein in plasma was 13.7/mg%, which indicated the presence of inflammation. Under the influence of destination antirheumatoid funds (methotrexate) content of C-reactive protein gradually decreased to 4.1 mg% on 18 March 1994 and to the level of 1.1 mg% on 27 June of the same year. However, in accordance with the classification Laresen (Laresen et al., Acta Radiol. Diag. 18, 481-491, 1977) REntgen and 27 June of the same year V the degree that clearly testified to the progression of joint destruction. And October 4, 1994, was conducted replacement surgery artificial joint prosthesis.

Based on the results of determining the concentration of OCIF in the synovial fluid in the above 2 cases, i.e. in the case of a minor progression of joint destruction and in another case with a high degradation of the joint, it was found that the concentration of OCIF in synovial fluid can be used to estimate the frequency of the risk of joint destruction rheumatic process and to assess the impact of treatment on the degradation of the joint.

Example 11
Set quantitative analysis for determination of Monomeric and homodimeric types of OCIF (80 samples)
1) 96-cell tablet with immobilized antibody 0I-19 with subsequent lock BlockAce in accordance with the method described in example 7: one tablet;
2) antibody 0I-4, labeled POD in accordance with the method of example 7: 10 ál (1000-fold dilution);
3) standard recombinant OCIF (monomer type): 0.5 ng/ml 400 ál;
4) the solution for dilution of the sample (0.01% tween-20 and 0.2 M buffer solution of Tris-HCl containing 40% BlockAce, pH 7,4): 10 ml;
5) reconstitution solution labeled antibody is the first tablet (S(-), containing 0.1% tween-20): 1l;
7) substrate solution for measuring the activity of enzymatic labels (TMB solution) and the reagent to stop the reaction (reagent to stop the reaction TMB): 10 ml each.

Set quantitative analysis to determine the dimeric and homodimeric types IF (80 samples)
1) 96-cell tablet with immobilized antibody 0I-26 with subsequent lock BlockAce in accordance with the method described in example 7: one tablet;
2) antibody 0I-4, labeled POD in accordance with the method of example 7: 10 ál (1000-fold dilution);
3) standard recombinant OCIF (dimeric type): 0.5 ng/ml 400 ál;
4) the solution for dilution of the sample (0.01% tween-20 and 0.2 M buffer solution of Tris-HCl containing 40% BlockAce, pH 7,4): 10 ml;
5) diluted solution of labeled antibody (0.1 M buffer solution of Tris-HCl containing 0.01% tween-20 and 25% BlockAce, pH 7.4): 10 ml;
6) the solution for washing 96-cell tablet (PBS ( -) containing 0.1% tween-20): 1 liter;
7) substrate solution for measuring the activity of enzymatic labels (TMB solution) and the reagent to stop the reaction (reagent to stop the reaction TMB): 10 ml each.

Method of quantitative analysis (set 1 and set 2)
In each cell in the tablet 1 respectively add 100 ál of the way the Le leaving for 2 h at room temperature, each well washed 5-6 times with 300 ál of solution 6. For that the washing procedure you can use automatic washing machine tablets. After washing in each of the wells add 100 ál of antibody solution 0I-4, labeled POD 2 and diluted 1000 times with a solution of 5, and the plate is left for 2 h at room temperature. Each cell in the tablet 5-6 times wash solution 6. For that the washing procedure you can use automatic washing machine tablets. In each of the wells add 100 ál of enzyme solution substrate 7 and the plate is left on for 20-30 min at room temperature. Then the enzymatic reaction is stopped by adding 100 μl of the reagent to stop the reaction 7.

Determine the optical density of each cell at 450 nm using an apparatus for reading a microplate. Using optical density at 450 nm in the case of addition in each cell serially diluted standard recombinant human OCIF 3 build a standard curve of recombinant human OCIF. Using this standard curve, you can determine the concentration of OCIF each sample.

Industrial applicability
By determining the concentrations of human factor, enzyme inhibition osteoclastogenesis, biologist is easily and accurately diagnose metabolic bone diseases especially osteoporosis and osteoarthritis. As for the diagnostics of the present invention uses the above monoclonal antibodies and kits for the quantitative analysis of OCIF with the use of monoclonal antibodies, as described above, it is possible to easily and accurately make the diagnosis of metabolic bone diseases, especially osteoporosis and osteoarthritis. The present invention can be used for the method for diagnosis of metabolic bone diseases, especially osteoporosis and osteoarthritis, or for reagent quantitative analysis for his research.

Reference microorganism
Name and address of the official Depository, which were deposited with the specified organisms present invention
Name: National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology Ministry of International Trade and Industry.

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan.

Date of transfer is deposited in an official Depository: October 16, 1997

Deposit number assigned official Depository - FERM BP-6419.

Name and address of the official Depository, in which these organisms present invention were deposited with the
Name: National Institute of Bioscience and Human-Technoloqy Agency of Industrial Science and Technology Ministry of International Trade and Indus16 October 1997

Deposit number assigned official Depository - FERM BP-6420.

Name and address of the official Depository, in which these organisms present invention were deposited with the
Name: National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology Ministry of International Trade and Industry.

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan.

Date of transfer is deposited in an official Depository: October 16, 1997

Deposit number assigned official Depository - FERM BP-6421.


Claims

1. Method for the diagnosis of metabolic bone diseases, including the study of biological fluid, characterized in that the individual biological fluid to determine the concentration of Monomeric and homodimeric types of factor that inhibits osteoclastogenesis (OCIF), using monoclonal antibodies, equally recognize Monomeric and homodimeric types of OCIF having a dissociation constant less than 10-9M antigen or selectively recognize dimeric type OCIF having a dissociation constant less than 210-7M antigen.

2. The method according to p. 1, characterized in that the biological fluid is blood disease is a osteoporosis.

4. The method according to p. 1 or 2, characterized in that the metabolic bone disease is a arthrosis.

5. The method according to p. 1 or 2, characterized in that the metabolic bone disease is a rheumatoid arthritis.

6. The method according to p. 3, characterized in that it includes a step for identifying a subject who suffers from osteoporosis or has the risk of its development if the specified entity in biological fluids have more OCIF than in healthy control subject.

7. The method according to p. 4 or 5, characterized in that it includes a step for identifying a subject who suffers from osteoarthritis or rheumatoid arthritis or has the risk of their development, if the specified entity in a biological fluid is present fewer OCIF than in healthy control subject.

8. Monoclonal antibody to quantify the concentration of OCIF, which is able to equally recognize Monomeric and homodimeric types of OCIF.

9. Monoclonal antibody under item 8, designated as 01-19 derived from hybridoma with the Deposit number FERM BP 6419, which is able to equally recognize Monomeric and homodimeric types of OCIF.

10. Monoclonal antibo to raspoznavat Monomeric and homodimeric types of OCIF.

11. Monoclonal antibody under item 8, characterized in that it is capable of recognizing the epitope present in Monomeric and homodimeric types of OCIF, and has a high affinity and exhibits a dissociation constant less than 10-9M antigen or capable of recognizing the epitope present only in the dimeric type OCIF and absent in the monomer type OCIF, and has a high affinity and exhibits a dissociation constant less than 210-7M antigen.

12. Monoclonal antibody to quantify the concentration of OCIF, which is able to selectively recognize dimeric type OCIF.

13. Monoclonal antibody according to p. 12, designated as 01-26 derived from hybridoma with the Deposit number FERM BP 6421, which is able to selectively recognize homodimeric type OCIF.

14. Monoclonal antibody under item 12, characterized in that it is capable of recognizing the epitope present in Monomeric and homodimeric types of OCIF, and has a high affinity and exhibits a dissociation constant less than 10-9M antigen, or capable of recognizing the epitope present only in the dimeric type OCIF and absent in the monomer type OCIF, and has a high affinity and exhibits design is to determine the concentration of OCIF, containing at least one monoclonal antibody that will reposne epitope present in Monomeric and homodimeric types of OCIF, has a high affinity and exhibits a dissociation constant of 10-9M with the antigen or at least one monoclonal antibody that recognizes an epitope present only in the dimeric type OCIF and absent in the monomer type OCIF, has a high affinity and exhibits a dissociation constant less than 210-7M antigen.

 

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