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WO1998029560A1 - Anticorps monoclonal contre la collagenase 3 et procede de dosage immunologique utilisant cet anticorps - Google Patents

Anticorps monoclonal contre la collagenase 3 et procede de dosage immunologique utilisant cet anticorps Download PDF

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Publication number
WO1998029560A1
WO1998029560A1 PCT/JP1997/004884 JP9704884W WO9829560A1 WO 1998029560 A1 WO1998029560 A1 WO 1998029560A1 JP 9704884 W JP9704884 W JP 9704884W WO 9829560 A1 WO9829560 A1 WO 9829560A1
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Prior art keywords
monoclonal antibody
antibody
latent
mmp
specifically
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PCT/JP1997/004884
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English (en)
Japanese (ja)
Inventor
Hironori Tamei
Isao Azumano
Shin-Ichi Yoshida
Carlos Lopez-Otin
Kazushi Iwata
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Fuji Yakuhin Kogyo KK
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Fuji Yakuhin Kogyo KK
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present invention relates to a monoclonal antibody against collagenase 3 (meta-mouth proteinase 13, MP-13) and an immunological quantification method using the monoclonal antibody. More specifically, the present invention relates to a method for immunologically quantifying latent MMP-13 and active class P-13 using a monoclonal antibody that specifically reacts with MMP13.
  • the present invention relates to an immunoassay for collagenase 3 (latent and active forms P-13) used in the medical and physiological fields, and a reagent used therefor. More specifically, the present invention relates to a method for immunologically quantifying latent and active MMP-13 using an anti-MMP-13 monoclonal antibody produced using purified ⁇ P-13, and a method therefor.
  • the extracellular matrix is composed of complex components such as collagen such as type IV collagen, and adhesive glycoproteins such as proteoglycan, elastin, fibronectin, laminin, and heparan sulfate (Marti) nez-Hernandez et a
  • MMP matrix meta-oral protease with different substrate specific 3 ⁇ 4fe
  • Type collagenase or gelatinase A country P2
  • stomach muracinin-1 , ⁇ -3
  • matrilysin ⁇ 7
  • neutrophil collagenase ⁇ 8
  • 92 kDa gelatinase IV Type collagenase or gelatinase B: Fig. P-9
  • Stromlysin-1 Fig. P-10
  • Stromlysin-1 3 MMP-11
  • Macrophage meta-oral elastase MMP-12
  • Collagenase 3 National P-13
  • Membrane type P MT Tsuyoshi P
  • Oral Bio 1. ed., 4, 197 250, 1993; SD Shapiro et al., J. Biol.
  • MPs are synthesized and produced inside cells and released as precursors (pro-forms or latent forms) outside the cells as needed.
  • Metastasis of cancer cells is established through the subsequent steps of destruction of the basement membrane, invasion of blood vessels, invasion, engraftment of secondary organs, and proliferation. It is a barrier to cancer cell metastasis.
  • type IV collagenase MMP2 and MP-9
  • MMP2 and MP-9 type IV collagenase
  • whose main substrate is type IV collagen, which is the main structure of the basement membrane is highly expressed in highly metastatic cancer cells.
  • Fig. P The activity expression regulation in Fig. P is at least at the transcription level, the stage of activation from the latent enzyme that does not show enzymatic activity to the active enzyme, and tissue-inhibit metabolic protease, a specific inhibitor of P. (TIMP), it is considered that the activity is regulated (M. Matrisian et al., Trends Genet., 6: 121-125, 1990). Although all ⁇ ⁇ ⁇ is secreted as an inactive latent form, in vitro experiments indicate that activation of, for example, goat P-1 and country P9 can be caused by serine proteases such as plasmin, trypsin, and forceepsin G.
  • serine proteases such as plasmin, trypsin, and forceepsin G.
  • TlMPs three types have been reported, and they are called 'III-1, T1MP-2 and T1MP-3, respectively. These TlMPs usually bind to active ViMPs and are thought to have physiological effects such as repairing tissues, preventing tissue destruction, suppressing cancer metastasis or promoting cell growth.
  • T1MP-1 binds to latent MMP-9
  • T1MP-2 binds to latent MMP-2
  • the activity of each MMPs and self-division It is thought to control sex.
  • these rigid Ps are not necessarily produced only from cancer cells, but also produce different MMPs from surrounding fibroblasts and inflammatory cells (K Tryggvason et al.
  • ⁇ P2 is particularly expressed in fibroblasts at various sites with alterations in tissue architecture.
  • MMP-9 the frequency of detecting active tt is low.
  • activated syrup 2 is localized at the tip of cancer cell infiltration (11 ⁇ (100 ( ⁇ 1)), suggesting the importance of cancer cell infiltration.
  • the measurement of free active rigid Ps can be a means of knowing the amount of active Ps directly involved in the decomposition of matrix components of MMPs present in tissues and body fluids. By quantifying the type, it will be possible to diagnose or monitor diseases in which MPs activity is enhanced, such as arthropathy, cancer invasion, metastasis, periodontal disease and pulmonary fibrosis. L'll be expected. Therefore, the measurement of the free active form ⁇ - ⁇ 3 in particular means that the amount of the active form P-13 directly involved in the decomposition of the matrix component of rigid P-13 present in tissues and body fluids can be determined. It can be a means to know and free activation type ⁇ -13 The quantification is expected to enable diagnosis or monitoring of diseases and conditions in which MMP13 activity is promoted.
  • An object of the present invention is to provide a method capable of separating and quantifying the amount of P-13 in each country quickly and with high sensitivity and accuracy using simple operations and reagents. It is also an object of the present invention to provide a reagent kit used in such a method.
  • an object of the present invention is to provide latent type P13, active type P-13, or both latent type and active type P-13 in a test sample using a monoclonal antibody that specifically reacts with P13.
  • An object of the present invention is to provide a method for measuring the complex of the active form P-13 and TlMPs with high sensitivity, high accuracy, and rapid quantification of P-13, respectively.
  • the present invention uses at least two types of monoclonal antibodies that specifically react with MMP 13 as a measurement reagent to immunologically measure latent P-13 or active P-13.
  • the present invention provides a method for quantifying P13, characterized in that it performs the measurement of both latent and active forms of ⁇ P-13.
  • the two types of monoclonal antibodies are monoclonal antibodies that specifically react with substantially different regions of VMP-13, respectively.
  • the monoclonal antibodies in the sandwich method are preferred.
  • Monoclonal antibodies that specifically react with one of the fractions P13 are used as the antibodies to be bound to the phase carrier, and monoclonal antibodies that specifically react with the other P13 are used as the antibodies to be labeled.
  • the immunoassay a complex using a monoclonal antibody specifically reacting with H-P-13 and a monoclonal antibody specifically reacting with MPs as assay reagents
  • the present invention provides a method for quantification of diagram P-13, characterized in that active P-13 is measured from P.
  • the method of the present invention provides an antibody that binds to a solid support or an antibody that imparts a label, each of which specifically reacts with a substantially different antigenic determinant of P-13. It is also characterized by the use of noclonal antibodies.
  • a monoclonal antibody which specifically reacts with a protein selected from the group consisting of a protein having MMP-13 activity or a salt thereof and a partial peptide of a protein having 13 activity or a salt thereof Antibodies;
  • Fig. 13 is an antibody against a protein that has substantially the same activity as P-13 or a salt thereof, or has substantially the same primary structure conformation.
  • a monoclonal antibody that specifically reacts with a protein selected from the group consisting of protein having solid P-13 activity or a salt thereof and partial peptide of protein having P13 activity or a salt thereof Used as a reagent to detect and measure at least one of latent MMP13, activated rigid P13, and MP13 bound to tissue 'inhibition of metalloprotease' from the others. Characteristic ⁇ Detection and measurement method of P-13;
  • the two antibodies used are monoclonal antibodies that specifically react with both the active and latent forms of MMP-13, and the latent and active forms of P- ⁇ 3 must be determined.
  • One of the two antibodies used is a monoclonal antibody that specifically reacts with both the active and latent forms of MP-13, and the other specifically reacts with the latent form of MMP-13
  • a method for measuring the latent form of MP13 comprising the steps of:
  • One of the two antibodies used is an antibody that specifically reacts with both the active and latent forms of MP-13 and the other is a monoclonal antibody that specifically reacts with the active form of P-13
  • the quantification method according to the above [7], wherein the quantification method is a primary antibody, and the activated form P1.3 is measured.
  • L 18 'The use of a sample selected from the group consisting of body fluids such as blood, serum, plasma, and synovial fluid, biological tissues, cultured tissues, culture solutions, and the like is used as a specimen. [16] the quantitative method according to any one of [1] to [12];
  • [22 A protein selected from the group consisting of a protein having P13 activity, a protein having substantially the same activity as the protein, or a partial peptide or a salt thereof as an antigen, and an antibody against the same.
  • the present invention provides a method for quantifying activated hidden P13, which is characterized in that it is measured biologically. According to the present invention, the following aspects are further provided:
  • Recombinant human pro-pro-P-13 is used as an immunogen and is prepared from a protein having MP-13 activity or a salt thereof and a partial peptide of a protein having MP13 activity or a salt thereof.
  • a monoclonal antibody which specifically reacts with one selected from the group consisting of:
  • the two antibodies used are an enzyme-labeled monoclonal antibody that specifically reacts to both the active and latent forms of P13 and a solid phase that specifically reacts to the latent form of hidden P-13
  • a monoclonal antibody produced and immobilized by the hybridoma of clone number 181 3C4 and a monoclonal antibody produced by the hybridoma of clone number 181 15-12 and enzyme-labeled were combined.
  • the quantification method according to the above [7] which is used together with the quantification method;
  • the two types of antibodies used are an enzyme-labeled monoclonal antibody that specifically reacts with the active form of 13 and an immobilized monoclonal antibody that specifically reacts with the active form of P13
  • the quantification method according to the above [7] which is characterized by:
  • a combination of a monoclonal antibody produced and immobilized by the hybridoma of clone number 181 1B7 and a monoclonal antibody produced by the hybridoma of clone number 18 and 15 ⁇ 12 and enzyme-labeled The quantification method described in [7] above is characterized by being used.
  • the invention relates to
  • a complex comprising the target antigen, the first antibody, and the second antibody by contacting the first antibody and the second antibody with a test sample containing the target antigen
  • the one of the first antibody and the second antibody is a monoclonal antibody against at least rigid P-13.
  • one of the second antibody and the second antibody is a monoclonal antibody having specificity only for latent type P-13, and the other is for a latent type MP-13 and an active type P-13. -1; a monoclonal antibody having specificity for both of the above;
  • the difference between the first antibody and the second antibody is a monoclonal antibody having specificity for both latent MP13 and active MMP. 4 0 ⁇ Described method:
  • test sample is combined with a first antibody (immobilized antibody) and a labeled second antibody (labeled antibody) bound to a solid phase carrier for the target antigen in the test sample.
  • first antibody immobilized antibody
  • labeled antibody labeled antibody
  • the target sample is body fluid such as whole blood, serum, blood
  • body fluid such as whole blood, serum, blood
  • Solid phase is glass, silica gel, silica-alumina, alumina, iron magnetized, magnetized alloy, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene fluoride, vinyl polyacetate, polymer , Polystyrene, styrene, bushyen copolymer, polyacrylamide, cross-linked polyacrylamide, styrene
  • the immunoassay reagent according to [51] or [52], wherein the solid-phased reagent is used for an antigen assay system by the sandwich method. provide.
  • the invention relates to (54; the above) to [5], [19] to [21], [25] and [26: L of L, deviation or monoclonal monoclonal antibody against P13 described
  • the label is selected from the group consisting of radioisotopes, enzymes, luminescent substances, fluorescent substances, metal colloids, pigment substances, and biotin. 5 8] is provided.
  • a subculture-capable hybridoma cell that produces an antibody that specifically reacts with a partial peptide of an active protein or a salt thereof, or an antibody selected from the group consisting of the following;
  • a recombinant protein having P-13 activity or a salt thereof and a partial peptide of the recombinant protein having 13 activity or a salt thereof are fused with cells that can be subcultured, and hybrid cells that can be subcultured and produce antibodies to proteins including MP-13 are selected.
  • MMP-13, latent MMP-13, and activated P-1: 3 that react (or recognize or bind) with the monoclonal antibody are substantially each of P-13 and latent fan.
  • 3 - 13, active Er - 13 equivalent biological activity, those having an enzymatic activity or immunological activity, t usually in the form that is present in vivo However, it may be in a form that can be isolated from a living body, or may be a mutant derived from the same gene.
  • Figure 1 shows an example of a standard curve for assay system A (solid phase clone No. 181-3C4, enzyme labeling clone No. 181 15A12) using recombinant human latent type 13 as a standard. ;:
  • Fig. 2 shows the standard curve of assay system B (solid phase clone No. 18 to 7F.1, enzyme labeling clone No. 181 -15A12) using recombinant human latent MP13 as a standard.
  • C showing an example of
  • Figure 3 shows the results of the measurement of MMP13 in joint fluid and serum in various diseases using assay system A (clone No. 181-3C4 for solid phase, clone No. 181-15A12 for enzyme labeling).
  • Figure 4 shows the results of measurement of P-13 in synovial fluid and serum in various diseases using assay system B (clone for solid phase No. 181-7F4, clone for enzyme labeling No. 181-15A12). .
  • Fig. 5 shows the volume of P-13 of each fraction obtained by gel filtration of the recombinant human latent pattern diagram P-13, and the amount of P-13 was measured using assay system A (solid phase clone No. 181-3C4, enzyme labeling -No. 181-15M2), assay system B (solid-phase clone No. 181 7F4, enzyme labeling The results are shown for the results obtained with Loan No. 181-15 ⁇ 12) and assay system C (Clone for solid phase ' ⁇ : ⁇ .18MB7, Clone No. 181-15A12 for enzyme labeling).
  • Figure 6 shows the fractions obtained by gel filtration of the product (active MMP_i3) obtained by activating recombinant human latent MMP-13 with 4-APMA, and measuring the amount of rigid P-13 in assay system A ( Clone for solid phase No. 181-3C4, Clone for enzyme labeling No. 18 to 15A12), Measurement system B (Clone for solid phase No. 181-7 ⁇ !, Clone for enzyme labeling No. 18 to 15A12) and measurement system C (solid-phase clone No. 181-1B7, enzyme labeling clone o. 18M5A12).
  • FIG. 7 is a photograph showing the morphology of a living biological tissue showing the results of tissue staining of a breast cancer tissue using clone Nos. 181-15-112.
  • Figure 8 shows that the active form of MMP-13 and T1MP-1 were mixed at a ratio of 1: 1 to 10: 1 to produce a P TM i3 TIMP1 reconstituted product.
  • 7-23G9 mouse anti-T1MP-1 antibody
  • enzyme labeling clone No. 181-15A12 BEST MODE FOR CARRYING OUT THE INVENTION
  • the present invention uses at least two types of monoclonal antibodies that specifically react with MMP-13 as measurement reagents, and immunologically measures latent MMP-13 or activates MMP-13.
  • the present invention provides a method for quantifying MMP-13, which comprises measuring 13 and measuring both latent and active P13.
  • the two types of monoclonal antibodies are preferably monoclonal antibodies that specifically react with different regions of MMP13.
  • a monoclonal antibody that specifically reacts with one MP-13 is used as an antibody to be bound to a solid phase in the sandwich method, and the other MMP-13 is used as an antibody to add a label. It is important to use a monoclonal antibody that specifically reacts with the antibody to perform its immunological measurement.
  • the method of the present invention is also characterized in that a monoclonal antibody that specifically reacts with a substantially different antigenic determinant of MMP-13 is used as an antibody to be bound to a solid support or an antibody to be labeled. It is assumed that.
  • an antibody such as a monoclonal antibody specifically reacting with the country P13 of the present invention is provided.
  • Antibodies such as the monoclonal antibodies according to the present invention This provides a useful research tool for research on cancer invasion and metastasis, as well as cancer diagnosis, and a research tool useful for research on the pathogenesis and diagnosis method of Alzheimer's disease.
  • the monoclonal antibody according to the present invention can be obtained by immunizing an animal by a known method using human P13 obtained according to the present invention as an immunogen. It can be produced by the method of Stein et al. (Nature, 256: 495-97, 1975). In this method, recombinant MP M-13 is used as an immunogen, and natural MP-13 can also be used as a power immunogen.
  • Human 1P-13 can be obtained from cells produced in and outside the living body, such as cultured cells, excised tissues, and cultured tissues.For example, it can be obtained from cells such as chondrocytes and breast cancer cells. .
  • human MMP-13 can be obtained as recombinant human P-13.
  • human MMP-13 can be obtained from human 13 producing cells such as chondrocytes and breast cancer cells by using gene recombination technology. Can be.
  • the recombinant heat MP-13 can be obtained by the method described in nauper et al., J. Biol. Cem., 271, 1544-1550, 1996, or by referring to the method.
  • Human MP13 prepared by the method of Knauper et al. Or one derived therefrom can be suitably used as an immunizing antigen.
  • P-13 of these editions can be prepared by a conventionally known method, for example, salting-out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion-exchange chromatography, electrophoresis, dialysis, ultrafiltration, and the like. It can be used after purification by affinity chromatography or high performance liquid chromatography.
  • the purified recombinant heat diagram P-13 can be suitably used as an immunizing antigen for producing a monoclonal antibody.
  • the monoclonal antibody can be appropriately labeled by a commonly used method.
  • luminescent substances such as enzymes, radioisotopes (radioactive substances) and chemiluminescent compounds, fluorescent substances, metal colloids, prosthetic molecules, pigment substances, biotin and the like can be used.
  • the monoclonal antibody of the present invention may be a monoclonal antibody obtained by utilizing cell fusion technology using myeloma cells.
  • the monoclonal antibody of the present invention can be produced, for example, by the following steps.
  • antigen for example, naturally occurring recombinant human pro-pro-Pi3 prepared according to the method described in ⁇ -13, Knauper et al., J. Biol. Chem., 271, 1544-1550, 1996 is used. No. 13 can use latent P-13 or active MP-13, and can also be used as an immunogenic conjugator. ⁇ It is possible to immunize animals by mixing them with an appropriate adjuvant. Can be used for Such antigens can be obtained from a variety of raw materials, such as cultured cells, cultured tissues, and other antigen-producing materials such as transformed cells, by a conventionally known method, for example, salting-out such as ammonium sulfate precipitation, gel filtration by Sephadex, etc.
  • an ion exchange chromatography method using a carrier having a acetyl group such as a getylaminoethyl group or a carboxymethyl group for example, using a carrier having a hydrophobic group such as a butyl group, an octyl group, or a phenyl group.
  • It can be obtained by purification by a method such as a chromatographic method, a dye gel chromatography method, an electrophoresis method, a dialysis method, an ultrafiltration method, an affinity chromatography method, or a high performance liquid chromatography method.
  • it can be purified and separated by treating with polyacrylamide gel electrophoresis or affinity chromatography in which an antibody specifically reacting with an antigen such as a monoclonal antibody is immobilized.
  • MMP-13 A characteristic sequence region is selected based on the amino acid sequence deduced from the cDNA sequence that has been fragmented or cloned and sequenced, and the polypeptide is chemically synthesized by designing it. It may be a polypeptide fragment, and the fragment is bound to various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as protein, protein, and protein. It can also be used to design monoclonal antibodies that can only react to sequences (some L can recognize only specific sequences).
  • a cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared.
  • the carrier proteins For binding to carrier proteins, the carrier proteins can first be activated.
  • the activation of an activation bonding group may be mentioned.
  • Examples of the active carboxylic acid bonding group include (1) an active carboxylic acid group or an active carboxylic acid group such as a nitrophenyl ester group, a pentafluorophenyl ester group, a 1-benzotriazole ester group, and ⁇ : — Succinimide ester group, etc. (2) Active dithio group, for example, 2-pyridyl dithio group, etc.
  • Examples of carrier proteins include keyhole 'Lindet Hemosinin (KLH)'. Polypeptides such as albumin (BSA), ovalbumin, globulin, polylysine, and bacterial cell components such as BCG.
  • Immunization is performed using animals such as mice such as BALB / c.
  • the dose of the antigen is, for example, about 1 to 400 ag Injection, generally intraperitoneally or subcutaneously in the host animal, and then every 1 to: every other week, preferably every 1 to 2 weeks, intraperitoneally, subcutaneously, intravenously, or intramuscularly. Repeat 2 to 10 times.
  • As the mouse for immunization in addition to BALBZc mouse, F1 mouse of BALBZc mouse and other mouse can be used. If necessary, an antibody titer can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity.
  • An infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin.
  • ⁇ 3—NS—1—Ag4—1 (NS—1, Eur. J. 1 unol., 6, 511-519, 1976), SP2 / 0-Ag14 (SP2, ⁇ ature, 276, 269-270, 1978), mouse myeloma M ⁇ ⁇ ⁇ PC—2
  • P3—X63—Ag8—U1 from one cell line P3U1, Current topics in Microbiol.
  • the 8-azaguanine-resistant mouse myeloma cell line can be obtained from Dulbecco's MEM medium (DME 4 medium), RPM1-I64 () medium or other cell culture medium, such as substances such as benicillin and amikacin, bovine fetal serum ( FCS), etc., and further subcultured in a medium supplemented with 8-azaguanine (for example, 5 to 45 g / m 1).
  • a cell line can be provided.
  • a cell line may be provided:
  • the immunized animal eg, mouse
  • a spleen cell suspension is obtained.
  • lymph node cells from various parts of the body can be obtained and used for cell fusion.
  • the spleen cell suspension obtained and the myeloma cell line obtained according to the above-described steps 3 and 3 are used, for example, in a minimal essential medium (MEM medium), a DMEM medium, RPMI-164 ⁇ ⁇ Place in cell culture medium and add cell fusion agent, eg, polyethylene glycol.
  • MEM medium minimal essential medium
  • DMEM medium DMEM medium
  • RPMI-164 ⁇ ⁇ Place in cell culture medium
  • cell fusion agent eg, polyethylene glycol.
  • the cell fusion agent those known in various fields in addition to the above can be used.
  • Examples of such a cell fusion agent include inactivated Sendai virus (HVJ: Hemagg1 utinating virus of Japan).
  • HVJ Hemagg1 utinating virus of Japan
  • 30 to 60% of polyethylene glycol can be added in an amount of 0.5 to 2 ml, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be strongly used, and furthermore, polyethylene glycol having a molecular weight of 1.000 can be added. ⁇ 000 polyethylene glycol can be more preferably used.
  • the concentration of polyethylene glycol in the fusion medium is preferably, for example, 30 to 60%. 3 If necessary, for example, a small amount of dimethyl sulfoxide or the like can be added to promote fusion.
  • the ratio of spleen cells (lymphocytes) to myeloma cell lines used for fusion is, for example, preferably 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. it can.
  • the fusion reaction is performed for 1 to 10 minutes, and then a cell medium such as RPMI-164 medium is added.
  • the fusion reaction treatment can be performed plural times. After the fusion reaction, the cells are separated by centrifugation and transferred to a selection medium.
  • the selection medium examples include FCS-containing MEM medium, hypoxanthine, aminopterin and thymidine, a medium such as RPMII640 medium, and so-called HAT ⁇ ground.
  • the method of replacing the selective medium is generally such that the same volume as the volume dispensed into the culture plate is added the next day, and then half of the HAT medium is replaced every i to 3 days. It can also be made with appropriate modifications. Also in the 8 th 1 6 days after fusion, dividing the completion aminopterin, was, as & feeder the medium may be changed every 1-4 days with a so-called HT medium, also be used, for example a mouse thymus cells Yes, it is preferred, sometimes.
  • HT medium also be used, for example a mouse thymus cells Yes, it is preferred, sometimes.
  • the culture supernatant of the culture medium which has a high level of hybridoma growth, can be analyzed by, for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA). Screening is performed by using P13 or its fragment peptide as an antigen or measuring the target antibody using a labeled anti-mouse antibody using a fixed system or a fluorescence-induced cell separation device (FACS). .
  • RIA radioimmunoassay
  • ELISA enzyme immunoassay
  • FACS fluorescence immunoassay
  • Cloning hybridomas producing the desired antibody can be accomplished by the ability to pick up colonies in an agar medium, or by limiting dilution. It can be more preferably performed by a limiting dilution method. Cloning is preferably performed multiple times. Production of monoclonal antibodies
  • the obtained hybridoma strain is cultured in an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium, and the desired monoclonal antibody can be obtained from the culture medium.
  • an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium
  • the desired monoclonal antibody can be obtained from the culture medium.
  • the ability to ascite ascites in a hybridoma is cited.
  • each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal that is syngeneic to the myeloma cell-derived animal, and the ability to proliferate, for example, each hybridoma is transplanted to a nude mouse or the like, proliferated, and Monoclonal antibodies produced in ascites can be recovered and obtained.
  • Animals can receive intraperitoneal injections of mineral oil such as pristane (2,6,10,14-tetramethylpentyldecane) after transplantation of Hypri-doma. It can be grown and ascites collected.
  • the ascites fluid may be used as it is or by a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity ⁇ Can be purified and used as a monoclonal antibody by chromatography or high performance liquid chromatography.
  • the ascites containing the monoclonal antibody is subjected to ammonium sulfate fractionation, and then purified by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as Tin A column. Can be separated.
  • an anion exchange gel such as DEAE-Sepharose
  • an affinity column such as Tin A column.
  • affinity chromatography in which an antigen or antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and an affinity in which protein A is immobilized.
  • Nichii ⁇ Chromatography it is also possible to determine the sequence of the antibody obtained in such a large amount, or to produce the antibody by a genetic recombination technique using a nucleic acid sequence encoding the antibody obtained from the hybridoma strain.
  • these antibodies can be treated with enzymes such as trypsin, papain, and pepsin and optionally reduced to produce antibody fragments such as Fab, Fab ', and F (ab'). Good.
  • the antibody to impart label as an example of I gG fraction further c labeling of these cases, which can be the use of specific binding portion F ab 'obtainable by reduction after pepsin digestion, the following As mentioned above, there are enzymes (heloxidase, alkaline phosphatase or / 3-D-galactosidase), chemical substances, fluorescent substances L, and radioisotopes.
  • the detection / measurement in the present invention can be carried out by immunostaining, for example, tissue staining, cell staining, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and radioimmunoassey.
  • ELISA or the like can be used, and the measurement can be performed with or without BF separation.
  • the immunoassay is a radioimmunoassay or an enzyme immunoassay, and a sandwich type assay is also preferable.
  • a sandwich type assay one of the antibodies against P-13 is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase.
  • the sample is subjected to an incubation treatment to cause the labeled antibody and the immobilized antibody to react sequentially if necessary. After separating the unbound antibody, the labeled substance is measured.
  • the amount of label measured is proportional to the amount of antigen, ie, rigid P-13.
  • this assay it is called a simultaneous sandwich type assay, a forward sandwich type assay, or a reverse sandwich type assay according to the order of addition of an insolubilized antibody or a labeled antibody. For example, washing, agitation, shaking, filtration or pre-extraction of the antigen, etc., are appropriately employed in the measurement process under specific circumstances.
  • measurement conditions such as the concentration of a particular reagent, buffer, etc., temperature, or incubation time can be varied according to factors such as the concentration of the antigen in the sample and the properties of the sample.
  • concentration of a particular reagent, buffer, etc., temperature, or incubation time can be varied according to factors such as the concentration of the antigen in the sample and the properties of the sample.
  • the person skilled in the art uses the usual experimental methods to appropriately select the optimal conditions effective for each measurement and perform the measurement. You can do it.
  • Many carriers that can immobilize antigens or antibodies are known, and in the present invention, they can be appropriately selected and used.
  • As the carrier various carriers used for an antigen-antibody reaction and the like are known. In the present invention, of course, any of these known carriers can be used.
  • glass for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride, and the like.
  • Polyvinylidene fluoride Polyvinyl methacrylate, Polymethacrylate, Polystyrene, Styrene-butene copolymer, Polyacrylamide, Cross-linked polyacrylamide, Styrene-methacrylate copolymer, Poly Glycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc., cross-linked albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, etc.
  • filter paper beads, inner walls of test containers, for example, test tubes, cells made of synthetic materials such as glass plates, glass bottles, glass cells, synthetic resin cells, glass rods, rods made of synthetic materials,
  • a solid material object
  • a rod with a thick or thin end such as a rod with a thick or thin end, a rounded end with a protrusion, or a rod with a flat protrusion, a copper-inverted rod, etc.
  • An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically reacts with the antibody obtained by the present invention can be bound.
  • the binding between the carrier and those involved in the antigen-antibody reaction can be performed by physical methods such as adsorption, chemical methods using condensing agents, activated substances, etc. It can be performed by a method using a chemical bonding reaction, etc.
  • Labels include enzymes, enzyme substrates, enzyme inhibitors, capture molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, coloring substances, luminescent compounds, luminescent substances, coloring substances, magnetic substances, Metal particles such as colloidal gold, radioactive materials and the like can be mentioned.
  • Enzymes include dehydrogenases, reductases, oxidases, and other oxidoreductases, such as transferases that catalyze the transfer of amino, carboxyl, methyl, acyl, and phosphate groups, such as ester bonds. And a hydrolytic enzyme that hydrolyzes a glycoside bond, an ether bond, a peptide bond, and the like, a lyase, an isomerase, and a ligase. Enzymes can be used in combination with multiple enzymes for detection. C, for example, cycling can be used.
  • radioisotope isotopes for labeling include: -P'J, VI], [1 :; 1 IJ, ⁇ ],::
  • Representative enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as Escherichia coli 3-D galactosidase, maleet 'dehydrogenase, and glucose 6-phosphate' dehydrogenase.
  • Alkaline phosphatases such as zeolites, gluco-soxidases, darcoamylases, acetylcholinesterases, lipases, algal phosphatases, and Escherichia coli lipophosphatases.
  • enamel fluorin derivatives such as 4-methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as nitro phenyl phosphate,> enzymatic cycling system using JADP, Using a substrate such as a lumferine derivative or a dioxetane derivative, or the like, it can be measured by the resulting fluorescence or luminescence. Norecyclin and luciferase systems can also be used.
  • the electrode may be a glass electrode, an ion electrode using a hardly soluble salt film, a liquid film electrode, a polymer film electrode, or the like.
  • Enzyme labeling can be replaced with biotin-labeled enzyme and enzyme-labeled avidin (streptavidin). Signs should use several different kinds of signs Can also. In such cases, it may be possible to make multiple measurements continuously or discontinuously and simultaneously or separately.
  • 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, and horseradish belvoxidase, umberyfurylgalactoside, nitrofurnidine and the like are used for signal formation.
  • Combinations of enzyme reagents such as rugalactoside and 1D-galactosidase, glucose-6-phosphate / dehydrogenase are also available, and quinol compounds such as hydroquinone, hydroxybenzoquinone, and hydroxyanthraquinone; Those capable of forming thiol compounds such as lipoic acid and glucan thione, phenol derivatives, and ferrocene derivatives by the action of enzymes and the like can be used.
  • fluorescent substance or chemiluminescent compound examples include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethyl octa-damin isothiocyanate, dansyl lip, dansyl fluoride, fluorescamine, and phycosamine.
  • fluorescein isothiocyanate for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethyl octa-damin isothiocyanate, dansyl lip, dansyl fluoride, fluorescamine, and phycosamine.
  • examples include pyriproteins, acridinium salts, noremiferin, noresipherase, luminols such as aequorin, imidazoles, oxalates, rare ⁇ page chelate compounds, coumarin derivatives and the like.
  • Labeling can be carried out using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between a chomino group and an aldehyde group, and the like.
  • the method can be applied by appropriately selecting from methods that can be easily performed by those skilled in the art, and methods modified from those methods.
  • a condensing agent that can be used for producing the immunogenic complex a condensing agent that can be used for binding to a carrier, and the like can be used.
  • condensing agent examples include glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, '-borimethylene bis-acetamide, N, N'- Tylene bismaleimide, ethylene glycol bis succinimidyl succinate, bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) sorbodimid, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N—succinimidyl 4-(— maleimide methyl) cyclohexane monocarboxylate (SMCC :) Ruphossuccinimidyl 4- (N-malemidmethyl) Cyclohexane 1—force ruboxylate, N-succinimidyl (4-Acetyl Acetate) amide benzoate, N-succinimidyl: 1— (1-Mley-midyl) ) Butyrate,
  • a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme or the like and an antibody bound to a carrier can be sequentially reacted, or simultaneously reacted. You can also.
  • the order in which the reagents are added depends on the type of carrier system chosen.
  • a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first placed in an appropriate test tube together with a sample containing the substance to be measured. Then, the measurement can be performed by adding beads such as the sensitized plastic.
  • the force used in the immunoassay is as follows:
  • the solid support is made of polystyrene, polycarbonate, or polypropylene, which adsorbs proteins such as antibodies well.
  • various materials and forms such as polyvinyl balls, microplates, sticks, and fine particles can be arbitrarily selected and used.
  • the measurement can be performed in an appropriate buffer system so as to maintain an optimum pH, for example, a pH of about 4 to 9.
  • buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer.
  • the antibody-antigen reaction is performed at a temperature between about 0 ° and 60 ° C.
  • EDTA ethylenediaminetetraacetate
  • Certain Ls commonly employed in the art may be subjected to a blocking treatment to prevent non-specific binding reactions known to those skilled in the art, for example, normal serum proteins such as mammals, albumin, etc. , Skim milk, fermented milk, collagen, gelatin, etc. These methods can be used without any particular limitation as long as the purpose is to prevent non-specific binding reactions.
  • Monoclonal antibodies that can be used can be used in labeled antibody methods, such as Shigeru Muramatsu, et al., Experimental Biology ⁇ ! , Showa 60, edited by the Biochemical Society of Japan, Seismic Chemistry Laboratory Course 5, Immunological Biochemistry Research Method, Tokyo Kagaku Dojin, 1996, etc.
  • Immunostaining may be carried out by reacting a labeled antibody directly with an antigen in a tissue or a cell, or first, an unlabeled monoclonal antibody of the present invention. And then react with a labeled antibody (secondary antibody) having specificity for the primary antibody (primary antibody), followed by an indirect method.
  • a labeled antibody secondary antibody having specificity for the primary antibody (primary antibody)
  • secondary antibody having specificity for the primary antibody
  • Examples include the peroxidase-antiperoxidase method (PAP method), the avidin-biotin complex method (ABC), and the protein A method.
  • the labeling method for tissues and cells used for staining is prepared, for example, by embedding tissues in OCT compound (Miles) and freezing using liquid nitrogen or dry ice / aceton solution. It is possible. Also, sample specimens can be prepared by embedding in paraffin or epoxy resin.
  • Examples include spinal fluid, saliva, amniotic fluid, urine, other body fluids, cell culture media, tissue culture media, tissue homogenates, biopsy samples, tissues such as breast cancer tissues, and cells.
  • the object of the present invention is to use a monoclonal antibody against MP13 and a monoclonal antibody against 13 or T! MPs for use as a solid phase carrier to fractionate and quantitate free active or latent P13 in a test sample.
  • An object of the present invention is to provide an excellent method for performing the method and a reagent kit therefor.
  • the monoclonal antibody, the fragment of the monoclonal antibody or the labeled product obtained by the present invention is particularly useful as an ffl reagent for measuring MP13, a cancer test reagent, and a reagent for immunostaining of 3. It is preferably useful as a cancer test agent, for example, a cancer test agent capable of specifically detecting breast cancer, etc.
  • an object of the present invention is a method and a reagent capable of monitoring disease states such as tissue destruction and cancer metastasis by differentially quantifying free active or latent MMP-13 using the above-described quantification method, Is to provide a diagnostic agent.
  • the use of the above reagents in the medical and physiological fields, the determination of the degree of tissue destruction or cancer metastasis, the degree of tissue repair, or the promotion of physiological effects such as promotion of cell growth It is understood that all uses of the above formulas as indicators are included in that embodiment of the present invention.
  • the present invention can be used as a diagnostic means useful for research relating to the diagnostic treatment of cancer, such as the presence or absence of cancer cells, diagnosis of cancer malignancy, or the like.
  • Various technical means applicable to other medical and physiological applications can be provided.
  • the present invention will be described in detail with reference to Examples, but it should be understood that the present invention is not limited to these Examples, and that various embodiments based on the concept of the present specification are possible. is there.
  • terms are based on IUPAC 1UB Communication on Biochemical Nomenclature or based on the meaning of terms commonly used in the art.
  • mouse-derived monoclonal monoclonal anti-human collagenase 3 antibody-producing hybridoma 18F7F4 has been deposited with N1BH on February 13, 1996 (deposit date) (accession number FERM P 16002).
  • FERM P 16002 accession number
  • a request was made to transfer the original deposit to a deposit based on the Budapest Treaty, and it is stored under the accession number FERM BP 6212.
  • mouse-derived monoclonal anti-collagenase anti-hypertensive antibody-producing hybridoma 7-23G9 was deposited on April 17, 1991 (accession number) as 7 accession number PERM BP-3469, and was deposited in ⁇ . (Transferred from the original deposit to a deposit based on the Budapest Treaty upon request on July i, 1991).
  • Monoclonal antibody-producing clones were obtained by using recombinant human I, Prokoku 13 (Knauper et al., J. Biol. Chem., 271, 1544 1550, 1996) as an immunogen.
  • RPMI-160 (Flow La b.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 UZm 1), and amikacin sulfate (100 g / m 1) ), Adjusted to pH 7.2 with dry ice, and sterilized and filtered through a 0.2 ⁇ m Toyo membrane filter.
  • FCS (M.A. Bioproducts), which had been sterilized and filtered, was added to the above RPM 1640 ⁇ 1 ground to a concentration of 15% (v / V).
  • Polyethylene glycol 4000 (PEG4000, Merk & Co.) was added to RPMI-1640 ⁇ ground to 50% (wZw) to prepare a serum-free solution.
  • the medium used is as follows.
  • HAT medium The NS-1 medium described in b) above was further supplemented with hivoxanthin (100 ⁇ M), aminopterin (0.4 ⁇ M) and thymidine (16 ⁇ M).
  • HT ⁇ Sound The same composition as HA ⁇ S above except that aminobuterin was removed.
  • cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas.
  • a cloning medium containing 107 mouse thymocytes as a feeder per ml of NS-1 medium was prepared, and 5 cells per 36 ⁇ l, 36 ⁇ l and 21 ⁇ l of 9 ⁇ -well microwells were prepared. One and 0.5 Hypri-doma were added. Fifth, 0.1 ml of NS-1 medium was added to all cells.
  • the EL1SA method was performed on the group in which the viability of the hybridoma was sufficient 11 to 13 days after the start of the cloning and the colony formation negative level was 50% or more. If all the tested wells are not positive, check the number of colonies in the antibody-hidden gel, and select 4 to 6 wells that have been confirmed to have .1 colonies in the gel, and re-clone. Finally, as shown in Table 1, a hybridoma producing a monoclonal antibody against human P13 was obtained. f) In vitro and in vivo growth of monoclonal antibodies
  • Propagation of the monoclonal antibody is performed by a standard method. That is, each obtained hybrid
  • the cells were cultured in an appropriate culture medium such as NS-1 medium (in vitro propagation), and a monoclonal antibody having a concentration of 10 to 100 ⁇ g / m 1 was obtained from the culture supernatant.
  • an appropriate culture medium such as NS-1 medium (in vitro propagation)
  • a monoclonal antibody having a concentration of 10 to 100 ⁇ g / m 1 was obtained from the culture supernatant.
  • Pristane Aldrich Chem. Co., Ltd.
  • a mouse anti-human MMP-13 IgG-HRP complex was prepared according to the method of Is ikavva. El al. Described in J. 1 Satsuno unoassay, .1, 209-327. 1983.
  • Monoclonal monoclonal antibody which was found to be reactive against human MMP13, was dialyzed against 0.1 M phosphate buffer (pH 6.5), and 100 mM against IgG contained in the solution. A 1-fold molar amount of S-acetyl mercaptosuccinic anhydride was added as a dimethylformamide solution, and the mixture was incubated at 30 C for 30 minutes.
  • HRP was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mgm1, and 25-fold molar amount of EMCS was added to dimethylformamide with respect to the amount of HRP.
  • the mixture was added as a solution and reacted at 0 ° C. for 30 minutes.
  • the reaction mixture was subjected to gel filtration with Sephadex G-25 column equilibrated with (). 1 M phosphate buffer (pH 6.0).
  • Preparation of maleimide-de target words ⁇ HRP fraction prep was c c) IgG-H P complex
  • IgG-HRP of each clone was prepared.
  • recombinant human rigid P13 activated with recombinant human latent form ⁇ P-13 or 4-aminophenylmercuric acetate (4-APA) was subjected to SDS PAGE, and transferred to nitrocellulose membrane. -Stained with HRP.
  • Recombinant human figure P-13 was reacted in a 96-well microphone plate on which each antibody was immobilized. After washing, it was reacted with each anti-lgG HRP, and unreacted IgG HRP was removed by washing. Then, HRP activity was measured using phenylenediamine as a substrate.
  • mouse anti-human MMP13 IgG was dissolved in 0.1 M phosphate buffer (pH 7.5), and 10 The concentration was adjusted to 0 ⁇ gm1. Transfer the monoclonal antibody solution to 96 wells 3. Add 100 ⁇ l per well to the black plate. It was left for 24 hours. Next, the monoclonal antibody solution was removed, and each was washed three times with a physiological saline solution. Then, a 30 mM phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA was used. 0, immersed in buffer A), and stored at 1 ° C c b) 1 step sandwich Search for EIA measurement system
  • Human MMP-13 was diluted with an immunoreaction buffer and added to a 96-well vinyl plate (Falcon) at a concentration of 20 ⁇ .
  • the enzyme-labeled antibody prepared from each monoclonal antibody was diluted with buffer A to 1 gZm 1, and added to each of the above-mentioned vinyl plates at 100/1 and mixed.
  • a buffer for immunological reaction (30 mM phosphate buffer containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA, pH 7.0), and the above-mentioned c)) were used. According to the methods of the measurement system A and the measurement system B, P1.3 in the body fluid was measured. When using a synovial fluid as a sample, if the stock solution itself has a large viscosity force and it is difficult to obtain an accurate measurement value, it is preferable to dilute with the above buffer solution A. e) Simultaneous reproducibility test
  • the standard antigen was subjected to the simultaneous-reproducibility test of the assay system A and the assay system B.
  • the CV value of each measured value of both standard antigen solutions was 10% or less.
  • the results are shown in Table 4.
  • Measurement system A is a combination of solid phase clone No. 181-3 C4 and enzyme labeling clone No. 181-15A12
  • measurement system B is solid phase clone No. 18 to 7 F4 and enzyme labeling. This is a measurement system in combination with Clone No. 181-A12.
  • Table 4 Simultaneous reproducibility
  • the measurement system ⁇ is a combination of the solid-phase clone No. 181-3C4 and the enzyme-labeled clone No. 181-15A12, and the results are shown in Fig. 3.
  • Figure 4 shows a measurement system for the combination of clone No. 18F 7F4 and clone No. 181-15A12 for enzyme labeling.
  • Interstitial collagenase (Rigid P1): The method of Zhang et al. Described in Clin. Chim. Purified according to
  • MP-2 Human neonatal dermal fibroblast B1RGB (RCB 222) according to the method of Fuji mo to et al. Described in Clin. Chim. Acta, 221, 91-103, 1993. Purified.
  • MMP-3 Purified from NB1RGB according to the method of Obata et al. Described in Clin. Chim. Acta, 211, 59-72, 1992.
  • Neutrophil collagenase (P-8) Purified from human placenta according to the method of atsuki et al. Described in Clin. Chim. Acta, 244, 129143, 1996.
  • EL1SA was performed using antibodies, eight clones: 181-1B7, 181-3C4, 181-4E1K 181 5-9, 181-7F4, 18th brain, 181-14G11, and 18th 15A12.
  • 9 Add 6 ng microtiter plate (Costai-) to 50 ng of the above-mentioned human MP1, human P-2, human MMP-3, human P-8, human P9, human MT P Alternatively, coating was carried out at 4 ° C with Human Go P-13. To this was added IgG HRP (I ⁇ / m 1) prepared from each anti-human MMP13 monoclonal antibody, and the mixture was incubated at room temperature for 1 hour.
  • IgG HRP I ⁇ / m 1
  • Human tissues were subjected to immunohistological staining using the mouse-derived monoclonal anti-human collagenase 3 antibody of the present invention.
  • Formalin-fixed paraffin sections (5 li) of human tissues were stained by the streptavidin-biotin method.
  • the monoclonal antibody 181 15-12 was diluted with 20 mM phosphate buffer (pH 7.2) and reacted with the tissue section at room temperature for 30 minutes.
  • the plate was reacted with a biotinylated antibody (Biogenex, San Ramon, Calif.) For 20 minutes at room temperature, followed by a reaction with streptavidin-alkaline phosphatase for 20 minutes at room temperature.
  • the cells were stained using a naphthol-containing phosphate thread. After counterstaining with hematoxylin, dehydrated and dried AquatexOlerck, Damstadt, Germany)
  • the latent and active form P-13 can be immunologically quantified using the P monoclonal antibody produced using the purification 13. Each amount can be separated and quantified. Measurement of latent MP-13 in the test sample using simple procedures and reagents with good sensitivity and accuracy, and rapid measurement of active MP-13 ⁇ latent and active P- 13 Both can be quantified respectively. Achieving P-13 immunoassays will accurately determine the active form of MMP13, which is thought to play an important role in inflammatory diseases such as rheumatoid arthritis, cancer, and neoplastic diseases. The ability to separate and quantify well and quickly allows the diagnosis of these inflammatory reactions, and is useful as a diagnostic agent for inflammatory diseases such as rheumatoid arthritis, neoplastic diseases, and metastatic cancer. :

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Abstract

Procédé dans lequel la MMP-13 latente et la MMP-13 active peuvent être dosées séparément, au moyen d'un anticorps monoclonal anti-MMP-13. L'anticorps monoclonal MMP-13 est préparé avec de la pro-MMP-13 humaine purifiée. Au moins trois types de ces anticorps peuvent être obtenus, notamment un type qui se lie spécifiquement à la fois à la MMP-13 active et à la MMP-13 latente, un type qui se lie spécifiquement à la MMP-13 latente et un type qui se lie spécifiquement à la MMP-13 active. Une utilisation combinée des anticorps monoclonaux de ces trois types avec des anticorps en phase solide, des anticorps marqués avec une enzyme (p. ex. EIA) etc. permet de doser séparément la MMP-13 latente et la MMP-13 active.
PCT/JP1997/004884 1996-12-26 1997-12-26 Anticorps monoclonal contre la collagenase 3 et procede de dosage immunologique utilisant cet anticorps Ceased WO1998029560A1 (fr)

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US7041787B2 (en) 2000-12-29 2006-05-09 Kimberly-Clark Worldwide, Inc. Design and use of advanced zinc chelating peptides to regulate matrix metalloproteinases
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JP2020521804A (ja) * 2017-06-02 2020-07-27 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングＭｅｒｃｋ Ｐａｔｅｎｔ Ｇｅｓｅｌｌｓｃｈａｆｔ ｍｉｔ ｂｅｓｃｈｒａｅｎｋｔｅｒ Ｈａｆｔｕｎｇ Ｍｍｐ１３結合免疫グロブリン
CN111733140A (zh) * 2020-04-04 2020-10-02 华中农业大学 一种抗犬基质金属蛋白酶的杂交瘤细胞株及其制备方法和一种单克隆抗体及其应用
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WO2000058502A3 (fr) * 1999-03-25 2000-11-16 Max Delbrueck Centrum Utilisation de collagenase 3 pour identifier les maladies de destruction articulaire, en particulier pour prevoir l'evolution de la maladie et pour determiner les predispositions genetiques a l'arthrite rhumatoide (ra)
US6756197B1 (en) 1999-03-25 2004-06-29 Max-Delbrück-Centrum für Molekulare Medizin Collagenase 3 as a prognostic marker for rheumatoid arthritis
US7041787B2 (en) 2000-12-29 2006-05-09 Kimberly-Clark Worldwide, Inc. Design and use of advanced zinc chelating peptides to regulate matrix metalloproteinases
WO2003087369A3 (fr) * 2002-04-16 2004-01-22 Veli-Matti Kaehaeri Nouveau ribozyme et utilisation de celui-ci
WO2004021007A1 (fr) * 2002-08-29 2004-03-11 Invitek Gesellschaft Für Biotechnologie & Biodesign Mbh Kits de dosage elisa pour la detection de la collagenase 3 en tant que proenzyme et sous forme activee dans des liquides corporels et des surnageants de cultures cellulaires
EP2186894A4 (fr) * 2007-07-10 2010-10-20 Shionogi & Co Anticorps monoclonal ayant une activité neutralisante contre le mmp13
WO2009008414A1 (fr) 2007-07-10 2009-01-15 Shionogi & Co., Ltd. Anticorps monoclonal ayant une activité neutralisante contre le mmp13
US8536313B2 (en) 2007-07-10 2013-09-17 Shionogi & Co., Ltd. Monoclonal antibody having neutralizing activity against MMP13
JP5354680B2 (ja) * 2007-07-10 2013-11-27 塩野義製薬株式会社 Ｍｍｐ１３に対する中和活性を有するモノクローナル抗体
JP2020521804A (ja) * 2017-06-02 2020-07-27 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングＭｅｒｃｋ Ｐａｔｅｎｔ Ｇｅｓｅｌｌｓｃｈａｆｔ ｍｉｔ ｂｅｓｃｈｒａｅｎｋｔｅｒ Ｈａｆｔｕｎｇ Ｍｍｐ１３結合免疫グロブリン
JP2023113141A (ja) * 2017-06-02 2023-08-15 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｍｍｐ１３結合免疫グロブリン
US11813307B2 (en) 2017-06-02 2023-11-14 Merck Patent Gmbh Polypeptides binding ADAMTS5, MMP13 and aggrecan
US12129308B2 (en) 2017-06-02 2024-10-29 Merck Patent Gmbh MMP13 binding immunoglobulins
CN111733140A (zh) * 2020-04-04 2020-10-02 华中农业大学 一种抗犬基质金属蛋白酶的杂交瘤细胞株及其制备方法和一种单克隆抗体及其应用
CN111733140B (zh) * 2020-04-04 2022-07-01 华中农业大学 一种抗犬基质金属蛋白酶的杂交瘤细胞株及其制备方法和一种单克隆抗体及其应用

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