WO2012014997A1 - 抗fdpモノクローナル抗体、それを用いたfdp測定用試薬及び試薬キット、並びにfdp測定方法 - Google Patents
抗fdpモノクローナル抗体、それを用いたfdp測定用試薬及び試薬キット、並びにfdp測定方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/586—Liposomes, microcapsules or cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/75—Fibrin; Fibrinogen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/224—Haemostasis or coagulation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Definitions
- the present invention relates to a novel anti-FDP monoclonal antibody that recognizes FDP which is a degradation product of fibrin and fibrinogen.
- the present invention also relates to a reagent and reagent kit for FDP measurement using the antibody, and a method for measuring FDP.
- a fibrinolytic reaction takes place in the body to remove it.
- the fibrinolytic reaction that dissolves the thrombus is called secondary fibrinolysis, and fibrin in the thrombus is decomposed by the action of an enzyme such as plasmin to produce a fibrin degradation product (FbnDP; secondary fibrinolysis) in the blood.
- FbnDP fibrin degradation product
- a fibrinolytic reaction called primary fibrinolysis that occurs without thrombus formation also occurs.
- fibrinogen is decomposed by the action of an enzyme such as plasmin to produce a fibrinogen degradation product (FbgDP; primary fibrinolysis) in the blood.
- FbgDP fibrinogen degradation product
- FDP fibrinogen and fibrin degradation products
- FDP fibrinolysis
- cardiovascular disorders cardiovascular disorders
- hyperfibrinolysis abnormal bleeding
- DIC disseminated intravascular coagulation syndrome
- it is required to measure the total amount of FDP regardless of the type of FDP.
- reagents using immunoturbidimetry are commercially available.
- an FDP measurement reagent there is a reagent using a polyclonal antibody such as an anti-human fibrinogen antibody.
- the FDP measurement value becomes a pseudo high value unless a biological sample from which fibrinogen is removed, such as serum, is used as a specimen.
- the work of preparing serum is complicated.
- blood coagulation tests such as PT (prothrombin time), APTT (activated partial thromboplastin time), and Fbg (fibrinogen concentration
- plasma is used as a specimen.
- the plasma FDP measurement reagent used is preferred (see Patent Documents 1 to 5).
- Reagent for FDP measurement using serum reacts equally to primary and secondary fibrinolysis.
- a difference is observed between the reactivity of the monoclonal antibody used to the primary fibrinolysis and the reactivity to the secondary fibrinolysis. That is, in the conventional reagent for measuring plasma FDP, the reactivity with respect to the secondary fibrinolysis tends to be higher than the reactivity with respect to the primary fibrinolysis.
- fibrinogen does not decompose under physiological conditions. Therefore, since the secondary fibrinolysis occupies most of the FDP, the above difference in reactivity does not cause a problem in normal measurement.
- the activity of plasmin in blood is excessive and primary fibrinolysis is enhanced.
- the biological sample obtained from the subject in such a state contains primary fibrinolysis. Therefore, the conventional plasma FDP measurement reagent with low reactivity to the primary fibrinolysis may not be able to accurately measure the total amount of FDP of the subject in the fibrinolysis-enhanced state.
- the present inventors have produced a plurality of types of anti-FDP monoclonal antibodies having different reactivity to FDP, and by using a reagent containing a carrier sensitized with these, primary fibrinolysis can be obtained.
- the present invention was completed by finding that FDP can be measured with high accuracy even in the contained biological sample.
- the following three monoclonal antibodies Reacts with the X, Y and D fractions of fibrinogen and FDP primary fibrinolytic and with the DD, DD / E and XDP fractions of FDP secondary fibrinolytic
- a first monoclonal antibody It does not react with fibrinogen, but reacts with the X, Y and E fractions of the primary fibrinolysis of FDP, and the DD, DD / E and XDP fractions of the secondary fibrinolysis of FDP
- a second monoclonal antibody that reacts with the fraction, and does not react with fibrinogen, but reacts with the Y and E fractions of the primary fibrinolysis of FDP, and the DD fraction of the secondary fibrinolysis of FDP, DD
- An anti-FDP monoclonal antibody selected from a third monoclonal antibody that reacts with the / E and XDP fractions is provided.
- a reagent for FDP measurement comprising a carrier sensitized with the first, second and third monoclonal antibodies.
- an FDP measurement reagent kit comprising a first reagent comprising a buffer and a second reagent comprising a carrier sensitized with the first, second and third monoclonal antibodies.
- the carrier produced by the antigen-antibody reaction, a step of mixing a suspension of carrier particles sensitized with the first, second and third monoclonal antibodies and a biological sample. And measuring the degree of particle aggregation.
- the anti-FDP monoclonal antibody of the present invention it is possible to provide a reagent, a reagent kit, and a measuring method capable of measuring FDP with high accuracy even for a biological sample containing a primary fibrinolysis.
- FDP means both fibrin degradation products and fibrinogen degradation products.
- the “fibrin degradation product” is also referred to as a secondary fibrinolysis, and stabilized fibrin, which is a polymer formed by coagulation of fibrinogen in blood by the action of an enzyme such as thrombin, It is a protein group that is generated by being degraded by enzymes.
- fibrin degradation products include DD fraction, DD / E fraction, XDP fraction and the like.
- a DD / E fraction multimer for example, a DD / EY fraction that is a trimer of the DD / E fraction, a YXY / DXXXD fraction that is a pentamer of the DD / E fraction, And DXXD / YXXY fraction, which is a 7-mer of the DD / E fraction.
- the XDP fraction is also collectively referred to as D dimer.
- the “fibrinogen degradation product” is also referred to as a primary fibrinolysis, and is a protein group generated by fibrinogen present in blood being degraded by an enzyme such as plasmin. Examples of fibrinogen degradation products include the X fraction, the Y fraction, the D fraction, and the E fraction.
- reactivity to FDP is defined by the type of FDP to which the anti-FDP monoclonal antibody specifically reacts. Therefore, when the types of FDP that can specifically react between one anti-FDP monoclonal antibody and another anti-FDP monoclonal antibody are different from each other, the reactivity of these antibodies with respect to FDP is determined to be different from each other.
- the anti-FDP monoclonal antibodies of the present invention are three types of antibodies having different reactivities to FDP, and include at least the Y fraction of the primary fibrinolysis of FDP, and the DD fraction of the secondary fibrinolysis of FDP, It reacts with the DD / E fraction and the XDP fraction.
- these three types of monoclonal antibodies are referred to as “first monoclonal antibody”, “second monoclonal antibody”, and “third monoclonal antibody”, respectively.
- the first monoclonal antibody reacts with the X, Y and D fractions of fibrinogen and FDP primary fibrinolysis, and the DD fraction and DD / E fraction of FDP secondary fibrinolysis. And react with the XDP fraction.
- the second monoclonal antibody does not react with fibrinogen, but reacts with the X, Y and E fractions of the primary fibrinolysis of FDP, and the DD fraction of the secondary fibrinolysis of FDP, DD It reacts with / E fraction and XDP fraction.
- the third monoclonal antibody does not react with fibrinogen, but reacts with the Y and E fractions of the primary fibrinolysis of FDP, and the DD fraction and DD / E fraction of the secondary fibrinolysis of FDP. And react with the XDP fraction.
- the above-mentioned first, second and third monoclonal antibodies may be antibodies derived from any mammals such as mice, rats, hamsters, rabbits, goats, horses, etc. Among them, mice are preferred.
- the antibody isotype may be IgG, IgM, IgE, IgA or the like.
- Antibodies also include antibody fragments and derivatives thereof. Specific examples include Fab fragments and F (ab ′) 2 fragments.
- the above first, second and third monoclonal antibodies can be obtained by immunological techniques known in the art. That is, FDP (primary fibrinolysis and / or secondary fibrinolysis) as an antigen and an adjuvant are arbitrarily mixed to immunize an animal, and B lymphocytes obtained from the animal and appropriate myeloma cells To produce a hybridoma, and purify the culture supernatant of the hybridoma to obtain a monoclonal antibody.
- FDP primary fibrinolysis and / or secondary fibrinolysis
- the first, second and third monoclonal antibodies used in the reagent of the present invention can be obtained by the following method.
- FDP used as an antigen can be obtained by acting an enzyme capable of degrading fibrin and fibrinogen such as plasmin on fibrin and fibrinogen.
- Fibrin and fibrinogen as raw materials for FDP are commercially available.
- Fibrin can be obtained by reacting fibrinogen with thrombin, factor XIII and calcium salt.
- An antigen can be immunized with an antigen solution obtained by arbitrarily mixing the antigen obtained as described above with an adjuvant and dissolving or suspending it in an appropriate buffer solution.
- concentration of the antigen in the antigen solution is preferably about 50 to 500 ⁇ g / ml.
- a carrier protein such as albumin or keyhole limpet hemocyanin may optionally be bound to the antigen.
- adjuvants known in the art can be used as the adjuvant.
- adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordetella pertussis vaccine), Muramyl Dipeptide (MPD), aluminum adjuvant (ALUM) and combinations thereof.
- FCA Freund's complete adjuvant
- FIA Freund's incomplete adjuvant
- MPL MPL
- TDM Ribi
- MPL + TDM Ribi
- pertussis vaccine Bordetella pertussis vaccine
- MPD Muramyl Dipeptide
- ALUM aluminum adjuvant
- the animal to be immunized may be a mouse, rat, hamster, horse, goat, rabbit or the like, preferably a mouse, more preferably a BALB / c mouse.
- the immunization method can be appropriately selected depending on the type of antigen used and the presence or absence of an adjuvant. For example, in the case of using a mouse, 0.05 to 1 ml of an adjuvant mixed antigen solution (antigen 10 to 200 ⁇ g) is injected intraperitoneally, subcutaneously, intramuscularly or into the tail vein, and boosted 1 to 4 times about every 4 to 21 days from the first immunization. The final immunization is performed after about 1 to 4 weeks.
- Immunization may be performed without using an adjuvant in the antigen solution by intraperitoneal injection with an increased amount of antigen. Blood is collected approximately 5-10 days after the booster and antibody titer is measured. The antibody titer can be measured according to a method known in the art such as an antibody titer assay described later. Approximately 3 to 5 days after the final immunization, the spleen can be removed from the immunized animal, and the spleen cells can be isolated to obtain antibody-producing cells.
- Monoclonal antibodies can be produced according to methods known in the art, such as those described in Kohler and Milstein, Nature, 256, 495-497 (1975).
- the myeloma cell to be used may be a cell derived from any mammal such as mouse, rat and human, for example, mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / o-Ag14, P3X63 -Examples include established myeloma cells such as Ag8 / 653.
- myeloma cells there is a myeloma cell type that produces an immunoglobulin light chain.
- the immunoglobulin heavy chain produced by the antibody-producing cell and this light chain are randomly bound. Sometimes. Therefore, it is preferable to use myeloma cells that do not produce an immunoglobulin light chain, such as P3X63-Ag8 ⁇ 653 and SP2 / o-Ag14.
- the antibody-producing cells and myeloma cells are preferably cells derived from the same species, particularly from the same strain.
- Examples of methods for producing hybridomas by fusing antibody-producing cells and myeloma cells include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device.
- PEG polyethylene glycol
- spleen cells and myeloma cells are placed in an appropriate medium or buffer containing about 30-60% PEG (average molecular weight 1000-6000), 1-10: 1, preferably 5-10: 1.
- the reaction may be carried out for about 30 seconds to 3 minutes under conditions of a temperature of about 25 to 37 ° C. and a pH of 6 to 8. After completion of the reaction, the cells are washed, removed from the PEG-containing solution, resuspended in a medium, seeded on a microtiter plate, and cultured.
- the hybridoma can be selected by culturing the cells fused as described above on a selective medium.
- the selective medium may be any medium that allows only fused cells to grow.
- hypoxanthine-aminopterin-thymidine (HAT) medium is used.
- Hybridomas are usually selected by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after cell fusion, and further culturing while repeating the medium exchange every 2 to 3 days. Thereafter, it can be carried out by selecting a well in which a hybridoma colony is grown by microscopic observation.
- Whether or not the hybridoma obtained in this way produces the desired antibody can be confirmed by collecting the culture supernatant of the hybridoma and performing an antibody titer assay.
- Antibody titer assays can be performed by methods known in the art. For example, a secondary antibody (antiglobulin antibody, anti-IgG antibody, anti-IgM) added with a culture supernatant diluted serially to an antigen protein immobilized on a solid phase and labeled with a fluorescent substance, an enzyme, or a radioisotope (RI) The antibody can be detected by reacting the antibody.
- the hybridoma confirmed to produce the desired antibody by the above antibody titer assay can be separated into single clones by a limiting dilution method, a soft agar method, a method using a fluorescence excitation cell sorter, or the like.
- a hybridoma producing the target antibody can be isolated by serially diluting a hybridoma colony with a medium so as to be about 1 cell / well.
- the method for obtaining a monoclonal antibody from a hybridoma can be appropriately selected depending on the required amount of the monoclonal antibody and the properties of the hybridoma. For example, a method of obtaining from the ascites of a mouse transplanted with the hybridoma, a method of obtaining from a culture supernatant by cell culture, and the like can be mentioned.
- a hybridoma capable of growing in the abdominal cavity of a mouse can obtain a high concentration monoclonal antibody of several mg / ml from ascites.
- monoclonal antibodies can be obtained from the culture supernatant of cell culture. In this case, although the amount of antibody production is low, purification is easy because there is little contamination with immunoglobulins and other contaminants.
- an antibody is obtained from the intraperitoneal area of a mouse transplanted with a hybridoma, for example, into the intraperitoneal area of a BALB / c mouse that has been pre-administered with an immunosuppressive substance such as pristane (2, 6, 10, 14-tetramethylpentadecane).
- an immunosuppressive substance such as pristane (2, 6, 10, 14-tetramethylpentadecane).
- a hybridoma (about 1 ⁇ 10 6 or more) is transplanted, and ascites collected after about 1 to 3 weeks is collected.
- hybridomas are cultured by a high-density culture method or spinner flask culture method to obtain a culture supernatant containing the antibody. be able to.
- serum is added to the culture medium, impurities such as other antibodies and albumin are contained, and antibody purification is often complicated. Therefore, it is preferable to reduce the amount of serum added to the culture medium as much as possible.
- the hybridoma is acclimated to a serum-free medium by a conventional method and cultured in a serum-free medium. This facilitates antibody purification.
- Purification of monoclonal antibodies from ascites and culture supernatant can be performed by known methods, such as fractionation by salting out using salts such as ammonium sulfate and sodium sulfate, polyethylene glycol fractionation, ethanol fractionation, Examples include methods based on DEAE ion exchange chromatography and gel filtration chromatography.
- the antibody can be purified by affinity chromatography using a protein A binding carrier or an anti-mouse immunoglobulin binding carrier.
- the anti-FDP monoclonal antibody of the present invention for example, as the accession number NITE BP-949, an independent administrative agency, Product Evaluation Technology Base Organization Patent Microorganism Depositary Center (postal number 292-0818, Kisarazu City, Chiba Prefecture, Japan)
- An antibody produced by the hybridoma “FDP3-2920” commissioned on June 1, 2010 (hereinafter, also referred to as “FDP3-2920 antibody”) is listed in Kazusa Kama feet 2-5-8).
- FDP3-797 antibody As the second monoclonal antibody, for example, under the accession number NITE BP-950, the National Institute of Technology and Evaluation Microbiological Deposits Center (Postal Code 292-0818, 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan) And antibodies produced by the hybridoma “FDP3-797” commissioned on June 1, 2010 (hereinafter also referred to as “FDP3-797 antibody”).
- the third monoclonal antibody for example, under the accession number NITE BP-951, the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Zip number 292-0818, 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan) And antibodies produced by the hybridoma “FDP3-2935” commissioned on June 1, 2010 (hereinafter also referred to as “FDP3-2935 antibody”).
- the concentration ratio of the first monoclonal antibody, the second monoclonal antibody, and the third monoclonal antibody in the FDP measurement reagent of the present invention (hereinafter also referred to as the reagent of the present invention) is not particularly limited, Can be determined.
- the reagent of the present invention includes a carrier sensitized with the first, second and third monoclonal antibodies.
- a carrier include organic polymer compounds, inorganic compounds, and red blood cells.
- Organic polymer compounds include insoluble agarose, insoluble dextran, cellulose, latex, polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid.
- Examples thereof include polymers, acrylic acid polymers, acrylonitrile butadiene styrene copolymers, vinyl chloride-acrylic acid ester copolymers, and polyvinyl acetate acrylate.
- examples of inorganic compounds include silica and alumina.
- the shape of the carrier particles is not particularly limited, and may be any shape such as a spherical shape or a planar shape.
- the average diameter of the carrier particles can be appropriately selected according to the measuring instrument and the like, but usually 0.05 to 0.5 ⁇ m is appropriate.
- Latex is particularly preferable as the material of the particles.
- the method for sensitizing the carrier particles with the first, second and third monoclonal antibodies may be any of physical adsorption methods and chemical binding methods known in the art, but is easy to operate. Therefore, the physical adsorption method is preferable.
- the carrier particle When the carrier is a particle, the carrier particle may be a carrier particle sensitized with the first, second and third monoclonal antibodies, or each monoclonal antibody may be individually sensitized. It may be a mixture of prepared carrier particles. When the sensitization conditions to the carrier particles are different between the first, second and third monoclonal antibodies, it is preferable to sensitize each antibody to the carrier particles individually.
- the carrier particles sensitized with the first, second and third monoclonal antibodies are suspended in an appropriate buffer.
- the concentration of latex particles in the suspension is preferably 0.5 to 10 mg / ml, more preferably 0.75 to 5 mg / ml.
- the total concentration of the monoclonal antibody in the suspension is preferably 10 to 100 ⁇ g / ml, more preferably 20 to 50 ⁇ g / ml.
- a buffer solution having a buffering action at pH 5 to 10, preferably pH 6 to 9 can be mentioned.
- Specific examples include phosphate buffer, imidazole buffer, triethanolamine-hydrochloric acid, Good buffer, and the like.
- Good buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, etc. .
- MOPSO is preferable.
- the above buffer solution includes a protein stabilizer (for example, BSA), a preservative (for example, sodium azide, phenylmethanesulfonyl fluoride, etc.), a pH adjuster, a sensitizer (for example, polyvinylpyrrolidone, polyanion, polyethylene glycol, Sugars, etc.), inorganic salts (eg, sodium chloride, calcium chloride, etc.), background inhibitors (eg, human anti-mouse antibody (HAMA) absorbers, etc.) and the like may further be included.
- a protein stabilizer for example, BSA
- a preservative for example, sodium azide, phenylmethanesulfonyl fluoride, etc.
- a pH adjuster for example, polyvinylpyrrolidone, polyanion, polyethylene glycol, Sugars, etc.
- inorganic salts eg, sodium chloride, calcium chloride, etc.
- background inhibitors eg, human anti-
- an FDP measurement reagent kit (hereinafter also referred to as the reagent kit of the present invention) can be mentioned.
- the reagent kit of the present invention includes a first reagent comprising a buffer solution and a second reagent containing a suspension of carrier particles sensitized with the first, second and third monoclonal antibodies.
- the reagent kit of the present invention is obtained by immunoassay, for example, an assay (latex agglutination method) in which latex particles sensitized with the first, second and third monoclonal antibodies are reacted with FDP in a biological sample. It is a reagent kit for detecting FDP in the inside.
- the first, second and third monoclonal antibodies used in the reagent kit of the present invention include FDP3-2920 antibody, FDP3-797 antibody and FDP3-2935 antibody, respectively.
- the form of the reagent kit of the present invention is a two-reagent type consisting of the first reagent and the second reagent as described above, but may be a one-reagent type consisting of one reagent.
- the reagent kit is preferably a two-reagent type consisting of a first reagent and a second reagent. More preferably, the reagent kit of the present invention is in a form including a first reagent containing a buffer solution and a second reagent comprising the above FDP measurement reagent of the present invention.
- Examples of the buffer solution that can be used for the first reagent constituting the reagent kit of the present invention include the same buffer solution that can be used for suspending carrier particles in the reagent of the present invention.
- the first reagent may contain additives such as the above-mentioned protein stabilizer, preservative, pH adjuster, sensitizer, and inorganic salt.
- the FDP measurement method of the present invention can be carried out by using the FDP measurement reagent or reagent kit of the present invention.
- a method for measuring FDP in a biological sample using the FDP measurement reagent kit of the present invention will be specifically described below.
- a first reagent containing a buffer solution and a biological sample are mixed and incubated.
- the biological sample include serum, plasma and urine obtained from a subject.
- the volume ratio for mixing the first reagent and the biological sample may be about 5: 1 to 50: 1.
- the incubation time may be about 1 to 10 minutes.
- a second reagent containing a suspension of carrier particles sensitized with the first, second and third monoclonal antibodies is added to the mixture of the first reagent and the biological sample.
- the volume ratio when mixing the mixture and the second reagent may be about 1: 0.05 to 1: 1.5.
- the degree of aggregation is measured as the amount of change in absorbance per minute.
- This measurement is preferably performed with an optical instrument capable of measuring scattered light intensity, absorbance, or transmitted light intensity.
- the measurement wavelength can be selected from the range of 300 to 2400 nm, preferably 300 to 1000 nm, more preferably 500 to 1000 nm.
- the concentration and / or amount of FDP in a biological sample can be calculated from the amount of change in absorbance measured using a calibration curve obtained by measuring an FDP standard substance having a known concentration.
- the reagent kit of the present invention can also be used for a method of optically measuring the degree of aggregation of carrier particles by mixing a first reagent and a second reagent and then adding a biological sample to the mixture of both reagents.
- first, second and third monoclonal antibodies used in the FDP measurement method of the present invention include FDP3-2920 antibody, FDP3-797 antibody and FDP3-2935 antibody, respectively.
- Example 1 Preparation of hybridoma producing anti-FDP monoclonal antibody and acquisition of the antibody (1) Preparation of immunogen (antigen) (1-1) Preparation of primary fibrinolytic antigen Fibrinogen (Sigma) 241 mg (39.7 mg / ml) was added with plasmin (Sigma) to a final concentration of 60 mU / ml and reacted at 37 ° C. for 8 hours. Thereafter, aprotinin was added to a final concentration of 1 U / ml to stop the degradation reaction.
- immunogen antigen
- (1-1) Preparation of primary fibrinolytic antigen Fibrinogen (Sigma) 241 mg (39.7 mg / ml) was added with plasmin (Sigma) to a final concentration of 60 mU / ml and reacted at 37 ° C. for 8 hours. Thereafter, aprotinin was added to a final concentration of 1 U / ml to stop the degradation reaction.
- the resulting reaction solution was centrifuged at 12000 ⁇ g for 20 minutes, and the resulting supernatant was loaded onto a lysine-sepharose 4B column (volume 8 ml) equilibrated with 50 mM Tris buffer (pH 7.4) and chromatographed. After performing the chromatography, Sepharose was removed with a spin column. The obtained solution was replaced twice with a sample buffer (62.5 mM Tris, 192 mM glycine, 1% SDS (pH 6.8)) using an ultrafiltration centrifuge tube (Amicon 1550K; Millipore).
- the obtained solution was filled in Sephacryl S-300 (GE Healthcare), and flowed at a flow rate of 70-80 ⁇ l / min using a peristaltic pump, and fractions were collected every 10 minutes.
- Each of the obtained fractions was analyzed by SDS-PAGE using a molecular weight marker, and fractions containing the X fraction, the Y fraction, and the D fraction were collected.
- the collected fraction was filled in Sephacryl S-300 (GE Healthcare), and flowed at a flow rate of 2 ml / min using a peristaltic pump, and the fraction was collected every 30 seconds.
- fibrin gel was crushed using a 50 ml syringe. Fibrin gel was resuspended in 4.6 ml of TBS (pH 7.4). Plasmin was added to the suspension to a final concentration of 75 mU / ml and reacted at 37 ° C. for 6 hours. Thereafter, aprotinin was added to a final concentration of 1 U / ml to stop the degradation reaction.
- the obtained reaction solution was centrifuged at 12000 ⁇ g for 20 minutes, and the resulting supernatant was loaded onto a lysine-sepharose 4B column (volume 3.5 ml) equilibrated with 50 mM Tris buffer (pH 7.4). After chromatography, Sepharose was removed with a spin column. The resulting solution was replaced with a sample buffer (62.5 mM Tris, 192 mM glycine, 1% SDS (pH 6.8)) three times by an ultrafiltration centrifuge tube (Amicon 1550K; Millipore).
- the obtained solution was filled in Sephacryl S-300 (GE Healthcare) and flowed at a flow rate of 2 ml / min using a peristaltic pump, and fractions were collected every 30 seconds.
- Each of the obtained fractions was analyzed by SDS-PAGE using a molecular weight marker, and fractions containing XDP fraction, DD / E fraction, DD fraction and E fraction were collected.
- the collected fraction was filled in Sephacryl S-300 (GE Healthcare), and flowed at a flow rate of 2 ml / min using a peristaltic pump, and the fraction was collected every 30 seconds.
- mice in total 20 ⁇ g of the above antigen prepared to 100 ⁇ g / ml with Ribi adjuvant was administered to mice in total, 4 times.
- the antibody titer of the mice was measured by the method described later.
- mice with high antibody titers were finally immunized by administering an antigen prepared with PBS to 100 ⁇ g / ml from the tail vein of the mice.
- the wells were washed three times with 0.05 mM Tween20-containing 10 mM phosphate buffer (pH 7.0) (hereinafter referred to as a washing solution). Subsequently, the wells were filled with 10% phosphate buffer containing 1% BSA (hereinafter referred to as blocking buffer) to obtain primary and secondary antigen solid phases. 20 ⁇ l of serum and 80 ⁇ l of blocking buffer were added to each well of the antigen solid phase, and reacted at room temperature for 1 hour.
- the wells were washed 5 times with a washing solution, and then 100 ⁇ l of anti-mouse IgG-POD labeled antibody (DAKO) diluted 2000 times with a blocking buffer was added to each well and reacted at room temperature for 30 minutes.
- DAKO anti-mouse IgG-POD labeled antibody
- the well was washed 5 times with a washing solution, and then 100 ⁇ l of ODP substrate solution (Kokusai Reagent Co., Ltd.) was added to each well and allowed to react at room temperature for 15 minutes.
- 100 ⁇ l of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 490 nm was measured.
- serum obtained from non-immunized mice was added instead of serum.
- X63 cells were subcultured in RPMI-1640 medium (containing 20 ⁇ g / ml 8-azaguanine) containing 10% of inactivated fetal calf serum (FCS). From 3 days before cell fusion, the cells were further cultured in RPMI-1640 medium containing 10% FCS without 8-azaguanine, and X63 cells in the logarithmic growth phase were used. After washing the X63 cells three times with RPMI-1640 medium, the number of cells was counted to obtain 7.0 ⁇ 10 7 cells.
- FCS inactivated fetal calf serum
- Polyethylene glycol-4000 is dissolved in RPMI-1640 medium so as to be 50 (w / v)%, and the ratio of the number of spleen cells to X63 cells is 10: 1 in the obtained medium. Mixed. Then, the cells were fused using an electric fusion device SSH-2 (manufactured by Shimadzu Corporation). This cell fusion was performed under the condition that a mixture of spleen cells and myeloma cells was energized with an AC voltage (40 V) for 10 seconds and then applied with a DC pulse (2.3 KV / cm, pulse width 40 ⁇ s).
- HAT selection medium containing 1 ⁇ 10 ⁇ 4 M hypoxanthine, 4 ⁇ 10 ⁇ 7 M aminopterin and 1.6 ⁇ 10 ⁇ 5 M thymidine (HAT) in RPMI-1640 medium containing 10% FCS
- the cells were suspended at 2.0 ⁇ 10 6 cells / ml.
- 50 ⁇ l of this cell suspension was dispensed into each well of a 96-well microtiter plate, and then cultured in a CO 2 incubator at a temperature of 37 ° C., a humidity of 95%, and an 8% CO 2 atmosphere.
- On the first and second days from the start of the culture one drop of the above HAT selection medium was added to each well.
- days 7 and 9 2 drops of HAT selection medium were added to each well for further culturing.
- the medium was changed to a medium containing no HAT to obtain a hybridoma.
- Hybridoma screening (-1) Primary screening Among the hybridomas obtained above, a strain producing an antibody reactive to each of primary and secondary fibrinolytic antigens is obtained by the following method. Selected by. To each well of the antigen solid phase used in (3) above, 20 ⁇ l of the culture supernatant of each hybridoma and 80 ⁇ l of blocking buffer were added and reacted at room temperature for 1 hour. After completion of the reaction, the wells were washed 5 times with a washing solution, and 100 ⁇ l of anti-mouse IgG-POD labeled antibody (DAKO) diluted 2000 times with a blocking buffer was added to each well and reacted at room temperature for 30 minutes.
- DAKO anti-mouse IgG-POD labeled antibody
- the antibody obtained from the hybridoma of FDP3-797 was used as the second monoclonal antibody (hereinafter referred to as FDP3-797 antibody).
- the antibody obtained from the hybridoma of FDP3-2935 was used as the third monoclonal antibody (hereinafter referred to as FDP3-2935 antibody).
- FbgDP solution After performing the chromatography, Sepharose was removed with a spin column to prepare an FbgDP solution. The protein concentration of the obtained FbgDP solution was measured using a protein quantification reagent (Bio-Rad). A part of the FbgDP solution was used to confirm the reactivity of the reagent for FDP measurement of the present invention described later to FDP. The obtained FbgDP solution was replaced twice with a sample buffer (62.5 mM Tris, 192 mM glycine, 1% SDS (pH 6.8)) using an ultrafiltration centrifuge tube (Amicon 15 50K; Millipore).
- the obtained solution was filled in Sephacryl S-300 (GE Healthcare), and flowed at a flow rate of 70-80 ⁇ l / min using a peristaltic pump, and fractions were collected every 10 minutes.
- Each of the obtained fractions was analyzed by SDS-PAGE using a molecular weight marker, and fractions containing the X fraction, the Y fraction, the D fraction, and the E fraction were collected. This was replaced twice with phosphate buffer (PBS) by Amicon 15 50K (Millipore) to obtain FbgDP antigen.
- PBS phosphate buffer
- Fibrin gel was crushed using a 50 ml syringe. Fibrin gel was resuspended in 4.6 ml of TBS (pH 7.4). Plasmin was added to the suspension to a final concentration of 75 mU / ml and reacted at 37 ° C. for 6 hours. Thereafter, aprotinin was added to a final concentration of 1 U / ml to stop the degradation reaction. The obtained reaction solution was centrifuged at 12000 ⁇ g for 20 minutes, and the resulting supernatant was loaded onto a lysine-sepharose 4B column (volume 3.5 ml) equilibrated with 50 mM Tris buffer (pH 7.4).
- FbnDP solution After chromatography, Sepharose was removed with a spin column to prepare an FbnDP solution. The protein concentration of the obtained FbnDP solution was measured using a protein quantification reagent (Bio-Rad). A part of the FbnDP solution was used for confirming the reactivity of the FDP measurement reagent of the present invention described later to FDP.
- the obtained FbnDP solution was replaced with a sample buffer (62.5 ⁇ mM Tris, 192 ⁇ mM glycine, 1% SDS (pH 6.8)) three times by an ultrafiltration centrifuge tube (Amicon 15-50K; Millipore).
- the obtained solution was filled in Sephacryl S-300 (GE Healthcare) and flowed at a flow rate of 2 ml / min using a peristaltic pump, and fractions were collected every 30 seconds.
- Each of the obtained fractions was analyzed by SDS-PAGE using a molecular weight marker, and the fractions containing the XDP fraction, the DD / E fraction, and the DD fraction were collected. This was replaced twice with phosphate buffer (PBS) by Amicon 15-50K (Millipore) to obtain FbnDP antigen.
- PBS phosphate buffer
- Table 1 The results obtained by the ELISA method are shown in Table 1.
- “+” indicates that the antibody reacts with the fraction, and “ ⁇ ” indicates that the antibody does not react with the fraction.
- the FDP3-2920 antibody, the FDP3-797 antibody, and the FDP3-2935 antibody have different reactivity to FDP. More details -The FDP3-2920 antibody reacts with the X, Y and D fractions of fibrinogen and the primary fibrinolysis of FDP, and the DD fraction and DD / E fraction of the secondary fibrinolysis of FDP.
- the FDP3-797 antibody does not react with fibrinogen but reacts with the X, Y and E fractions of the primary fibrinolysis of FDP, and the DD fraction of the secondary fibrinolysis of FDP, DD / Reacts with E and XDP fractions;
- -The FDP3-2935 antibody does not react with fibrinogen but reacts with the Y and E fractions of the primary fibrinolysis of FDP and the DD fraction, DD / E fraction of the FDP secondary fibrinolysis and Reacts with XDP fraction.
- Example 2 Manufacture of FDP measurement reagent and reagent kit (1) Manufacture of first reagent containing buffer solution A buffer solution prepared by mixing each reagent so as to have a final concentration shown in Table 2 was used as a 1M sodium hydroxide aqueous solution. After adjusting the pH to 7.1, the buffer solution was prepared by measuring up to 1 liter with ultrapure water.
- Example 3 Examination of FDP Reactivity of FDP Measurement Reagent Kit of the Present Invention Measurement of FDP in plasma using the FDP measurement reagent kit of the present invention produced in Example 2 above and another company's product did.
- the first reagent of the reagent kit for FDP measurement of the present invention the reagent produced in Example 2 (1) above was used.
- the reagent manufactured in Example 2 (2) above was used as the second reagent.
- the mixing ratio (volume ratio) of the latex particle suspension in which each antibody was individually sensitized is 1: 1: 1.
- sample buffer for SDS-PAGE 10% glycerol, 2% SDS, 0.01% BPB-containing 62.5 mM Tris-HCl (pH 6.8)
- FDP GoldenoldWest Biologicals, Inc.
- Samples were prepared. This was separated by SDS-PAGE, and the separated protein was transferred to a PVDF membrane, followed by Western blotting using an anti-human fibrinogen rabbit polyclonal antibody. From the results of Western blotting, the amount of the primary fibrinolysis fraction containing the D fraction was analyzed, and the group with a high primary fibrinolysis fraction was designated as “high fibrinolysis enhancement”, and the other groups were designated “ Panel plasma was classified into 2 groups as "low fibrinolysis degree".
- the sample group H represents a group with a high fibrinolysis degree
- the sample group L represents a group with a low fibrinolysis degree.
- ⁇ indicates the median.
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Abstract
Description
フィブリノゲン、並びにFDPの一次線溶物のX画分、Y画分及びD画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第1のモノクローナル抗体、
フィブリノゲンとは反応しないが、FDPの一次線溶物のX画分、Y画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第2のモノクローナル抗体、及び
フィブリノゲンとは反応しないが、FDPの一次線溶物のY画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第3のモノクローナル抗体
から選択される抗FDPモノクローナル抗体が提供される。
また、本発明によれば、緩衝液からなる第1試薬、及び上記の第1、第2及び第3のモノクローナル抗体を感作した担体を含む第2試薬を含むFDP測定用試薬キットが提供される。
さらに、本発明によれば、上記の第1、第2及び第3のモノクローナル抗体を感作した担体粒子の懸濁液と、生体試料とを混合する工程と、抗原抗体反応により生じる、前記担体粒子の凝集の度合を測定する工程とを含むFDP測定方法が提供される。
本明細書において「フィブリン分解産物」とは、二次線溶物とも呼ばれ、トロンビンなどの酵素の作用により血液中のフィブリノゲンが凝固されて形成されるポリマーである安定化フィブリンが、プラスミンなどの酵素によって分解されて生じるタンパク質群である。フィブリン分解産物としては、DD画分、DD/E画分、XDP画分などが挙げられる。XDP画分としては、DD/E画分の多量体、例えばDD/E画分の3量体であるDXD/YY画分、DD/E画分の5量体であるYXY/DXXD画分、DD/E画分の7量体であるDXXD/YXXY画分などが挙げられる。また、当該技術においては、XDP画分はDダイマーとも総称される。
本明細書において「フィブリノゲン分解産物」とは、一次線溶物とも呼ばれ、血液中に存在するフィブリノゲンがプラスミンなどの酵素によって分解されて生じるタンパク質群である。フィブリノゲン分解産物としてはX画分、Y画分、D画分及びE画分が挙げられる。
第1のモノクローナル抗体は、フィブリノゲン、並びにFDPの一次線溶物のX画分、Y画分及びD画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応することを特徴とする。
第2のモノクローナル抗体は、フィブリノゲンとは反応しないが、FDPの一次線溶物のX画分、Y画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応することを特徴とする。
第3のモノクローナル抗体は、フィブリノゲンとは反応しないが、FDPの一次線溶物のY画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応することを特徴とする。
(抗原の取得)
抗原として用いるFDPは、プラスミンのようなフィブリン及びフィブリノゲンを分解できる酵素をフィブリン及びフィブリノゲンに作用させて得ることができる。なお、FDPの原料となるフィブリン及びフィブリノゲンは市販されている。また、フィブリンは、フィブリノゲンにトロンビン、第XIII因子及びカルシウム塩を作用させて得ることができる。
上記のようにして得られる抗原を、アジュバントと任意に混合し、適当な緩衝液に溶解又は懸濁して得られる抗原液で、動物を免疫することができる。該抗原液中の抗原の濃度は、50~500μg/ml程度が好ましい。抗原の免疫原性が低い場合は、アルブミン、キーホールリンペットヘモシアニンのようなキャリアータンパク質を任意に抗原と結合させてもよい。
免疫法は、使用する抗原の種類やアジュバントの有無により適宜選択することができる。例えばマウスを用いる場合、アジュバント混合抗原液0.05~1ml(抗原10~200μg)を腹腔内、皮下、筋肉内又は尾静脈内に注射し、初回免疫から約4~21日毎に1~4回追加免疫を行い、さらに約1~4週間後に最終免疫を行う。抗原量を多くして腹腔内注射することにより、抗原液にアジュバントを用いずに免疫を行ってもよい。追加免疫の約5~10日後に血液を採取して抗体価を測定する。抗体価は、後述する抗体価アッセイのような当該技術において公知の方法にしたがって測定できる。最終免疫から約3~5日後に、免疫された動物から脾臓を摘出し、脾臓細胞を分離して抗体産生細胞を得ることができる。
モノクローナル抗体は、当該技術において公知の方法、例えばKohler及びMilstein, Nature, 256, 495-497 (1975)に記載の方法にしたがって作製できる。
用いる骨髄腫細胞は、マウス、ラット、ヒトなどいずれの哺乳動物に由来する細胞であってもよく、例えばマウスミエローマP3X63-Ag8、P3X63-Ag8-U1、P3NS1-Ag4、SP2/o-Ag14、P3X63-Ag8・653などの株化骨髄腫細胞が挙げられる。骨髄腫細胞の中には免疫グロブリン軽鎖を産生する種類の骨髄腫細胞があり、これを融合対象として用いると、抗体産生細胞が産生する免疫グロブリン重鎖とこの軽鎖とがランダムに結合することがある。したがって、免疫グロブリン軽鎖を産生しない骨髄腫細胞、例えばP3X63-Ag8・653やSP2/o-Ag14などを用いるのが好ましい。抗体産生細胞と骨髄腫細胞とは、同種動物、特に同系統の動物由来の細胞が好ましい。
第2のモノクローナル抗体としては、例えば受託番号NITE BP-950として独立行政法人製品評価技術基盤機構特許微生物寄託センター(郵便番号292-0818、日本国千葉県木更津市かずさ鎌足2-5-8)に2010年6月1日付けで受託されたハイブリドーマ「FDP3-797」により産生される抗体(以下、「FDP3-797抗体」ともいう)が挙げられる。
第3のモノクローナル抗体としては、例えば受託番号NITE BP-951として独立行政法人製品評価技術基盤機構特許微生物寄託センター(郵便番号292-0818、日本国千葉県木更津市かずさ鎌足2-5-8)に2010年6月1日付けで受託されたハイブリドーマ「FDP3-2935」により産生される抗体(以下、「FDP3-2935抗体」ともいう)が挙げられる。
本発明の試薬キットは、イムノアッセイ、例えば上記の第1、第2及び第3のモノクローナル抗体を感作したラテックス粒子と、生体試料中のFDPとを反応させるアッセイ(ラテックス凝集法)などにより該試料中のFDPを検出するための試薬キットである。
本発明の試薬キットに用いられる第1、第2及び第3のモノクローナル抗体の一例としては、それぞれFDP3-2920抗体、FDP3-797抗体及びFDP3-2935抗体が挙げられる。
まず、緩衝液を含む第1試薬と生体試料とを混合してインキュベートする。ここで、生体試料としては被験者から得られる血清、血漿、尿などが挙げられる。第1試薬と生体試料とを混合する際の容量比は、5:1~50:1程度であればよい。また、インキュベート時間は1~10分間程度であればよい。
生体試料中のFDPの濃度及び/又は量は、濃度既知のFDP標準物質の測定により得られる検量線を用いて、測定した吸光度の変化量から算出できる。
なお、本発明のFDP測定方法に用いられる第1、第2及び第3のモノクローナル抗体の一例として、それぞれFDP3-2920抗体、FDP3-797抗体及びFDP3-2935抗体が挙げられる。
(1)免疫原(抗原)の調製
(1-1)一次線溶の抗原の調製
フィブリノゲン(Sigma社)241 mg(39.7 mg/ml)に、プラスミン(Sigma社)を終濃度60 mU/mlとなるように添加して、37℃で8時間反応させた。その後、アプロチニンを終濃度1U/mlとなるように加えて、分解反応を停止させた。得られた反応液を12000×gで20分間遠心し、得られた上清を、50 mMトリス緩衝液(pH7.4)で平衡化したリジン-sepharose 4Bカラム(ボリューム8ml)に充填してクロマトグラフィーを行った後、スピンカラムにてセファロースを除いた。
得られた溶液を、限外ろ過用遠心チューブ(アミコン15 50K;ミリポア社)によりサンプル緩衝液(62.5 mM Tris、192 mMグリシン、1%SDS(pH6.8))で2回置換した。得られた溶液をSephacryl S-300(GE Healthcare社)に充填し、ペリスタポンプを用いて流速70~80μl/分で流して10分ごとにフラクションを回収した。得られた各フラクションについて分子量マーカーを用いるSDS-PAGEにより解析して、X画分、Y画分及びD画分が含まれるフラクションを回収した。
回収したフラクションをSephacryl S-300(GE Healthcare社)に充填し、ペリスタポンプを用いて流速2ml/分で流して30秒ごとにフラクションを回収した。得られた各フラクションについて、上記と同様にSDS-PAGEにより解析して、一次線溶の高分子画分(X画分及びY画分)が含まれるフラクションを回収した。これをアミコン15 50K(ミリポア社)によりリン酸緩衝液(PBS)で2回置換して、一次線溶の抗原とした。
フィブリノゲン(Sigma社)92 mg(23 mg/ml)に、塩化カルシウム、ヒトトロンビン(三菱ウェルファーマ社)及び第XIII因子(ニプロ社)をそれぞれ終濃度25 mM、4U/ml及び0.05 U/mlとなるよう加え、37℃で一晩反応させて、フィブリノゲンをフィブリンに変換させた。反応液中に生じたフィブリンゲルをトリス緩衝液(TBS(pH7.4))50 mlで洗浄し、4℃、3000×gで10分間遠心し、フィブリンゲルを回収した。この操作を2度繰り返した後、50 mlシリンジを用いてフィブリンゲルを砕いた。フィブリンゲルをTBS(pH7.4)4.6 mlに再懸濁した。懸濁液にプラスミンを終濃度75 mU/mlとなるように添加して、37℃で6時間反応させた。その後、アプロチニンを終濃度1U/mlとなるように加えて、分解反応を停止させた。
得られた溶液を、限外ろ過用遠心チューブ(アミコン15 50K;ミリポア社)によりサンプル緩衝液(62.5 mM Tris、192 mMグリシン、1%SDS(pH6.8))で3回置換した。得られた溶液をSephacryl S-300(GE Healthcare社)に充填し、ペリスタポンプを用いて流速2ml/分で流して30秒ごとにフラクションを回収した。得られた各フラクションについて、分子量マーカーを用いるSDS-PAGEにより解析して、XDP画分、DD/E画分、DD画分及びE画分が含まれるフラクションを回収した。
回収したフラクションをSephacryl S-300(GE Healthcare社)に充填し、ペリスタポンプを用いて流速2ml/分で流して30秒ごとにフラクションを回収した。得られた各フラクションについて上記と同様にSDS-PAGEにより解析して、DD/E画分及びDD画分が含まれるフラクションを回収した。これをアミコン15 50K(ミリポア社)によりリン酸緩衝液(PBS)で2回置換して、二次線溶の抗原とした。
被免疫動物として、5週齢の近交系BALB/c系雌性マウス(日本チャールズリバー株式会社)を用いた。該マウスを動物飼育チェンバー内(23±1℃、湿度70%)で標準ペレットを使用し、任意に給水して飼育した。
上記(1-1)及び(1-2)で調製した各抗原をPBSで希釈して、200μg/mlの抗原溶液とした。各抗原溶液0.5 mlと同量のフロイント完全アジュバント(Difco社製)とを混合して乳化した。この乳化状の抗原を、4匹の5週齢のBALB/c系雌性マウスに200μlずつ腹腔内投与した。さらに、2週間毎に、Ribiアジュバントで100μg/mlとなるように調製した上記抗原をマウスに20μgずつ、計4回投与した。さらに1ヶ月後、Ribiアジュバントで100μg/mlとなるように調製した上記抗原を追加免疫した後、マウスの抗体価を、後述する方法により測定した。抗体価の高いマウスには、2週間後、PBSで100μg/mlに調製した抗原をマウスの尾静脈より投与して最終免疫した。
免疫開始時より定期的にマウス眼底網膜より少量の血液を採取し、これらの血液から血清を分離した。得られた血清について、フィブリン及びフィブリノゲン分解産物に対する抗体価を、以下の方法により調べた。
上記(1-1)及び(1-2)で調製した各抗原溶液をPBSで10μg/mlに希釈し、それぞれ100μlずつ96穴マイクロタイタープレートのウェルに分注し、4℃で18時間静置した。その後、0.05%Tween20含10 mMリン酸緩衝液(pH7.0)(以下、洗浄液と称する)でウェルを3回洗浄した。続いて、ウェルに1%BSA含10 mMリン酸緩衝液(以下、ブロッキング緩衝液と称する)を満たし、一次線溶及び二次線溶の抗原固相を得た。
該抗原固相の各ウェルに、血清20μl及びブロッキング緩衝液80μlを加え、室温で1時間反応させた。反応終了後、ウェルを洗浄液で5回洗浄した後、ブロッキング緩衝液で2000倍希釈した抗マウスIgG-POD標識抗体(DAKO社)100μlを各ウェルに加え、室温で30分間反応させた。反応終了後、ウェルを洗浄液で5回洗浄した後、ODP基質液(国際試薬株式会社)を100μlずつ各ウェルに加え、室温で15分間反応させた。続いて、2N硫酸を100μlずつ各ウェルに加えて反応を停止し、490 nmにおける吸光度を測定した。
なお、陰性コントロールとして、血清の代わりに非免疫マウスから得た血清を添加した。
最終免疫から3日後にBALB/cマウスから脾臓を摘出し、EMEM培養液中で脾臓細胞を分散させた。次いで、脾臓細胞をEMEM培養液で4回洗浄した後、細胞数を計測し、7.0×108個の脾臓細胞を得た。
細胞融合のための親細胞株としては、8-アザグアニン(2-アミノ-6-オキシ8-アザプリン)耐性のBALB/cマウス由来骨髄腫培養細胞株(P3X63-Ag8・653;以下、「X63細胞」という)を用いた。X63細胞は、非働化した牛胎仔血清(FCS)を10%含むRPMI-1640培地(20μg/ml 8-アザグアニン含有)で継代培養した。細胞融合の3日前より8-アザグアニンを含まない10%FCS含有RPMI-1640培地でさらに培養し、対数増殖期にあるX63細胞を用いた。X63細胞をRPMI-1640培地で3回洗浄した後、細胞数を計測し、7.0×107個の細胞を得た。
(5-1)1次スクリーニング
上記で得られたバイブリドーマのうち、一次線溶及び二次線溶の抗原のそれぞれに反応性を示す抗体を産生する株を、以下の方法により選択した。
上記(3)で用いた抗原固相の各ウェルに、各ハイブリドーマの培養上清20μl及びブロッキング緩衝液80μlを加え、室温で1時間反応させた。反応終了後、ウェルを洗浄液で5回洗浄した後、ブロッキング緩衝液で2000倍希釈した抗マウスIgG-POD標識抗体(DAKO社)を100μlずつ各ウェルに加え、室温で30分間反応させた。反応終了後、ウェルを洗浄液で5回洗浄した後、ODP基質液(国際試薬株式会社)を100μlずつ各ウェルに加え、室温で15分間反応させた。続いて、2N硫酸を100μlずつ各ウェルに加えて反応を停止し、490 nmにおける吸光度を測定した。なお、陰性コントロールとして、ハイブリドーマの培養上清の代わりに培養液のみを添加した。
測定結果に基づいて、一次線溶の高分子画分に反応する抗体を産生するハイブリドーマ、並びにDD/E画分及びDD画分に反応する抗体を産生するハイブリドーマを選択した。
1次スクリーニングで選択した各ハイブリドーマを限界希釈法によりクローニングし、一次線溶の高分子画分に反応する抗体を安定に産生するハイブリドーマ(1クローン;以下、「FDP3-2920」という)と、DD/E画分及びDD画分に反応する抗体を安定に産生するハイブリドーマ(2クローン;以下、それぞれ「FDP3-797」及び「FDP3-2935」という)を選択した。
なお、上記のFDP3-2920、FDP3-797及びFDP3-2935は、それぞれ受託番号NITE BP-949、受託番号NITE BP-950及び受託番号NITE BP-951として、独立行政法人製品評価技術基盤機構特許微生物寄託センター(郵便番号292-0818、日本国千葉県木更津市かずさ鎌足2-5-8)に2010年6月1日付けで受託された。
(6-1)腹水の作製
6週齢、雌性BALB/cヌードマウス(日本チャールズリバー株式会社)の腹腔内に、上記の各ハイブリドーマ(1×107個/匹)を接種した。接種から1週間後、該マウスにハイブリドーマ(1×107個/匹)を追加接種した。追加接種から2週間後、該マウスから注射器を用いて腹水を採取した。なお、各ハイブリドーマにつき、上記のマウスを5匹ずつ使用した。
得られた腹水17.5 mlに、50%飽和となる量の硫酸アンモニウムを少量ずつ加え、冷却しながら撹拌して沈殿を得た。次いで、この沈殿を回収し、PBSで溶解して溶液にした。そして、該溶液を透析チューブに入れ、4リットルのPBSで12日間透析した。透析後、チューブ内の溶液を0.45μmフィルターで濾過することにより、精製された各モノクローナル抗体を得た。
FDP3-2920のハイブリドーマにより得られた抗体を第1のモノクローナル抗体として用いた(以下、FDP3-2920抗体と称する)。FDP3-797のハイブリドーマにより得られた抗体を第2のモノクローナル抗体として用いた(以下、FDP3-797抗体と称する)。FDP3-2935のハイブリドーマにより得られた抗体を第3のモノクローナル抗体として用いた(以下、FDP3-2935抗体と称する)。
第1、第2及び第3のモノクローナル抗体としてのFDP3-2920抗体、FDP3-797抗体及びFDP3-2935抗体のフィブリン及びフィブリノゲン分解産物に対する反応性の違いを、以下のようなELISA法により検討した。
フィブリノゲン(Sigma社)241 mg(39.7 mg/ml)に、プラスミン(Sigma社)を終濃度60 mU/mlとなるように添加して、37℃で8時間反応させた。その後、アプロチニンを終濃度1U/mlとなるように加えて、分解反応を停止させた。得られた反応液を12000×gで20分間遠心し、得られた上清を、50 mMトリス緩衝液(pH7.4)で平衡化したリジン-sepharose 4Bカラム(ボリューム8ml)に充填してクロマトグラフィーを行った後、スピンカラムにてセファロースを除き、FbgDP溶液を調製した。得られたFbgDP溶液のタンパク質濃度を、タンパク質定量試薬(Bio-Rad社)を用いて測定した。また、FbgDP溶液の一部を、後述する本発明のFDP測定用試薬のFDPへの反応性の確認に用いた。
得られたFbgDP溶液を、限外ろ過用遠心チューブ(アミコン15 50K;ミリポア社)によりサンプル緩衝液(62.5 mM Tris、192 mMグリシン、1%SDS(pH6.8))で2回置換した。得られた溶液をSephacryl S-300(GE Healthcare社)に充填し、ペリスタポンプを用いて流速70~80μl/分で流して10分ごとにフラクションを回収した。得られた各フラクションについて分子量マーカーを用いるSDS-PAGEにより解析して、X画分、Y画分、D画分、およびE画分が含まれるフラクションをそれぞれ回収した。これをアミコン15 50K(ミリポア社)によりリン酸緩衝液(PBS)で2回置換して、FbgDP抗原とした。
フィブリノゲン(Sigma社)92 mg(23 mg/ml)に、塩化カルシウム、ヒトトロンビン(三菱ウェルファーマ社)及び第XIII因子(ニプロ社)をそれぞれ終濃度25 mM、4U/ml及び0.05 U/mlとなるよう加え、37℃で一晩反応させて、フィブリノゲンをフィブリンに変換させた。反応液中に生じたフィブリンゲルをトリス緩衝液(TBS(pH7.4))50 mlで洗浄し、4℃、3000×gで10分間遠心し、フィブリンゲルを回収した。この操作を2度繰り返した後、50 mlシリンジを用いてフィブリンゲルを砕いた。フィブリンゲルをTBS(pH7.4)4.6 mlに再懸濁した。懸濁液にプラスミンを終濃度75 mU/mlとなるように添加して、37℃で6時間反応させた。その後、アプロチニンを終濃度1U/mlとなるように加えて、分解反応を停止させた。得られた反応液を12000×gで20分間遠心し、得られた上清を、50 mMトリス緩衝液(pH7.4)で平衡化したリジン-sepharose 4Bカラム(ボリューム3.5 ml)に充填してクロマトグラフィーを行った後、スピンカラムにてセファロースを除き、FbnDP溶液を調製した。得られたFbnDP溶液のタンパク質濃度をタンパク質定量試薬(Bio-Rad社)を用いて測定した。また、FbnDP溶液の一部を後述する本発明のFDP測定用試薬のFDPへの反応性の確認に用いた。
フィブリノゲンをTBSに溶解して得たフィブリノゲン溶液(50 mg/ml)を、フィブリノゲン抗原とした。
各抗FDPモノクローナル抗体溶液をPBSで0.5μg/mlに希釈し、それぞれ100μlずつ96穴マイクロタイタープレートのウェルに分注し、4℃で18時間静置した。その後、0.05%Tween20含10 mMリン酸緩衝液(pH7.0)(以下、洗浄液と称する)でウェルを3回洗浄した。続いて1%BSA含10 mMリン酸緩衝液(以下、ブロッキング緩衝液と称する)を満たし、抗FDPモノクローナル抗体の固体固相を得た。
ウェルを洗浄液で3回洗浄した後、該抗体固相の各ウェルに、上記で調製したFbgDP各抗原及びFbnDP各抗原を100μlずつ加え、室温で30分間反応させた。反応終了後、ウェルを洗浄液で3回洗浄した後、ペルオキシダーゼ標識抗フィブリノゲン抗体(DAKO社)を100μlずつ各ウェルに加え、室温で1時間反応させた。反応終了後、ウェルを洗浄液で3回洗浄した後、ODP基質液(国際試薬株式会社)を100μlずつ各ウェルに加え、室温で15分間反応させた。続いて2N硫酸を100μlずつ各ウェルに加えて、反応を停止し、490 nmにおける吸光度を測定した。
より詳しくは、
- FDP3-2920抗体は、フィブリノゲン、並びにFDPの一次線溶物のX画分、Y画分及びD画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する;
- FDP3-797抗体は、フィブリノゲンとは反応しないが、FDPの一次線溶物のX画分、Y画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する;
- FDP3-2935抗体はフィブリノゲンとは反応しないが、FDPの一次線溶物のY画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する。
(1)緩衝液を含む第1試薬の製造
各試薬を表2に示される終濃度となるように混合した緩衝液を、1M水酸化ナトリウム水溶液でpHを7.1に調整した後、超純水で1リットルにメスアップすることにより、緩衝液を製造した。
(2-1)FDP3-2920抗体のラテックス粒子への感作
FDP3-2920抗体の終濃度が1mg/mlとなるように、50 mM 2-ヒドロキシ-3-モルホリノプロパンスルホン酸/150 mM NaCl溶液に混合した。そして、この混合液と20%(重量比)ラテックス溶液(粒径0.238μm;JSR株式会社)とを混合した。
得られた混合液に、50 mM 2-ヒドロキシ-3-モルホリノプロパンスルホン酸/150 mM NaCl溶液/2%BSA溶液を等量加えて混合した後、10℃、38400×gで30分間遠心した。上澄みを除去し、得られた沈殿物に、上澄みと等量の50 mM 2-ヒドロキシ-3-モルホリノプロパンスルホン酸/150 mM NaCl溶液/2%BSA/1.5%シュークロース溶液を添加して混合した。
得られた混合液を、氷冷条件で超音波破砕機(大岳社製)、超音波処理装置(Dr. Hielscher Gmbh UP-200S相当品)を用いてソニケーションを実施し、FDP3-2920抗体を感作したラテックス粒子の懸濁液(抗体濃度39μg/ml)を得た。
FDP3-797抗体の終濃度が1mg/mlとなるように、50 mM 2-ヒドロキシ-3-モルホリノプロパンスルホン酸/150 mM NaCl溶液に混合した。以下、上記の(2-1)FDP3-2920抗体のラテックス粒子への感作において述べたことと同様にして、FDP3-797抗体を感作したラテックス粒子の懸濁液(抗体濃度39μg/ml)を得た。
FDP3-2935抗体の終濃度が1mg/mlとなるように、50 mM 2-ヒドロキシ-3-モルホリノプロパンスルホン酸/150 mM NaCl溶液に混合した。以下、上記の(2-1)FDP3-2920抗体のラテックス粒子への感作において述べたことと同様にして、FDP3-2935抗体を感作したラテックス粒子の懸濁液(抗体濃度39μg/ml)を得た。
FDP3-2920抗体、FDP3-797抗体及びFDP3-2935抗体の各抗体を感作したラテックス粒子の懸濁液を、それぞれ等量混合することにより、3種類のモノクローナル抗体を感作した担体粒子を含むFDP測定用試薬を得た。以下、このFDP測定用試薬を第2試薬とした。
また、FDP3-797抗体とFDP3-2935抗体とのFDPに対する反応性の違いを検討するために、FDP3-797抗体及びFDP3-2935抗体の各抗体を感作したラテックス粒子の懸濁液を、それぞれ等量混合して、2種類のモノクローナル抗体を感作した担体粒子を含むFDP測定用試薬を得た。
上記の実施例2で製造した本発明のFDP測定用試薬キットと、他社製品とを用いて血漿中のFDPを測定した。
本発明のFDP測定用試薬キットの第1試薬として、上記の実施例2(1)で製造した試薬を用いた。また、第2試薬として、上記の実施例2(2)で製造した試薬を用いた。第2試薬における、各抗体をそれぞれ個別に感作したラテックス粒子の懸濁液の混合率(体積比)は、1:1:1である。
よって、本発明のFDP測定用試薬及び試薬キットは、他社製品に比べて、線溶亢進状態にある被験者から得られた検体についてもFDP濃度をより正確に測定できることが示された。
Claims (10)
- 以下の3種のモノクローナル抗体:
フィブリノゲン、並びにFDPの一次線溶物のX画分、Y画分及びD画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第1のモノクローナル抗体、
フィブリノゲンとは反応しないが、FDPの一次線溶物のX画分、Y画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第2のモノクローナル抗体、及び
フィブリノゲンとは反応しないが、FDPの一次線溶物のY画分及びE画分と反応し、且つFDPの二次線溶物のDD画分、DD/E画分及びXDP画分と反応する第3のモノクローナル抗体
から選択される抗FDPモノクローナル抗体。 - 前記第1のモノクローナル抗体が、受託番号NITE BP-949として独立行政法人製品評価技術基盤機構特許微生物寄託センターに2010年6月1日付けで受託されたハイブリドーマにより産生される抗体である請求項1に記載の抗FDPモノクローナル抗体。
- 前記第2のモノクローナル抗体が、受託番号NITE BP-950として独立行政法人製品評価技術基盤機構特許微生物寄託センターに2010年6月1日付けで受託されたハイブリドーマにより産生される抗体である請求項1または2に記載の抗FDPモノクローナル抗体。
- 前記第3のモノクローナル抗体が、受託番号NITE BP-951として独立行政法人製品評価技術基盤機構特許微生物寄託センターに2010年6月1日付けで受託されたハイブリドーマにより産生される抗体である請求項1~3のいずれか1項に記載の抗FDPモノクローナル抗体。
- 請求項1~4のいずれか1項に定義される第1、第2及び第3のモノクローナル抗体を感作した担体を含むFDP測定用試薬。
- 前記担体が粒子である請求項5に記載のFDP測定用試薬。
- 前記担体粒子が、前記第1、第2及び第3のモノクローナル抗体を個別に感作した担体粒子の混合物である請求項6に記載のFDP測定用試薬。
- 緩衝液からなる第1試薬、及び
請求項1~4のいずれか1項に定義される第1、第2及び第3のモノクローナル抗体を感作した担体粒子の懸濁液を含む第2試薬
を含むFDP測定用試薬キット。 - 請求項1~4のいずれか1項に定義される第1、第2及び第3のモノクローナル抗体を感作した担体粒子の懸濁液と、生体試料とを混合する工程と、
抗原抗体反応により生じる、前記担体粒子の凝集の度合を測定する工程と
を含むFDP測定方法。 - 前記凝集の度合の測定が、吸光度の変化の測定である請求項9に記載のFDP測定方法。
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EP11812586.3A EP2599865B1 (en) | 2010-07-30 | 2011-07-28 | Anti-fdp monoclonal antibody, fdp measurement reagent and reagent kit using same, and fdp measurement method |
JP2012526573A JP5903381B2 (ja) | 2010-07-30 | 2011-07-28 | 抗fdpモノクローナル抗体、それを用いたfdp測定用試薬及び試薬キット、並びにfdp測定方法 |
US13/752,035 US8865424B2 (en) | 2010-07-30 | 2013-01-28 | Anti-FDP monoclonal antibody, FDP measurement reagent and reagent kit using same, and FDP measurement method |
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CN104458367A (zh) * | 2014-11-06 | 2015-03-25 | 上海长岛生物技术有限公司 | 一种d-二聚体和fdp复合质控品及其制备方法 |
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CN103026231B (zh) * | 2010-07-30 | 2015-06-24 | 希森美康株式会社 | Fdp测定用试剂及试剂盒、以及测定方法 |
CN103954758A (zh) * | 2014-05-15 | 2014-07-30 | 海南世济医学技术有限公司 | 一种用于纤维蛋白和纤维蛋白原及其降解产物的检测方法和试剂盒 |
JP6640494B2 (ja) * | 2015-08-28 | 2020-02-05 | シスメックス株式会社 | 血液検体分析方法、血液検体分析装置、及びコンピュータプログラム |
CN114149507A (zh) * | 2021-12-31 | 2022-03-08 | 武汉华美生物工程有限公司 | 抗fdp单克隆抗体、抗体对及其制备方法和用途 |
CN117089529B (zh) * | 2023-10-16 | 2024-03-15 | 深圳普门科技股份有限公司 | 杂交瘤细胞及其制备方法、单克隆抗体及试剂盒 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60166698A (ja) * | 1984-02-09 | 1985-08-29 | Dai Ichi Pure Chem Co Ltd | モノクロ−ナル抗体 |
JPS60185800A (ja) * | 1983-11-14 | 1985-09-21 | ニユ−ヨ−ク ブラツド センタ−,インコ−ポレイテイド | モノクロナ−ル抗体 |
JPH0538906A (ja) | 1991-08-07 | 1993-02-19 | Yokohama Rubber Co Ltd:The | 乗用車用ラジアルタイヤ |
JPH0746104A (ja) | 1994-02-04 | 1995-02-14 | Kawasaki Steel Corp | プログラマブル入力回路 |
JPH11349600A (ja) * | 1998-06-02 | 1999-12-21 | Teikoku Hormone Mfg Co Ltd | モノクローナル抗体及びfdpの測定方法 |
JP2001021557A (ja) * | 1999-07-12 | 2001-01-26 | Iatron Lab Inc | Dダイマー及びdd/e複合体の免疫学的分析方法及び分析用キット |
JP2001354700A (ja) | 2000-06-12 | 2001-12-25 | Bio Links Kk | モノクロ−ナル抗体及びヒトフィブリン分解産物の測定方法 |
JP2002372536A (ja) | 2001-06-15 | 2002-12-26 | Dai Ichi Pure Chem Co Ltd | 免疫学的測定法 |
JP2006105633A (ja) * | 2004-09-30 | 2006-04-20 | Sysmex Corp | Dダイマー測定用試薬およびこれに用いるモノクローナル抗体 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4208479A (en) * | 1977-07-14 | 1980-06-17 | Syva Company | Label modified immunoassays |
US4851334A (en) | 1984-01-03 | 1989-07-25 | The New York Blood Center, Inc. | Monoclonal antibodies specific to in vivo fragments derived from human fibrinogen, human fibrin I or human fibrin II |
WO1988001514A1 (en) * | 1986-08-25 | 1988-03-10 | American Biogenetic Sciences, Inc. | Monoclonal antibodies to fibrin |
US5091512A (en) * | 1988-06-13 | 1992-02-25 | American Biogenetic Sciences, Inc. | Fibrinogen-specific monoclonal antibody |
DE4242736A1 (de) * | 1992-12-17 | 1994-06-23 | Behringwerke Ag | Synthetische Peptide, Antikörper dagegen und ihre Verwendung |
US5821068A (en) * | 1993-11-02 | 1998-10-13 | Iatron Laboratories, Inc. | Anti-human soluble fibrin antibody, hybridoma, and immunoassaying method |
JP3889084B2 (ja) * | 1996-05-15 | 2007-03-07 | 株式会社三菱化学ヤトロン | 新規なモノクローナル抗体並びにe−Dダイマー及びe−DD/E複合体の免疫学的測定法 |
EP1832654B1 (en) * | 2004-12-28 | 2011-07-27 | Sekisui Medical Co., Ltd. | Anti-human soluble fibrin monoclonal antibody and immunological assay method using the antibody |
ES2655099T3 (es) * | 2010-04-01 | 2018-02-16 | Lsi Medience Corporation | Nuevos anticuerpos monoclonales y procedimiento de análisis inmunológico del dímero D |
-
2011
- 2011-07-28 JP JP2012526573A patent/JP5903381B2/ja active Active
- 2011-07-28 EP EP11812586.3A patent/EP2599865B1/en active Active
- 2011-07-28 CN CN201180036439.4A patent/CN103025871B/zh active Active
- 2011-07-28 WO PCT/JP2011/067327 patent/WO2012014997A1/ja active Application Filing
-
2013
- 2013-01-28 US US13/752,035 patent/US8865424B2/en active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60185800A (ja) * | 1983-11-14 | 1985-09-21 | ニユ−ヨ−ク ブラツド センタ−,インコ−ポレイテイド | モノクロナ−ル抗体 |
JPS60166698A (ja) * | 1984-02-09 | 1985-08-29 | Dai Ichi Pure Chem Co Ltd | モノクロ−ナル抗体 |
JPH0538906A (ja) | 1991-08-07 | 1993-02-19 | Yokohama Rubber Co Ltd:The | 乗用車用ラジアルタイヤ |
JPH0746104A (ja) | 1994-02-04 | 1995-02-14 | Kawasaki Steel Corp | プログラマブル入力回路 |
JPH11349600A (ja) * | 1998-06-02 | 1999-12-21 | Teikoku Hormone Mfg Co Ltd | モノクローナル抗体及びfdpの測定方法 |
JP3472138B2 (ja) | 1998-06-02 | 2003-12-02 | 帝国臓器製薬株式会社 | モノクローナル抗体及びfdpの測定方法 |
JP2001021557A (ja) * | 1999-07-12 | 2001-01-26 | Iatron Lab Inc | Dダイマー及びdd/e複合体の免疫学的分析方法及び分析用キット |
JP2001354700A (ja) | 2000-06-12 | 2001-12-25 | Bio Links Kk | モノクロ−ナル抗体及びヒトフィブリン分解産物の測定方法 |
JP2002372536A (ja) | 2001-06-15 | 2002-12-26 | Dai Ichi Pure Chem Co Ltd | 免疫学的測定法 |
JP2006105633A (ja) * | 2004-09-30 | 2006-04-20 | Sysmex Corp | Dダイマー測定用試薬およびこれに用いるモノクローナル抗体 |
Non-Patent Citations (3)
Title |
---|
KOHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497 |
NAOKO TODA: "Kiki Shiyaku Seminar B(l) Kesshochu FDP Sokutei Shiyaku 'Nanopia P-FDP' ni Tsuite", JAPANESE JOURNAL OF CLINICAL LABORATORY AUTOMATION, vol. 27, no. 4, 2002, pages 327 * |
See also references of EP2599865A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104458367A (zh) * | 2014-11-06 | 2015-03-25 | 上海长岛生物技术有限公司 | 一种d-二聚体和fdp复合质控品及其制备方法 |
CN104458367B (zh) * | 2014-11-06 | 2019-03-26 | 上海长岛生物技术有限公司 | 一种d-二聚体和fdp复合质控品及其制备方法 |
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CN103025871A (zh) | 2013-04-03 |
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CN103025871B (zh) | 2016-05-18 |
US8865424B2 (en) | 2014-10-21 |
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EP2599865A4 (en) | 2014-03-05 |
JPWO2012014997A1 (ja) | 2013-09-12 |
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