WO1994004700A1 - Monoclonal antibody recognizing fk506-binding protein, method of assaying fk506-binding protein level, and kit thereof - Google Patents
Monoclonal antibody recognizing fk506-binding protein, method of assaying fk506-binding protein level, and kit thereof Download PDFInfo
- Publication number
- WO1994004700A1 WO1994004700A1 PCT/JP1993/001136 JP9301136W WO9404700A1 WO 1994004700 A1 WO1994004700 A1 WO 1994004700A1 JP 9301136 W JP9301136 W JP 9301136W WO 9404700 A1 WO9404700 A1 WO 9404700A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fkbp
- binding protein
- antibody
- monoclonal antibody
- enzyme
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- 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
-
- 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
-
- 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
Definitions
- the present invention relates to a monoclonal antibody recognizing a protein that binds to FK506 having immunosuppressive activity (FK506 binding protein), a method for measuring the concentration of the FK506 binding protein, and a kit for measuring the concentration. And can be used in the medical field.
- FK506 or FR-905056 have potent immunosuppressive effects and can often be used as prophylactic or therapeutic agents for rejection and autoimmune diseases during organ transplantation. It is known (for example, Japanese Patent Application Laid-Open No. 61-14881). However, its action is so powerful that determining the optimal dose is an important issue.It is important to administer an amount that can exert an effective immunosuppressive activity without causing side effects. Very important.
- FKBP intracellular FK506-binding protein
- FKBP-12 molecular weight 12KDa
- FKBP-13 molecular weight 13KDa
- FKBP-25 molecular weight 25KDa
- FKBP-56 molecular weight 56KDa
- FKBP-80 molecular weight 8OKDa
- FK506 binds most strongly to FKBP-112, and its affinity constant (Kd) is 0.4 nM.
- Kd affinity constant
- the inventors of the present invention have succeeded in producing a monoclonal antibody that recognizes the antigenic determinant of FKBP and does not inhibit the binding of FKBP to FK506, A method for accurately measuring the concentration of medium FKBP and a kit for measurement were established.
- the present invention provides a monoclonal antibody that recognizes an antigenic determinant in FKBP and does not inhibit the binding between FKBP and FK506,
- An FKBP concentration measurement method comprising reacting FK506 labeled with an enzyme with FKBP in a test sample with a clonal antibody, and reacting FK506 labeled with an enzyme with FKBP in a test sample with immobilized FKBP
- a kit for measuring the concentration of FKBP comprising the monoclonal antibody, FKBP, and FK506 labeled with an enzyme.
- Anti-FKBP antibodies Monoclonal antibodies that recognize the antigenic determinant of FKBP and do not inhibit the binding of FKBP to FK506 (hereinafter referred to as anti-FKBP antibodies) include, for example, Kohler and Milstein (Kohler and Mistein). )) Can be produced by a conventional cell fusion method such as CN Nature, 256, 495 (1975)].
- a spleen cell obtained from a mouse immunized with FKBP and a mouse myeloma cell are subjected to cell fusion to produce a hybridoma, and a hybridoma is prepared from the hybridoma as described in Example 1 or Example 2 below.
- a monoclonal antibody that recognizes FKBP but does not inhibit the binding between FKBP and FK506 can be prepared. More preferably, it is a class of IgG or IgM, and most preferably, a subclass such as IgG, ⁇ or IgG, /.
- an anti-FKBP antibody which specifically reacts with only 12 kd FKBP or reacts with both 12 kd and 30 to 35 kd FKBP is obtained.
- the FKBP concentration measurement method in the present invention is a two-step method of selectively measuring only the target FKBP using an anti-FKBP antibody, and a one-step method of non-selectively measuring all FKBPs to which FK506 binds.
- the law can be broadly divided.
- an anti-FKBP antibody that recognizes an antigenic determinant in the target type of FKBP but does not affect the binding between FKBP and FK506 is immobilized like imnolate.
- the phase is immobilized using an IgG such as anti-mouse IgG (H + L), 2) and a test sample containing FKBP (a serially diluted FKBP when preparing a calibration curve) and peroxidase (hereinafter FK506 labeled with an enzyme such as POD [denoted as POD] 3)
- FK506 labeled with an enzyme such as POD [denoted as POD]
- o-Fdylenediamine and FKBP bound to anti-FKBP antibody using an enzyme substrate solution such as hydrogen peroxide solution are reacted with the prepared FK506-C32 (LA) -POD).
- the FKBP concentration can be determined by measuring the degree of color development of the substrate observed in accordance with the amount of the enzyme-labele
- FKBP such as FKBP-12 is immobilized on a solid phase such as immnobrate
- a test sample containing FKBP (when preparing a calibration curve, (Stepwise diluted FKBP) and FK506-C32 (LA) -react with enzyme-labeled FK506, such as POD.3) Perform a competitive reaction between FKBP in the test sample and immobilized FKBP.
- the test was performed by measuring the degree to which the enzyme-labeled FK506 captured by the converted FKBP was reduced in accordance with the amount of FKBP in the test sample using an enzyme substrate solution such as the two-step method. You can know the FKBP concentration in the sample.
- the anti-FKBP antibody of the present invention can be used as a FKBP concentration measurement kit consisting of the anti-FKBP antibody, an FKBP standard product, and enzyme-labeled FK506, and subjected to the concentration measurement by the above two-step method.
- the concentration of only a specific FKBP (for example, FKBP-12) can be measured selectively, and the total concentration of all FKBPs can be measured. It is now possible to fine-tune the dose of FK506 according to the condition of each patient.
- FKBP-12 FKBP-12 Phosphate buffer hereinafter referred to as PBS 250 ⁇ .
- G / m 1 0.1 ml and an equal volume of Freimd's Complete Adjuvant (FCA) were mixed and immunized intraperitoneally into mouse BALB / c did.
- FCA Freimd's Complete Adjuvant
- the same amount of FKBP-12 was mixed with Freund's Incomplete Adjuvant (F [A]) and immunized intraperitoneally several times approximately every 10 days.
- 101 blood was collected from the tail vein of the mouse, mixed with PBS 990 ⁇ 1, and used as a measurement sample.
- anti-mouse IgG (H + L) -POD [Vector (Vector Lab.)] 1001 / well is added, and the mixture is further reacted at room temperature for 1 hour. After washing, the color was developed with a 0-phenylenediamine system by a conventional method.
- mice showing an increase in antibody titer were further injected with 0.2 ml of FKBP-125 Ug / ml (PBS) through the tail vein as a final immunization.
- PBS FKBP-125 Ug / ml
- the spleen was extracted, and spleen cells (1.44 ⁇ 10 cells) were prepared.
- Aqueous solution containing 0.08% trifluoroacetic acid of FKBP-12 (847 it g / m 1) 460 ⁇ 1 was added with 12 mg of urea, and mixed and stirred at room temperature to give FKBP-12 (600 / gZm l ) Was prepared. This was used for immunization as it was.
- FKBP- 1 2 1 032 ig (11118/13 1 used 10 32 1) and lobulin (Sigma, L 0 t No. 76 F—8040) in PBS (2 mg / m 1) 1 032 1 Mix.
- PBS 2 mg / m 1
- 0.62 ml of a 0.13 M glutaraldehyde-P83 solution is added dropwise. The mixture was stirred at room temperature for 14 hours, then dialyzed against PBS 11 three times, and used as an immunogen.
- the mixing ratio of FKBP-12 and ovalbumin was FKBP-121032 ⁇ g: 2064 g of ovalbumin, and the solution volume was 2.7 ml.
- Antiserum diluent ⁇ Then, the plate was washed three times with a 0.05% Tween 20 / PBS solution. Add 100 ⁇ l of an anti-mouse IgG (H + L) solution diluted with 100% dilution with 0.2% milk / 0.05% Tween 20 / PBS solution to each well, add 1001 to each well, and add room temperature For 2 hours.
- the anti-mouse IgG (H + L) solution labeled with ALPHA's alkaline phosphatase was removed by suction and washed five times with a 0.05% Tween 20ZPBS solution. Then, 0. ImM 4-methylumbelliferyl phosphate: buffer (.
- hybridoma 2C 1-87 obtained by screening and cloning, or hybridoma 3F4-70 was inoculated into each F75 flask so as to have 4 ml of 5xlO ml (50mlZF75). After culturing for one day, the culture was centrifuged, washed twice with a serum-free medium, and then suspended in 0.5 ml each. Two weeks before, 0.5 ml of Hypridoma suspension was transplanted into the abdominal cavity of a BALBZC mouse (BALBZC, flat, 6-week-old) injected with 0.5 ml of pristane intraperitoneally.
- BALBZC mouse BALBZC, flat, 6-week-old
- mice were laparotomized, and 5 ml of ascites was obtained from each mouse.
- Each ascites was purified using the Affigel Protein A MAPS-II kit (Bio-Rad), and the anti-FKBP-12 monoclonal antibody 2C1-87 (subtype: IgG, ⁇ ) and 3F4-70 (Sub-Eve: IgG, ⁇ ) A purified antibody was obtained.
- an anti-FKBP-12 monoclonal antibody 31-3-D4-C6 (subtype: IgM) was obtained.
- Example 3 Measurement of FKBP-12 concentration (two-step method)
- FKBP- 12 (501 / ⁇ ⁇ ) serially diluted with 0.05% Tween 20-PBS was added at room temperature for 2.5 hours. Let react. After washing with 0.05% Tween 20-PBS three times, add FK506-C32 (LA) -POD solution (501Zell) diluted in 500-fold PBS and leave at room temperature for 2 hours. . After washing 5 times with 0.05% Tween 20-PBS, color is developed with o-diphenylamine Z hydrogen peroxide solution according to a conventional method, and the degree of color change that changes according to the amount of FKBP-12 is measured. .
- the FKBP-12 throat was about 10 OngZml.
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Description
Claims
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU47613/93A AU667743B2 (en) | 1992-08-12 | 1993-08-11 | Monoclonal antibody recognizing FK506-binding protein, method of assaying FK506-binding protein level, and kit thereof |
DK94908103T DK0672756T3 (da) | 1992-08-12 | 1993-08-11 | Monoklonalt antistof, som genkender FK506-bindende protein, fremgangsmåde til måling af niveauet af FK506-bindende protein og kit dertil |
EP94908103A EP0672756B1 (en) | 1992-08-12 | 1993-08-11 | Monoclonal antibody recognizing fk506-binding protein, method of assaying fk506-binding protein level, and kit thereof |
KR1019950700510A KR100243536B1 (ko) | 1992-08-12 | 1993-08-11 | Fk506-결합 단백질을 인식하는 모노클로날 항체, fk506-결합 단백질의 농도를 분석하는 방법 및 이를 위한 키트 |
JP50610194A JP3185221B2 (ja) | 1992-08-12 | 1993-08-11 | Fk506結合蛋白質を認識するモノクローナル抗体,fk506結合蛋白質の濃度測定法,および測定用キット |
DE69331585T DE69331585T2 (de) | 1992-08-12 | 1993-08-11 | Monoklonaler antikörper spezifisch für das fk-506-bindende protein, methode zur bestimmung des gehaltes an fk-506-bindendem protein in einer probe und reagenzsatz dafür |
US08/379,563 US5576183A (en) | 1992-08-12 | 1993-08-11 | Monoclonal antibody recognizing FK506-binding protein, method for assaying FK506-binding protein level, and kit therefor |
CA002142197A CA2142197C (en) | 1992-08-12 | 1993-08-11 | Monoclonal antibody recognizing fk506-binding protein method for assaying fk506-binding protein level, and kit therefor |
AT94908103T ATE213272T1 (de) | 1992-08-12 | 1993-08-11 | Monoklonaler antikörper spezifisch für das fk-506-bindende protein, methode zur bestimmung des gehaltes an fk-506-bindendem protein in einer probe und reagenzsatz dafür |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4/214967 | 1992-08-12 | ||
JP21496792 | 1992-08-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1994004700A1 true WO1994004700A1 (en) | 1994-03-03 |
Family
ID=16664530
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1993/001136 WO1994004700A1 (en) | 1992-08-12 | 1993-08-11 | Monoclonal antibody recognizing fk506-binding protein, method of assaying fk506-binding protein level, and kit thereof |
Country Status (12)
Country | Link |
---|---|
US (1) | US5576183A (ja) |
EP (1) | EP0672756B1 (ja) |
JP (1) | JP3185221B2 (ja) |
KR (1) | KR100243536B1 (ja) |
AT (1) | ATE213272T1 (ja) |
AU (1) | AU667743B2 (ja) |
CA (1) | CA2142197C (ja) |
DE (1) | DE69331585T2 (ja) |
DK (1) | DK0672756T3 (ja) |
ES (1) | ES2169068T3 (ja) |
PT (1) | PT672756E (ja) |
WO (1) | WO1994004700A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996018102A1 (fr) * | 1994-12-07 | 1996-06-13 | Fujisawa Pharmaceutical Co., Ltd. | Methode de mesure de la concentration en proteines de fixation de fk506 |
WO2001049738A1 (fr) * | 1999-12-29 | 2001-07-12 | Fudan University | Nouveau polypeptide, proteine fkbp humaine 11, et polynucleotide codant pour ce polypeptide |
US7078495B1 (en) * | 1999-08-03 | 2006-07-18 | Dade Behring Inc. | Monoclonal antibodies to tacrolimus and immunoassay methods for tacrolimus |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999035160A1 (en) * | 1998-01-09 | 1999-07-15 | Human Genome Sciences, Inc. | Human fk506 binding proteins |
US6492325B1 (en) * | 1998-05-22 | 2002-12-10 | Boys Town National Research Hospital | Use of α1β1 integrin receptor inhibitors and TGF-β1 inhibitors in the treatment of kidney disease |
US7186518B2 (en) * | 2003-11-21 | 2007-03-06 | Dade Behring Inc. | Method and composition useful for determining FK 506 |
WO2006108130A2 (en) * | 2005-04-06 | 2006-10-12 | Abbott Laboratories | Methods to measure immunosuppressive tacrolimus, sirolimus, and cyclosporin a complexes in a blood sample |
KR101158362B1 (ko) | 2011-04-20 | 2012-06-22 | 한국과학기술원 | 세포 환경 내에서의 단일 분자 수준의 단백질-단백질 상호작용 분석 방법 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4894366A (en) * | 1984-12-03 | 1990-01-16 | Fujisawa Pharmaceutical Company, Ltd. | Tricyclo compounds, a process for their production and a pharmaceutical composition containing the same |
WO1993007269A1 (en) * | 1991-10-11 | 1993-04-15 | Vertex Pharmaceuticals Incorporated | ISOLATION OF AN Mr 52,000 FK506 BINDING PROTEIN AND MOLECULAR CLONING OF A CORRESPONDING HUMAN cDNA |
-
1993
- 1993-08-11 CA CA002142197A patent/CA2142197C/en not_active Expired - Fee Related
- 1993-08-11 KR KR1019950700510A patent/KR100243536B1/ko not_active IP Right Cessation
- 1993-08-11 US US08/379,563 patent/US5576183A/en not_active Expired - Fee Related
- 1993-08-11 DE DE69331585T patent/DE69331585T2/de not_active Expired - Fee Related
- 1993-08-11 JP JP50610194A patent/JP3185221B2/ja not_active Expired - Fee Related
- 1993-08-11 DK DK94908103T patent/DK0672756T3/da active
- 1993-08-11 EP EP94908103A patent/EP0672756B1/en not_active Expired - Lifetime
- 1993-08-11 AT AT94908103T patent/ATE213272T1/de not_active IP Right Cessation
- 1993-08-11 ES ES94908103T patent/ES2169068T3/es not_active Expired - Lifetime
- 1993-08-11 AU AU47613/93A patent/AU667743B2/en not_active Ceased
- 1993-08-11 PT PT94908103T patent/PT672756E/pt unknown
- 1993-08-11 WO PCT/JP1993/001136 patent/WO1994004700A1/ja active IP Right Grant
Non-Patent Citations (3)
Title |
---|
Journal of Biological Chemistry, Vol. 267, No. 14, (1992), T. JAYARAMAN et al., "FK506 Binding Protein Associated with the Calcium Release Channel (Ryanodine Receptor)", p. 9474-9477. * |
Nature, Vol. 346, (1990), R.F. STANDAERT et al., "Molecular Cloning and Overexpression of the Human FK506-Binding Protein, FKBP", p. 671-674. * |
Transplantation Proceedings, Vol. 23, No. 6, (1991), S.L. ROSBOROUGH et al., "Identification of FKBP-Related Protein with Antibodies of Predetermined Specificity and Isolation by FK506 Affinity Chromatography", p. 2890-2893. * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996018102A1 (fr) * | 1994-12-07 | 1996-06-13 | Fujisawa Pharmaceutical Co., Ltd. | Methode de mesure de la concentration en proteines de fixation de fk506 |
US7078495B1 (en) * | 1999-08-03 | 2006-07-18 | Dade Behring Inc. | Monoclonal antibodies to tacrolimus and immunoassay methods for tacrolimus |
US8030458B2 (en) | 1999-08-03 | 2011-10-04 | Siemens Healthcare Diagnostics Inc. | Monoclonal antibodies to tacrolimus and immunoassays methods for tacrolimus |
WO2001049738A1 (fr) * | 1999-12-29 | 2001-07-12 | Fudan University | Nouveau polypeptide, proteine fkbp humaine 11, et polynucleotide codant pour ce polypeptide |
Also Published As
Publication number | Publication date |
---|---|
ATE213272T1 (de) | 2002-02-15 |
DK0672756T3 (da) | 2002-03-25 |
AU667743B2 (en) | 1996-04-04 |
DE69331585D1 (de) | 2002-03-21 |
KR950703061A (ko) | 1995-08-23 |
CA2142197C (en) | 2004-06-22 |
AU4761393A (en) | 1994-03-15 |
ES2169068T3 (es) | 2002-07-01 |
EP0672756B1 (en) | 2002-02-13 |
PT672756E (pt) | 2002-07-31 |
KR100243536B1 (ko) | 2000-02-01 |
CA2142197A1 (en) | 1994-03-03 |
US5576183A (en) | 1996-11-19 |
DE69331585T2 (de) | 2002-08-08 |
JP3185221B2 (ja) | 2001-07-09 |
EP0672756A4 (en) | 1996-10-09 |
EP0672756A1 (en) | 1995-09-20 |
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