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WO1996018102A1 - Methode de mesure de la concentration en proteines de fixation de fk506 - Google Patents

Methode de mesure de la concentration en proteines de fixation de fk506 Download PDF

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Publication number
WO1996018102A1
WO1996018102A1 PCT/JP1995/002427 JP9502427W WO9618102A1 WO 1996018102 A1 WO1996018102 A1 WO 1996018102A1 JP 9502427 W JP9502427 W JP 9502427W WO 9618102 A1 WO9618102 A1 WO 9618102A1
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WO
WIPO (PCT)
Prior art keywords
antibody
fkbp
binding protein
enzyme
concentration
Prior art date
Application number
PCT/JP1995/002427
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English (en)
Japanese (ja)
Inventor
Masakazu Kobayashi
Kazuyuki Ohtsuka
Original Assignee
Fujisawa Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fujisawa Pharmaceutical Co., Ltd. filed Critical Fujisawa Pharmaceutical Co., Ltd.
Priority to AU39936/95A priority Critical patent/AU3993695A/en
Publication of WO1996018102A1 publication Critical patent/WO1996018102A1/fr

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Classifications

    • 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 method for detecting or measuring the concentration of FK506-binding protein, and a kit for detecting or measuring the concentration of FK506, and can be used in the medical field.
  • FK506 or FR-900506 have potent immunosuppressive effects and are used as prophylactic or therapeutic agents for organ transplant rejection and autoimmune diseases. This is well known (for example, JP-A-61-148181).
  • FKBP binding protein
  • FKBP-12 molecular weight 12KDa
  • FKBP-13 molecular weight 13KDa
  • FKBP -25 molecule ⁇ 25KDa
  • FKBP-52 molecular weight 56KDa
  • FKBP which has a molecular weight of 11,819 daltons and is composed of 107 amino acids
  • FKBP-12 which has an affinity constant (Kd) of 0.4 ⁇
  • Kd affinity constant
  • FKBP-12 when FKBP-12 is added together with a certain amount of FK506 in a mouse MLR system, the immunosuppressive effect of FK506 is inhibited in proportion to the amount of FKBP-12 added. Therefore, if FKBP-12 is present in the blood, it will be bound to FKBP-12 in the FK506 blood plasma, and its immunosuppressive effect will be as expected from its plasma concentration. , It will not be obtained. It is considered that the patient's red blood cells are lysed after surgery, etc., and that the red blood cells FKBP-12 circulates in the plasma, and that there is a difference in the sensitivity of the patient to FK506. For example, to estimate the FK506 sensitivity of a patient and to set a more desirable FK506 plasma concentration, There is a need for a technique for accurately measuring the FKBP concentration.
  • International Publication No. 94Z04700 discloses two methods for measuring FKBP concentration.
  • One is to immobilize an anti-FKBP antibody that recognizes an antigenic determinant in FKBP but does not affect the binding of FKBP to FK506, and (1) Reacting FKBP in a test sample, then reacting (2) FK506 labeled with an enzyme, and further (3) measuring the enzyme activity.
  • the other is (1) the FKBP immobilized by allowing (1) the test sample and FK506 labeled with an enzyme to act on the immobilized FKBP and the FKBP immobilized on the test sample. And (2) measuring the enzymatic activity of the enzyme label FK506 captured by the immobilized FKBP. It has been disclosed.
  • the FKBP concentration in the test sample in which FK506 coexists is determined by using the binding ability of FKBP to FK506 and quantifying the FKBP portability. It was difficult to measure accurately.
  • the inventors of the present invention have been able to detect FKBP in a test sample such as serum or plasma using a monoclonal antibody that recognizes an antigenic determinant of FKBP. Is a method for accurately measuring its concentration, and for detecting or measuring its concentration Established kit.
  • an object of the present invention is to provide an enzyme-linked immunosorbent assay for detecting FKBP in a test sample or accurately measuring the concentration thereof.
  • Another object of the present invention is to provide a measuring kit for measuring the concentration.
  • a first antibody (a monoclonal antibody that recognizes an antigenic determinant in FKBP) immobilized on a solid phase;
  • Second antibody (a monoclonal antibody that recognizes an antigenic determinant in FKBP but recognizes a different antigenic determinant from the first antibody)
  • the first antibody and the second antibody that recognize the antigenic determinant in FKBP can be obtained, for example, by the basic method of Kohler and Minorestein [ature, 256.495 (1975)]. Can be produced by a conventional cell fusion method such as that described above.
  • a hybridoma is produced by fusing cell spleen cells obtained from a mouse immunized with FKBP with mouse myeloma cells, and a hybridoma is produced from the hybridoma as described below.
  • a monoclonal antibody recognizing FKBP can be prepared by the method described in Example 1 or Production Example 2. More preferably, 0 ⁇ ⁇ 1 ⁇ Ri class der, most rather than the good or is, 186 1 scan Ya 18. It is a subclass that is one of the best. According to the method described in the present specification, it is possible to obtain an anti-FKBP monoclonal antibody which reacts only with FKBP of 12 KDa alone or with both 12 Kd and 30 to 35 Kd FKBP. It is. The method is described in International Publication WO 94Z04700 and Transpl. Proc. 25.655-657 (1993).
  • Particularly preferred monoclonal antibodies for the first antibody and the second antibody are not affected even when FKBP is bound to FK506.
  • Monoclonal antibodies that recognize different antigenic determinants in FKBP can be mentioned.
  • second antibody in the present invention refers to a monoclonal antibody that recognizes an antigenic determinant in FKBP different from the first antibody as described above. In addition, it also means the above-mentioned monoclonal antibody having an appropriate label used in the detection or quantification of the complex.
  • the label in the “second antibody having a label” according to the present invention may be any as long as its presence can be detected.
  • an enzyme may be used.
  • Compounds that have an affinity for a certain protein for example, piotin
  • tertiary antibodies antibodies that recognize second antibodies
  • radioisotopes fluorescent substances, luminescent substances, etc. No.
  • carbohydrase eg, glucose Sidases (eg, -galactosidase, ⁇ -gluconosidase,> 5-glucuronidase, 5-phenolectosidase, or-galactosidase, ⁇ -gnorecosidase Sigma, sigma-mannosidase), amylase (eg, ⁇ -amylase,> 8—amylase, iso-amylase, Darco Lase, Takaamilase A), senorelase, lysozyme, etc.), (2) amidase (eg, perlase, asparaginase), (3) S Thelase [e.g., colistellase (e.g., acetate colonase)], phosphatase (e.g., e.g.
  • Nuclease eg, deoxyribonuclease, ribonuclease
  • iron porphyrin enzyme eg, lipase, peroxydidase, cytochrome oxidase
  • copper enzyme eg,
  • Piotin a compound known as vitamin H, has an extremely high affinity for avidin, a basic protein found in egg white. ing. It is known that an avidin is composed of four subunits. Gins also include these subunits and avidin labeled with the above-mentioned enzymes (preferably, phenolic phosphatase).
  • Examples of the third antibody which is an antibody that recognizes the second antibody, include anti-immunoglobulin such as an anti-mouse lambda chain antibody and an anti-mouse copper chain antibody.
  • Anti-immunoglobulin such as an anti-mouse lambda chain antibody and an anti-mouse copper chain antibody.
  • Blind antibodies, etc. which may have the above-mentioned label, especially those having an anti-mouse having an enzyme (alli phosphatase).
  • Lambda chain antibodies are preferred.
  • radioisotopes for example, 125 I, 131 I, 3 H,
  • Fluorescent substances such as fluorescamine and fluorescein isothiocyanate are luminous substances, and luminophores and luminomines are luminous substances. Examples include monoleic derivatives, noresiferin, and noresigenin.
  • a known method for binding the antibody to the label can be used. Loramin T method, periodic acid method, maleimide method, etc. are used. More specifically, the method of the embodiment described later can be used.
  • the FK506-binding protein (FKBP) in the present invention means FKBP-12, FKBP-13, FKBP-25, FKBP-52 and the like as described above.
  • FKBP-12 the strongest binding of FK506 is FKBP-12, and FKBP-12 is abundant in all tissues. Considering this fact, FKBP-12 is particularly preferred. In this case, the first antibody and the second antibody are not affected at all even if FKBP-12 binds to FK506, and are different from each other in FKBP-12. Monoclonal antibodies that recognize antigenic determinants.
  • various conventional methods can be used for quantifying the complex consisting of the first antibody, FKBP and the second antibody.
  • the following methods can be considered.
  • the second antibody is labeled with an appropriate enzyme in advance, and after forming the complex, an appropriate enzyme-substrate reaction is performed according to the enzyme.
  • C or an enzyme-labeled antibody that recognizes the second antibody in advance (for example, an alkaline phosphatase-labeled anti-mouse lambda chain antibody)
  • an enzyme-labeled antibody that recognizes the second antibody in advance for example, an alkaline phosphatase-labeled anti-mouse lambda chain antibody
  • the substrate used in the enzyme-substrate reaction can be selected according to the type of the enzyme. For example, 4- Preferable ability to use the reference, phosphate, chromagen, 0-fuel range, etc. Or, it is 4-methino-rember ferrino-phosphate.
  • the amount of change in the substrate generated by the enzyme-substrate reaction reflecting the amount of the formed complex is measured by a conventional method as absorbance or fluorescence intensity.
  • test sample is human serum, plasma, or tissue exudates
  • sodium ethylenediaminetetraacetate (EDTA.2Na) and sodium citrate are used.
  • EDTA.2Na sodium ethylenediaminetetraacetate
  • sodium citrate sodium citrate
  • the method for immobilizing the first antibody on the solid phase is carried out by a conventional method such as leaving the first antibody together with the solid phase for an appropriate time.
  • a conventional one can be used as long as it can immobilize an antibody such as a monoclonal antibody and can easily perform the subsequent separation work from the reaction solution.
  • an immoplate preferably.
  • the reaction in forming a complex between the first antibody and the FKBP in the test sample or further with the second antibody in the present invention was suitable for ordinary antigen-antibody reactions.
  • the reaction may be carried out under conditions, and preferably, shaking at room temperature for several hours.
  • a more specific procedure for detecting FKBP or measuring the concentration is performed in the same manner as in the examples described later, except that a complex is formed.
  • the order of addition of the first antibody, FKBP and the second antibody at the time of the addition is not limited to the order of Examples described later.
  • a monoclonal antibody capable of recognizing an antigenic determinant in FKBP without being affected by FKBP binding to FK506 (for example, see Examples below).
  • the anti-FKBP-12 monoclonal antibody 2C1-87 and the anti-FKBP-12 monoclonal antibody 3F4-70) used in the above were used as the first antibody and the second antibody, respectively.
  • FKBP can be accurately and conveniently detected or removed from serum or plasma of patients who have already received FK506 without first removing FK506. It is particularly useful because it can measure the concentration of
  • Production Example 1 Production of anti-FKBP antibody
  • the expression vector PFKBP333 was obtained by incorporation into a plasmid expressed under the control of a tryptophan promotor. This was transformed into E.coliHB101 to obtain an expressing bacterium HB101Z pFKBP (AT) 311. This is cultured in L-amp. Broth for 19 hours, and protein synthesis is induced by adding IAA (Indol — Acrylic acid) to a concentration of 90 g / ml (final concentration). Here I went. The E.
  • FKBP-12 was purified by dialysis [20mM Tris-HC1 (pH 7.4), 4 ° C overnight] -DEAE-Toyo PEARL 650M-reverse phase HPLC (C4).
  • PBS Phosphate buffer solution of FKBP-12
  • FCA Freund's Comlete Adjuvant
  • mice in which the antibody titer was increased were further injected with 0.2 ml of FKBP-12 250 ⁇ g / ml (PBS) via the tail vein as a final immunization.
  • PBS FKBP-12 250 ⁇ g / ml
  • the spleen was extracted, and spleen cells were prepared at 1.44 ⁇ 10 8 cels for 1 s.
  • mouse myeloma cells P3X63Ag8U.1 were adjusted to 2.9 ⁇ 10 7 cells and cell fusion was performed in 50% PEG4000 (final concentration). After that, the fused cells were screened with 10 6 cells Zmlximl in a HAT medium on a 24 ⁇ l plate.
  • the FKBP-12501 was obtained using the following (6).
  • FK506-C33 (LA) -POD (1000-fold dilution) 501 was coexisted.
  • FKBP-12 bound by anti-mouse IgG (H + L) was converted to FK506-C32 (LA) due to the disappearance of color development when FK506 of lOyu gZral was coexisted.
  • Mono-succinate FR-900506 substance Half Esthenol (230 mg) obtained in the same manner as in Example 1 of JP-A-11-92659, N-hydroxysuccinimide ( 35 mg) and 1-ethylenol 3 — (3-dimethylaminoaminopropyl) canolebodiimid hydrochloride (43 mg) in methylene chloride (10 ml) were added at room temperature. After stirring for lower 5 hours, the reaction mixture was washed with water and further dried. After distilling off the solvent, the residue obtained (250 mg) was diluted with 11-aminomindecanoic acid (120 rag) and triethylenamine (60 mg).
  • the mixing ratio of FKBP-12 and ovovanorebmin was 1032 ⁇ g of FKBP-12: 2064 g of ovoalbumin, and the solution volume was 2.7 ml.
  • mice were immunized as described in (1) and (2) of Production Example 1.
  • FKBP-12 PBS solution 501 20 ⁇ gZml of FKBP-12 PBS solution 501 was dispensed into each well of a 96-well plate for ELISA, and left at ⁇ 4 ° C.
  • the FKBP-12 solution of phenol was removed by sucking I and washed three times with a 0.05% Tween20Z PBS solution.
  • To each well of the plate was added 250% of 0.2% MINOLEC / PBS solution 2501, and the mixture was allowed to stand at room temperature for 30 minutes.
  • Aspirate 0.2% of PBS solution of PBS and aspirate add each antiserum diluted with 0.2% of Z-0.05% Tween20Z PBS solution to ⁇ ⁇ , and add at room temperature. Left for 2 hours.
  • Hybridomas 2C 1-87 obtained from screening and cloning, some hybridomas 3F4-70, each with F75 Seed the cells at 5 x 10 4 cells / "ml (50 ml F75) into Lasco, culture for 4 days, centrifuge the culture, wash twice with serum-free medium, Two weeks ago, 0.5 ml of Pristane was injected intraperitoneally into the abdominal cavity of a BALB mouse C (BALB / C, female, 6-week-old) injected intraperitoneally. After transplantation of 0.5 ml of hybridoma suspension, 10 days later, the mouse was laparotomized and 5 ml of ascites was obtained from each mouse.
  • Monoclonal antibody 2C1-87 or 3F4-70 in PBS (5 ug / ml) was added to each plate (50 each) for ELISA plate (Maxisorp F96, Dispense into each of the kits (manufactured by Nippon Oil Co., Ltd.), and let it stand still with 4. Wash the plate three times with 0.05% Tween 20 in PBS.
  • the enzyme substrate solution is prepared immediately before use.
  • Table 1 shows the results of the above measurement methods.
  • Monoclonal antibody 2C1-187 or 3F4-70 in PBS solution (S / ugZml) 50 / il was added to each plate for ELISA (Maxisorp F). 96, Nunc Co., Ltd.). Leave at C. Wash the plate three times with 0.05% Tween 20 in PBS.
  • a 1000-fold diluted force-phosphatase-labeled avidin solution (manufactured by Vector, Code A-2100) was applied to each plate of the above plate. Dispense and incubate at room temperature for 30 minutes while shaking with a plate mixer. Wash the plate 7 times with 0.05% Tween 20 in PBS.
  • Monoclonal antibody 3 F4-170 in PBS (5 ⁇ g / m1) (50 each) was used as a plate for ELISA (Maxisorp F 96. ) Dispense into each of the tubes in, and leave them at 4 ° C. Wash the plate three times with 0.05% Tween 20 in PBS.
  • aqueous solution of 4% block ace (described above) is dispensed at 250 i into each of the above plates, left at room temperature for 30 minutes, and the plate is then diluted with a 0.05% Tween 20 PBS solution. Wash once.
  • Monoclonal antibody 2 C 1 — 87 is diluted with a 1% block-acetate 0.1% Tween 20 aqueous solution to a concentration of 5 ⁇ gZml and 5,000 Make sure that the enzyme is diluted with a double-diffusion anti-mouse lambda chain antibody solution (Susan Biotechnology, Inc., code 1060). — 04) and leave for 2 hours. ) Binding antibody reaction
  • the plate is washed 7 times with a 0.05% zinc 20 PBS solution, and the conjugated antibody prepared in 4) is dispensed in 100 1 portions to each plate of the above plate, and the plate is mixed with a plate mixer. Leave for 2 hours at room temperature with shaking. Wash the plate seven times with 0.05% Tween 20 in PBS.
  • the site flow (trade name, trade name) was prepared by adding a 4% block ace / 0.1% tween 20 aqueous solution instead of the standard FKBP-12 solution. Measure the fluorescence intensity (Ex. 360 nm, Em. 460 nm) using a micropore (Millipore).
  • Table 3 shows the results of the above measurement methods.
  • Table 3 Enzyme-linked immunosorbent assay for FKBP-12
  • Example 2 In each plate of the plate after the specific adsorption prevention operation was completed in 4) of Example 2, 50/1 0.4% ethylenediamine tetrasodium nitrate was added to each well. , Dihydrate (EDTA-2Na), 4% block ace, and aqueous solution containing 0.1% Tween 20 (hereinafter referred to as Atsushi buffer). After that, FKBP-12 diluted with an aqueous solution of 4% block ace and 0.1% Tween 20 (hereinafter referred to as Atsushi diluent) was used as a standard solution. Bring human serum (control) or FKBP-12 to 25ngZml Add 50 ⁇ l of the human serum added to the above. The concentration of added FKBP-12 in serum was calculated from the standard curve. Table 4 shows the results.
  • Example 5 Measurement of blood FKBP-12 concentration in patients with different blood sampling methods Blood samples were collected from patients (A, B, C) with different pathological conditions and blood FKBP-12 levels were measured. did. In Example 3-4), after adding 501 atseno and * buffers to each of the plates of the plate for which the specific adsorption prevention operation has been completed, Standard FKBP-12 diluted with Hesse diluent or serum collected from the same patient, heparin blood plasma, and EDTA-Na blood plasma tripled with Atsey diluent. 50 ⁇ l of the diluted sample was added to each sample, and the measurement was performed according to Example 3. The concentration of FKBP-12 in the patient's serum was calculated from the standard curve. Table 5 shows the results. Table 5. Measurement of FKBP-12 concentration in blood using different blood collection methods

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Abstract

Méthode immunoenzymatique permettant de détecter les protéines de fixation de FK506 ou de mesurer leur concentration par la détection ou la quantification d'un complexe comprenant une protéine de fixation de FK506 et les premier et second anticorps reconnaissant respectivement les différents déterminants antigéniques de la protéine.
PCT/JP1995/002427 1994-12-07 1995-11-29 Methode de mesure de la concentration en proteines de fixation de fk506 WO1996018102A1 (fr)

Priority Applications (1)

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AU39936/95A AU3993695A (en) 1994-12-07 1995-11-29 Method of measuring the concentration of fk506-binding protein

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JP30339594 1994-12-07
JP6/303395 1994-12-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6709873B1 (en) 1997-04-09 2004-03-23 Isodiagnostika Inc. Method for production of antibodies to specific sites of rapamycin
JP2008536126A (ja) * 2005-04-06 2008-09-04 アボット・ラボラトリーズ 血液検体中の免疫抑制性タクロリムス、シロリムスおよびシクロスポリンａ複合体の測定方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987003602A1 (fr) * 1985-12-06 1987-06-18 Teijin Limited Anticorps monoclonal humain contre le virus megalocytique et procede de preparation dudit anticorps
JPS6349095A (ja) * 1986-08-15 1988-03-01 Unitika Ltd 抗ｃｋ−ｍモノクロ−ナル抗体及びその製造法
JPS6459068A (en) * 1987-08-31 1989-03-06 Tosoh Corp Method for measuring human thyroxin binding globulin
WO1994004700A1 (fr) * 1992-08-12 1994-03-03 Fujisawa Pharmaceutical Co., Ltd. Anticorps monoclonal reconnaissant la proteine se liant au fk506, procede de titrage du niveau de proteine se liant au fk506 et trousse de titrage

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987003602A1 (fr) * 1985-12-06 1987-06-18 Teijin Limited Anticorps monoclonal humain contre le virus megalocytique et procede de preparation dudit anticorps
JPS6349095A (ja) * 1986-08-15 1988-03-01 Unitika Ltd 抗ｃｋ−ｍモノクロ−ナル抗体及びその製造法
JPS6459068A (en) * 1987-08-31 1989-03-06 Tosoh Corp Method for measuring human thyroxin binding globulin
WO1994004700A1 (fr) * 1992-08-12 1994-03-03 Fujisawa Pharmaceutical Co., Ltd. Anticorps monoclonal reconnaissant la proteine se liant au fk506, procede de titrage du niveau de proteine se liant au fk506 et trousse de titrage

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6709873B1 (en) 1997-04-09 2004-03-23 Isodiagnostika Inc. Method for production of antibodies to specific sites of rapamycin
JP2008536126A (ja) * 2005-04-06 2008-09-04 アボット・ラボラトリーズ 血液検体中の免疫抑制性タクロリムス、シロリムスおよびシクロスポリンａ複合体の測定方法
JP4862038B2 (ja) * 2005-04-06 2012-01-25 アボット・ラボラトリーズ 血液検体中の免疫抑制性タクロリムス、シロリムスおよびシクロスポリンａ複合体の測定方法

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