WO1996018102A1 - Method of measuring the concentration of fk506-binding protein - Google Patents

Abstract

An enzyme immunoassay method for detecting FK506-binding proteins or measuring the concentration thereof by detecting or quantifying a complex comprising an FK506-binding protein and the first and second antibodies that recognize respectively different antigenic determinants of the protein.

Description

 Specification

 FK506 binding protein concentration measurement method

"Technical field"

 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.

 "Background technology"

 Compounds described as 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).

 However, since its action is so powerful, determination of the optimal dose is an important issue, and it does not cause any side effects, etc., and has an effective immunosuppressive activity. It is extremely important to administer an efficacious dose.

 On the other hand, subsequent studies show that the immunosuppressive effect of FK506 is similar to that of peptidylprolinoresin transisomerase. It is thought to be exerted by binding to a binding protein (hereinafter referred to as FKBP) [eg, ', Nature, 357, 692—694 and 695— 697 (1992)] o

It is known that there are several types of FKBP depending on their molecular weight, such as FKBP-12 (molecular weight 12KDa), FKBP-13 (molecular weight 13KDa), FKBP -25 (molecule 鼋 25KDa), FKBP-52 (molecular weight 56KDa), etc., and their structures have already been determined.

 For example, Nature, 341, 755-757 and 758-760 (1989). J. Am. Chem. Soc. 113. 1409-1411 (1991). Proc. Natl. Acad. Sc i. 88 , 6229-6233 (1991).

 In addition, a method for producing FKBP, which has a molecular weight of 11,819 daltons and is composed of 107 amino acids, by using genetic engineering technology has already been reported [Nature, 346, 671- 674 (1990)].

 On the other hand, among the FKBPs, FK506 binds most strongly to FKBP-12, which has an affinity constant (Kd) of 0.4ηΜ, and furthermore, FKBP It has been reported that -12 is present not only in lymphocytes but also in many tissues including erythrocytes [Transpl. Proc. 23 (6) , 2760—2762 (1991)].

Here, 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.

 As a technique for measuring FKBP concentration in plasma, 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.

 However, in these methods, 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.

 Therefore, there has been a demand for the development of a technology capable of accurately measuring the FKBP concentration in a test sample even when FK506 is present.

 "Disclosure of the invention"

As a result of intensive studies, 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.

 Accordingly, 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.

 That is, the present invention

 (1) a first antibody (a monoclonal antibody that recognizes an antigenic determinant in FKBP) immobilized on a solid phase;

 (2) FKBP in the test sample, and

(3) Second antibody (a monoclonal antibody that recognizes an antigenic determinant in FKBP but recognizes a different antigenic determinant from the first antibody)

After forming such a complex, detection of FKBP by measuring the complex, or a method for measuring its concentration, and the first antibody, second antibody and Regarding kits containing FKBP as a standard product.

 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.

Preferably, 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 ゃ ^ 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).

 From the monoclonal antibodies obtained as described above, two types of monoclonal antibodies recognizing different antigenic determinants in FKBP were selected. It can be used as the first antibody and the second antibody, respectively.

 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.

 The term "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. For example, 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.

 As the enzyme, a stable enzyme having a large specific activity is preferable. For example, (1) carbohydrase [eg, glucose Sidases (eg, -galactosidase, β-gluconosidase,> 5-glucuronidase, 5-phenolectosidase, or-galactosidase, α-gnorecosidase Sigma, sigma-mannosidase), amylase (eg, α-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. Lipase, lipase, etc.), (4) Nuclease (eg, deoxyribonuclease, ribonuclease) ), (5) iron porphyrin enzyme (eg, lipase, peroxydidase, cytochrome oxidase), (6) copper enzyme (eg, tyrosinase) , Ascorbic acid oxidase), (7) dehydrogenases (eg, alkanol dehydrogenase, lactate dehydrogenase, sulphate dehydrogenase, isoclosidase) Enolate dehydrogenase), and in particular, phenol-reactive phosphatase is preferred.

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. 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.

As radioisotopes, for example, ¹²⁵ I, ¹³¹ I, ³ H,

¹⁴ C and the like.

 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.

 In preparing the second or third antibody having a label, 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.

However, 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.

 In the present invention, various conventional methods can be used for quantifying the complex consisting of the first antibody, FKBP and the second antibody. For example, the following methods can be considered.

(A) 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.

 (B) After forming a complex with the first antibody, FKBP and the second antibody labeled with biotin in which biotin is bound in a conventional manner, the antibody is labeled with an appropriate enzyme. The reaction of the bigin is carried out, and an appropriate enzyme-substrate reaction is performed according to the labeling 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) A conjugate with the second antibody is prepared, and after forming a complex with the first antibody, FKBP and the conjugate, an appropriate enzyme-substrate reaction is performed.

1 o

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.

 Then, 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.

 When the test sample is human serum, plasma, or tissue exudates, sodium ethylenediaminetetraacetate (EDTA.2Na) and sodium citrate are used. By adding a chelating reagent such as trium, the measurement accuracy can be further improved.

 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. As the solid phase, 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. However, it is possible to use 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.

 ADVANTAGE OF THE INVENTION According to this invention, it became possible to easily and more accurately detect or measure the concentration of FKBP in a test sample in various blood vessels and blood. Thus, for example, it has become possible to provide important information for determining the optimal administration S of FK506.

 Then, if necessary, selectively detect only a specific FKBP (for example, FKBP-12), measure its concentration, or detect all FKBPs. In addition, the ability to measure the total concentration enables the precise setting of the FK506 dose according to the condition of each individual patient in an actual medical setting. It became so.

 In particular, 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. In each case, 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

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Examples, but the present invention is not limited thereto. Production Example 1 Production of anti-FKBP antibody

 (1) Production and features of FKBP-12

 Nature. 346, 671-674 (1990) in Harvard University S.

L. (: 11 616 61 "et al., Report, 185 569 61 ^ 6, and DNA 48 me r ^ Aplied Biosystems, Inc. It was synthesized using DNA synthesizer.

 5 '-C C A C A T G C C A C T C T C G T C T T C G A T G T G G A G C T T C T A A A A C T G G A A T G A-3'

The end of this 48- mer and La Benore at ²⁵ P, probe and to CL0NTECH's human T one cel l cDNA La I bra Li one HL1016b 50 Man Pla over click the scan click rie two in g in At that time, I got one positive plaque. A fragment containing FKBP-12 cDNA was subcloned from this plaque [pUC-23 (Ec)]. When this pUC-23 (Ec) was sequenced, the portion corresponding to N-terminal DNA sequence Nos. 1 to 32 was missing. Along with complementing this part, an AT rich silent mutant N-terminal DNA of about 80 bp synthesized to enhance expression in E. coli was used for E. coli and EcoRI—BamHI site. As a result, 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. coli was collected, the cells were crushed in French Press in 50 mM Tris-HC1, 2 mM β-ME, 2 mM CaCl ₂ , 10 mM MgCl, 5% glycerol, and centrifuged (4 ° C, Heat the supernatant at 60'C for 15 minutes-Centrifuge (4 ° C, 6,000 X g, 45 minutes + 4'C, 18, 000 g, 20 minutes x 2 times) FKBP-12 was purified by dialysis [20mM Tris-HC1 (pH 7.4), 4 ° C overnight] -DEAE-Toyo PEARL 650M-reverse phase HPLC (C4).

 (2) Immunization of FKBP-12 mouse

 Phosphate buffer solution of FKBP-12 (hereinafter referred to as PBS) solution 250 gZ ml 0.1 ml and an equal volume of Freund's Comlete Adjuvant (FCA) were mixed and immunized intraperitoneally with mouse BALBZc. O The same amount of FKBP-12 was mixed with Freund's Incomplete Adjuvant (FIA) and immunized intraperitoneally approximately several times every 10 days.

 (3) Measurement of blood antibody titer

1 was collected from the mouse tail vein, mixed with PBS 990β1, centrifuged, and used as a measurement sample. For the measurement, add 10 g / ml FKBP-12 PBS solution 501 to an immunoplate and allow it to react at room temperature for 3 hours to bind to the surface. After that, wash the gel, block the non-specific binding site with 0.2% Milk Blocker PBS, wash again, add 501 of the sample diluent, and allow to wash at room temperature. Let react for hours. After washing, add 100 µl of anti-mouse IgG (H + L) -POD [Vector Lab.] 100 and react for 1 hour at room temperature. Let it go. After washing, color was developed with a 0-phenylenediamine system according to a conventional method.

 (4) Production of anti-FKBP-12 antibody

The mouse in which the antibody titer was increased was further injected with 0.2 ml of FKBP-12 250 βg / ml (PBS) via the tail vein as a final immunization. Four days later, the spleen was extracted, and spleen cells were prepared at 1.44 × 10 ⁸ cels for 1 s. On the other hand, 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 μl plate. Two weeks later, the production of anti-FKBP-12 antibody was screened for 144% of the cells in which cell growth was confirmed in HAT medium by a method of measuring the antibody titer in blood. I did it. That is, in the method for measuring the antibody titer in blood, when a sample containing an anti-FKBP-12 antibody is added and reacted, 5 gZ ml of FKBP-2 is co-present in the presence of FKBP-12. The one that lost the color development in was selected. Furthermore, 32 clones with high antibody titer against FKBP-12 were selected.

 (5) Selection of anti-FKBP-12 antibody that recognizes FKBP-12 and does not inhibit the binding of FK506.FK506-C32 (LA) -POD to FKBP-12

After binding anti-mouse IgG (H + L) to the solid phase and further binding a screening antibody, the FKBP-12501 was obtained using the following (6). FK506-C33 (LA) -POD (1000-fold dilution) 501 was coexisted. 0-phenylene A clone having an increase in absorbance at 490 nm, which was generated by the reaction with diamine, was selected. At this time, 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. Recognizing the POD FK506, the ability, the strength, and the ability. As a result, the immunization method using FKBP-12 as the antigen as it was showed that five types of antibodies (5-11-A5, 5-1-B3, 5—1-C5,5 -2-Dl (IGj), 5-4 I D2), an immunoassay using an antigen obtained by denaturing FKBP-12 with urea as described in (7) below. From IA 4 (IgM), 3 A 8

_{(IgG 2), IF 7,} 3 B 8, or, Shitagen已(8) of the immune method or et al., Which had use as an antigen in the jar good of the FK BP- 12 is coupled with Obuarubu Mi emissions As a result, a total of 10 monoclonal antibodies of 4F8 were obtained.

(6) FKBP506-C32 (LA) One POD production method

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 mixture was stirred at room temperature for 6 hours in a solvent of water (1: 1, 20 ml). The reaction mixture is washed with water After drying, the solvent was distilled off, and the obtained residue was applied to silica gel column using chloroform as a developing solvent. 14-Hydroxy 12-[2-(4-N-(10-Power box)-(4-1 Amino 1, 4-Gioxo 1 1 1-3-meth oxy hex) 1-1-methyl vinyl 2) 1, 23-25-dim. —Tetramethylen 11,28—Zoxer 41 Azatori cyclo [22,3 .1 .4 ⁴ ' ⁹ ] Octa cos 1 18 — Gen 2, 3 , 10, 16—Tetraon (120 rag) were obtained.

 NMR (CDC1 a): 1.2-1.4 (m), 5.9-6.1 (1H, m)

* FAB MASS: 1109 (M + Na) ⁺

 Furthermore, by using the above compound and reacting horseradish peroxidase in the same manner as in Example 1-3) of JP-A-11-92659, FK506 was obtained. — A C32 (LA) -POD solution was obtained.

 (7) Preparation of urea-modified FKBP-12

 Add urea (312 mg) to 460 μl of 0.08% trifluoroacetic acid aqueous solution (847 β g / ml) of FKBP-12, mix at room temperature with FKBP-12 (600; ugZ inl ) Was prepared. This was used for immunization as it was.

 (8) Preparation of ovalbumin-bound FKBP-12

Mix 1032 g of FKBP-12 (use 1032 1 with 1 mgZ ml) and 1032 1 of PBS (2 mg / ml) of o-norebumin (Sigma, Lot No. 76F-8040) . To this, add 0.62 ml of 0.13 M glutaraldehyde-PBS solution dropwise. Room ^ bottom, The mixture was stirred for 14 hours, then dialyzed three times against PBS11, and used as an immunogen.

 The mixing ratio of FKBP-12 and ovovanorebmin was 1032 µg of FKBP-12: 2064 g of ovoalbumin, and the solution volume was 2.7 ml.

Production Example 2 Production of anti-FKBP-12 antibody

 (1) The mice were immunized as described in (1) and (2) of Production Example 1.

 (2) Measurement of blood antibody titer

20 μgZml 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 ΙΟΟμ ゥ, and add at room temperature. Left for 2 hours. Next, a small amount of the antiserum diluent was removed by suction, and the plate was washed three times with 0.05% Tween 20 PBS solution. 0.2% milk / 0.05% Twisted 20Z PBS diluted 1000 times with phenolic anti-mouse IgG (H + L) solution 100 1 was added to the gel and left at room temperature for 2 hours. The anti-mouse IgG (H + L) solution labeled with anophore phosphatase was removed from the solution, and the solution was pre-cleaned 5 times with a 0.05% Tween20 / PBSi solution. . After that, 0.1m Μ 4 メ Μ Μ Μ (4 MU-P; Sigma) buffer solution (10 mM diethanolamine 70.5% MgCl./0), add 100 1 to each well and release for 15 minutes at room temperature. did. The fluorescence intensity (Exitation; 360 nm, Emission; 460 nm) of each cell was measured with a halo plate reader.

 (3) Select the mouse with the strongest antibody titer, and 10 days after the fourth injection, as a final immunization, add 0.2 ml of 1.5 mgZ ml of FKBP-12 PBS solution through the tail vein of the mouse. Injected.

 (4) Cell fusion

 Cell fusion was performed in the same manner as in Production Example 11 (4) to obtain hybridomas (3-3-D4—C6, 2C1—87, and 3F4-70).

 (5) Antibody production and purification

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. Purification was performed using the gel protein A MAPS-II kit (BioRad), and the anti-FKBP-12 monoclonal antibody 2C187 (subtype: I) g G j λ) and 3F4-170 (subtype: IgGi) purified antibodies were obtained. Similarly, after culturing hybridoma 3-3—D4—C6, anti-FKBP-12 monoclonal antibody 3—3—D4—C6 (subtype: IgM) was added. Obtained.

Example 1. Enzyme immunoassay for FKBP-12 (AP-! HcAb method)

1) Phosphorylation of monoclonal antibodies

 1 mg of purified anti-FKBP-12 monoclonal antibody 2C-187, 3F4-170 is immobilized. Tarsee Labeling Kit (Cambridge Biochem Canole Inc., Code CK07-1000) Perform enzyme labeling to obtain 1 ml of each labeled antibody.

 2) Absorption operation of antibody on solid phase

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.

3) Specific adsorption prevention operation

An aqueous solution of 4% block ace (Snow Brand Co., UK-B80) was injected into each of the above plates for 250 minutes and left at room temperature for 30 minutes. Wash once with 0.05% Tween 20 in PBS. ) Antigen-antibody reaction

 Dispense 100% of the standard FKBP-12 solution 100 diluted with an aqueous solution of 0.1% Tween 20 into each of the plates in the above plate. Leave at room temperature for 2 hours while shaking with a mixer. Wash the plate 7 times with 0.05% Tween 20 in PBS.

) Labeled antibody reaction

 100% 1% Bac Ace 0.1 0.1% Alfa phosphatase-labeled monoclonal antibody diluted 1000 times with 1% Tween 20 aqueous solution Aliquots into each plate of the above plate, and place at room temperature for 2 hours while shaking with a plate mixer (3F4 for 2C1-87 solid phase). -70-labeled antibody, 2C1-87 labeled antibody for 3F4-170 solid phase). Wash the plate 7 times with 0.05% Tween 20 in PBS.

) Enzyme

 Dispense the enzyme substrate solution (100 // 1) prepared as described below into each plate of the above plate and incubate at room temperature for 30 minutes. The enzyme substrate solution is prepared immediately before use.

 Enzyme substrate solution:

 0.5 m Μ 4 メ ー ー ー 10 10 ml ml ジ ジ ml 10 10 ml 10 ml

 Magnesium chloride monohydrate

(MgCl ₂ -H 0) 10 mg

Distilled water 90 ml 7) Measurement

 Compared to the standard FKBP-12 solution, a 4% Bubble Ace / 0.1% Tween 20 aqueous solution was added instead of the standard solution. The fluorescence intensity (Ex. 360 nm, Em. 460 nm) is measured by the following method.

 Table 1 shows the results of the above measurement methods.

 Table 1 Enzyme-linked immunosorbent assay for FKBP-12

 (A P — M c A b method) Result

Example 2. Enzyme-linked immunosorbent assay for FKBP-12 (Piotin-McAb method) 1) Piotin labeling of monoclonal antibodies

 1 mg of purified 2 mg anti-FKBP-12 monoclonal antibody 2 C 1-87 or 3F4-170 in 7.5% sodium bicarbonate buffer solution (pH 8.5) Dissolve the solution in 1 and add lOmgZ ml of BNHS (N-hydroxysuccinimidyl-6-1 (biot inamido) hexanoate, manufactured by Vector, Code SP-1200) DMS0 solution 201, and stir occasionally. After reacting at room temperature for 2 hours, dialyze against PBS overnight to obtain 1 ml of a biotin-labeled antibody.

2) Absorption operation of antibody on solid phase

 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.

 3) Specific adsorption prevention operation

 To each of the above plates, inject 250 µl of 4% block ace (previously described) aqueous solution for 1 minute, let stand at room temperature for 30 minutes, and place the plates in 0.05% Tween 20. Wash once with PBS solution.

4) Antigen-antibody reaction

Dispense 100% of standard FKBP-12 solution diluted with 0.1% aqueous solution of 0.1% Tween 20 in each of the above plates. Incubate at room temperature for 2 hours while shaking with a plate mixer. Wash the plate seven times with 05% Tween 20 in PBS. ) Labeled antibody reaction

 100% of the above-mentioned biotin-labeled monoclonal antibody diluted 1000 times with an aqueous solution of 0.1% Tween 20 Dispense into each of the tubes and shake for 2 hours at room temperature while shaking with a plate mixer.

(3F4-170 labeled antibody for 2C1-187 solid phase, 2C1-87 labeled antibody for 3F4-170 solid phase). Wash the plate 7 times with 0.05% Tween 20 in PBS.

) Piotin-avidin reaction

 1% Block Ace-0.1% Tween 20 in water

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.

) Enzyme

 Dispense the enzyme substrate solution (100 / ul) prepared as described below into each plate of the above plate and incubate at room temperature for 30 minutes. Prepare the enzyme substrate solution immediately before use.

 Enzyme substrate solution:

 0.5mM 4 -Methyl benzoyl phenyl phosphone phosphone gently 1min 10ml

 Magnesium chloride, monohydrate

_{(MgCl 2 · H 2 0)} 10 mg

Distilled water 90 ml ) Measurement standard FKBP-12 solution was replaced with a 4% block ace / 0.1% ot Tween 20 aqueous solution.

 n i

 Then, measure the fluorescence intensity (Ex. 360 nm, Era. 460 nm) using a site flow (trade name, manufactured by Millipore). Table 2 shows the results of the above measurement methods. Table 2 Enzyme-linked immunosorbent assay for FKBP-12

 (Piotin-McAb method) Result Primary antibody (fluorescence intensity)

3 F 4 — 7 0 2 C 1-8 7

1000 2 3 9 1 7 1 3

500 1 9 1 3 4 1 7

250 1 2 8 8 2 4 1

125 7 3 6 1 2 5

62.5 3 8 2 6 0

31.25 1 7 5 2 4

7 8

7. 8125 1 4 Example 3. Enzyme-linked immunosorbent assay for FKBP-12 (AP-antiA-McAb method) 1) Adsorption of antibody to solid phase

 Monoclonal antibody 3 F4-170 in PBS (5 μg / 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.

 2) Specific adsorption prevention operation

 An 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.

 3) Antigen-antibody reaction

 Dispense 100% of the standard FKBP-12 solution diluted with an aqueous solution of 0.1% Tween 20 into each of the above plates. – Leave at room temperature for 2 hours while shaking with a mixer.

 ) 2 C 1 -87, AP-anti i λ

Monoclonal antibody 2 C 1 — 87 is diluted with a 1% block-acetate 0.1% Tween 20 aqueous solution to a concentration of 5 μgZml 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.

) Enzyme R

 Dispense the enzyme substrate solution (100 / l) prepared as described below into each of the plates described above, and incubate at room temperature for 30 minutes. The enzyme substrate solution is prepared immediately before use.

 Enzyme substrate solution:

 0.5mM 4-methyl phenyl phosphine

 Magnesium chloride monohydrate

(MgCl ο-H ₂ 0) 10 rag

 Distilled water 90 ml

) Measurement

 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

 (A P — an t i Λ-M c A b method)

Example 4 FKBP in Human Serum—Measurement at 12 滠

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 µl 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.

 Table 4. Measurement of FKBP—12 concentration in serum

 F K B P-1 2 Concentration measurement

 (ng / m 1)

1 2 9, 4 5

2 2 3. 3 2

3 3 2, 9 3

4 3 3. 7 7

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 μl 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

Example 6. Measurement of FKBP-12 concentration in liver transplant patients

Blood was collected from a liver-transplant patient and blood plasma was obtained. The FKBP-12 concentration in the blood was measured in the same manner as in Example 5. The results are shown in Table 6.

6. Measurement of blood FKBP--12 concentration in all liver transplant patients Blood FKBP-12 concentration Days after surgery

 (ng / m 1) thousand nit

Na rrJ U ri ^ 3 ΛJ Π \ J Π \ J

 A P ^

 t »Q 4 ^.

 7 R 2 ς η η V

1 1 says 4 4 3 .5

14 4 15 3. 3

17 7 2 70.4

1 8 says 8 1. 1 1

Claims

The scope of the claims  1. The first antibody (monoclonal antibody that recognizes the antigenic determinant in the FK506 binding protein) immobilized on the solid phase, the FK506 binding protein and the second antibody in the test sample. After forming a complex consisting of an antibody (a monoclonal antibody that recognizes the FK506 binding protein with a different antigenic determinant than the first antibody), the presence or amount of the complex is determined. A method for detecting or measuring the concentration of an FK506-binding protein, which is characterized by detecting or measuring.  2. The claim that the monoclonal antibodies, which are the first antibody and the second antibody, do not inhibit the binding between FK506 binding protein and FK506. The method described in Paragraph 1  3. The method according to claim 1 or 2, wherein the FK506-binding protein FKBP-12 is used. 4. The second antibody is a labeled second antibody, and by using the label, the presence or amount of the complex can be detected or measured. The method according to claim 1, 2, or 3, which is a feature.  5. The method according to claim 4, wherein the label is an enzyme, and the request utilizes the enzyme-substrate reaction.  6. The label is piotin, and the avidin that reacts with the piotin is allowed to act to detect or measure the presence or amount of bound avidin. The method according to claim 4, characterized in that: 7. Detect or measure the presence or amount of bound avidin 7. The method according to claim 6, wherein the method uses an enzyme that labels avidin.  8. The method according to claim 4, wherein the label is a third antibody (an antibody that recognizes the second antibody).  9. The method according to claim 8, wherein the third antibody is an anti-mouse lambda chain antibody.  10. The method according to claim 9, wherein the anti-mouse lambda chain antibody is a labeled anti-mouse lambda chain antibody.  11. The method according to claim 10, wherein the labeled anti-mouse lambda chain antibody is an anti-mouse lambda chain antibody labeled with an enzyme.  12. The method according to claim 5, 7, or 11, wherein the enzyme is alkaline phosphatase.  13. The method according to claim 4, wherein the test sample is a test sample to which a chelating reagent has been added.  14. The first antibody (monoclonal antibody that recognizes the antigenic determinant of FK506 binding protein) immobilized on the solid phase contains (1) FK506 binding protein or contains it The test sample suspected of causing the FK506-binding protein to act, and then (2) a second antibody (a monoclonal antibody that recognizes an antigenic determinant of the FK506 binding protein different from the first antibody) is allowed to act, and The method according to claim 1, wherein after (3), the presence or amount of the formed complex is detected or measured.  15. Kit containing FK506 binding protein, primary and secondary antibodies „