JPH05502101A - FK-506 receptor analysis method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to a method for analyzing drugs in biological fluids, and more particularly to methods for analyzing drugs in biological fluids. of the epidemic suppressant F K-506 and its biologically active metabolites, derivatives and analogues. Concerning competitive receptor protein binding analysis method.

Description of background technology Cyclosporine, a fungal-derived cyclic undecapeptide immunosuppressant, It is widely used as an anti-rejection drug for transplant patients. cyclosporine (CsA) appears to be T-cell specific and promotes interleukin-2 production and 1°Cs induces its immunosuppressive effect by inhibiting receptor expression. A. At the dosage necessary to prevent rejection in the patient's organs, the gastrointestinal and including metabolic problems, hypercholesterolemia, neurological signs and symptoms, and nephrotoxicity. As a result, CsA is known to exhibit severe toxic side effects. Immunosuppressants that have high efficacy at low doses and have few toxic side effects Research on substitutes is actively being conducted. Known as PK-506 Compounds are such agents.

FK-506 is manufactured by Fujisawa Pharmaceutical Co., Ltd. in Japan. uticalCo) is Streptomyces tsukubaensis (StrepLom ycestsukubaensis) The 7° structure of CsA, which is a CsA antibiotic, is completely different from that of CsA (see structure), but PK -506 exhibits similar, if not the same, anti-lymphocyte activity. but, The immunosuppressive activity of FK-506 is approximately 100 times higher than that of CsA, and it is effective in solid organ transplant patients. 1゜ has been used with good results, especially in liver transplant patients. The toxicity of FK-506 in human patients and experimental animals is reported to be lower than that of CsA. However, at the dosage required for IjlQ, anti-rejection activity, such There are reports of undesirable side effects of FK-506 in various species. 0° Balance dosing requirements to prevent rejection with avoidance of severe toxic side effects. As a result of the need to 11. To do this, we must carefully and constantly monitor the According to report 11, not only FK-506 is produced by metabolism in the liver. It is necessary to monitor plasma concentrations of biologically active FK-506 metabolites.

Therefore, not only FK-506 and its bioactive metabolites, but also pharmacokinetic Bioactive analogs and drugs to be used in research and in establishing patient doses. Accurate, sensitive, specific, rapid and inexpensive analysis methods for is necessary.

Tamura et al. 13 and Cadoff et al. Rabbit polyclonal antibodies raised against serum albumin-FK-506 complex antigens Fixed antibody against FK-506 using natural antibody or mouse monoclonal antibody. Discloses one-stage and two-stage enzyme-linked immunoassays (ELIS). child Several problems with this method have been reported: (1) Its sensitivity is dependent on the patient's blood FK-506 plasma required before drug extraction from plasma with organic solvents such as benzene highly concentration dependent; (2)F K-506 and immunologically active but (3) Since the concentration of the drug in the plasma is low, Moreover, since ELISA is inaccurate, the accuracy is low14. and (4) this analysis. Is there a correlation between the dose measured by the method and the plasma concentration?

Zeevi et al. 1S have developed a bioanalytical method for plasma FK-506. , this assay is based on the inhibition of the proliferative response of alloreactive T cell clones by this drug. Based on +s, these authors found that for all plasma samples tested, F The values obtained for K-506 using the ELISA method 13.14 described above are similar to those obtained using the bioanalytical method. reported values that were consistently much larger than those obtained. This means that the patient's blood Bioinactive FK- detected by ELISA but not by bioanalytical methods 506 metabolites. In other words, ELISA is Because it is an immunoassay that detects any molecule that has a pitope site, it metabolites may cross-react with anti-FK-506 antibodies.

There are several problems with this bioanalytical method for FK-506. This analysis The law does not identify immunosuppressants. Therefore, to prevent rejection of allogeneic transplants steroids such as prednisone administered concurrently with FK-506 (or C5A) ids are immunosuppressive and are not suitable for any bioanalytical method based on suppression of T cell proliferation. Even if it is, the reaction will be positive. Additionally, bioanalytical methods are commonly used in hospital clinical laboratories. Not easily adapted for use: Bioanalytical methods require low data throughput; The analytical method is slow, sensitive, and sensitive to biological changes between cells in tissue culture. oxidation makes such cells less than desirable as reagents.

Therefore, FK-506 and its biologically active metabolites, derivatives and There is a great need for rapid, inexpensive, accurate, sensitive, and specific methods for the analysis of analogs. It is considered essential. Such an analytical method has now been discovered and is disclosed below.

Summary of the invention Determining FK-506 and FK-506-like functional activity in biological fluid samples A new method of quantification has been discovered. In this method, the binding reagent is Competitor protein binding is purified soluble receptor protein isolated from target tissue Consists of analytical methods.

Therefore, we specifically discuss FK-506 and its biologically active metabolites, derivatives, and analogs. An objective of the present invention is to provide purified water-soluble receptor proteins for use in competitive binding assays. It is the purpose of light.

Furthermore, using the above-mentioned purified receptor protein, FK-50 is also targeted. .

We present a camphor FK-506 molecule suitable for use in the competitive receptor binding assay of the present invention. It is yet another object of the invention to provide.

These and other objects of the invention will be realized in the following discussion of preferred embodiments thereof. It will be clear from the description and claims.

Brief description of the drawing Figure 1 shows that FK-5 against CsA-binding protein in the cytosol of CEM lymphocytes. 06 and CsA are compared.

Figure 2 shows the specific binding of FK-506 to cytosolic proteins of CEM lymphocytes. Indicates the

Figure 3 shows the HPLC molecular weight on a BioRad TSK column. The sieve fraction was separated from the CEM lymphocyte cytosol by chromatography. The elution profile of the FK-506 binding protein fraction is shown.

Figure 4 shows BioRad BioSil S E C JU by Beckman HPLC on 125 column FK-506 (・-・) and C5A (○ −○) Shows fractionation of binding proteins. Also shown are molecular weight markers.

Figure 5, MaLrex Gel Blue (MaLrex Gel Blue) FK-506 (m) and Cs of JURKAT lymphocytes from the affinity column A (*) Shows the elution profile of binding protein as a function of salt concentration.

Figure 6A-B shows Matrex Blue A affinite. CsA and FK-50 isolated by chromatography and pooled BioRad S E C125 Molecular Weight Exclusion Column for 6 Bound Fractions The HPLC fractionation above is shown. Figure 6A shows the sample of cyclophilin (approximately 17 kDa). Figure 6B shows the FK-506 receptor of approximately 8-12 kDa. protein.

Figure 7 shows the weak cation exchange chromatography of the 17 kDa encrophilin of Figure 6A. -HPLC column [Beclvan TS KCM-25W Figure 2 shows fractionation on Spherogel.

Figure 8 shows the weak cation exchange protein of the FK-5068~10 kDa binding protein in Figure 6B. ROMATOGRAPHY HP L C column [Beckman TS The fractionation on K CM-25W Sepha 0 gel (Spherogel) is shown.

Figure 9 shows the weak cation exchange of approximately 5QkDa protein eluted from the size exclusion column. The fractionation on the exchange column is shown.

Figure 10 shows the purified f knee JU RK AT about 8-12 k D a rece Figure 2 shows analytical 5DS-PAGE of protein protein.

Figures 11A-C show that approximately 5 QkDa protein preparations (Figure 11A) bind to the same preparations. FK-506 (FIG. 11B) and its approximately 8-12 kDa receptor Scanning of CsA binding to protein-bound FK-506 (Figure 1IC). 1 shows a ScaLChard plot.

Figures 12A-B show purified 8-12 kDa JLJRKAT cell receptor protein. F in water (Figure 12A) and human whole blood (Figure 12B) using as binding reagent Figure 2 shows a semicircular curve for receptor analysis of K-506.

Detailed description of preferred embodiments The term "receptor" refers to a type of ligand that recognizes and selectively binds to it. After binding the ligand, an event that results in a biological response Can generate some kind of chemical or physical signal that starts the chain It is generally accepted that T4 refers to the large molecule T4 or glycoprotein. Ru. Blecher, M. et al., [Receptor and ``Human Diseases'', Williams and Wilkens l1lkens), Baltimore, 1981, No. 1*.

Therefore, FK-506 and its biologically active metabolites, analogs and derivatives (hereinafter referred to as , collectively referred to as FK-506). One of the characteristics of natural receptor proteins is that the water-soluble binding protein used as have specific, saturable, and reversible binding properties. , is an important aspect of the invention.

Water-soluble specific receptor protein suitable for CRBA carried out according to the invention are mammalian lymphocytes, including human or animal peripheral blood lymphocytes (PBL); - Next mixed lymphocyte culture, proliferative cell culture grown from organ transplant biopsies! Anti-different race Responsive T cells + sjI? , and established cell lines, e.g. DQwl-specific allogeneic Alloreactive T cell Rirone D B 291 @, transformed T helper cell line (CEM) and interleukin-2 producing JtJRKAT human lymphocytic leukemia T Target groups for FK-506 action such as cell lines (JURK AT 77.6.8) Advantageously prepared from extracts of the vine. Furthermore, FK-506 has a 17kDa rank. Does not bind to peptidyl-prolyl cis-trans isomerase in lofilin fraction , does not inhibit, but approximately 8-12 kDa FK-506 binding of JURKAT T cells This is done for the isomerase activity of protein 11. This enzyme is Cs It is very sensitive to CsA and is considered to be the primary severator of CsA action. 16-11゜Specific details of the receptor protein used in the CRBA of the present invention It must be emphasized that the source of the protein is not important; All that is required is to demonstrate the physiological receptor binding properties detailed above. be.

destroys cells and soluble proteins (cytosolic proteins) are recognized in the art They may be separated according to the method used. Typically, lymphocytes are collected, washed, And count. Cells were stored as pellets at -70°C until use. You may do so. Thaw cells and water using a Teflon or ground glass homogenizer. Homogenize at bath temperature. Completeness of cell destruction is tested by trypan blue removal method. can be done. the homogenate under cooling vacuum at least 20.000×g; Preferably at least 100. at OOOXg for at least 0.5 hours Centrifuge. The supernatant contains FK-506 receptor protein and cyclophyll. It contains both phosphorus and is analyzed for protein content and binding activity and used immediately. or at about -20°C or lower! - Freeze and store Ru.

Cytosol can be used directly for CRBA performed according to the present invention. Wear. However, preferably the FK-506 receptor protein is cytosolic or It is then purified and rolled before use.

F K-506 receptor protein was purified by affinity chromatography. can be partially purified. Preferred affinity columns are matrix , Kusu Gel Blue (Matrix Gel Blue) A [Amifun Co., Ltd. (A -icon corp, ), Danvers, M^], and using this, F K-506 and CsA-binding proteins are easily separated by interstitial gradients of salt buffer. can do. The cytosol can also be analyzed using molecular weight exclusion methods, including HPLC columns. Therefore, it can be separated. For example, a preferred molecular sieving method is Beckman ・Instrument Co., Ltd. (Beckman In5tru+*enL Co.) HPLC equipment and appropriately sized preparative BioRad Biosil d　Biogil)　SE　C125 column (see Example 6 below) (see). In addition, FK-506 receptor protein can be analyzed using cation exchange chromatography. For example, weak cation exchange Beckman TSK C Advantageously purified by M-25W Spherogel You can also do it. Hydrophobic interaction chromatography matrices can also be used according to the invention. Suitable for purifying FK-506. Adjacent to these areas where appropriate refining takes place. Any arrangement or combination of alternative systems can be used in accordance with the present invention. . For example, cytosol, affinity matrices, molecular weight exclusion gels and weak Particular preference is given to purification by thione exchanger sequences. For analysis of the present invention, In the case of chromatography, a single peak represents both protein and binding activity. If it represents the elution pattern, or in the case of 5DS-PAGE, the main protein If only one band appears, the receptor protein is considered purified. Kara Fractions of the sample eluate were collected, pooled, and subjected to rotary evaporation under reduced pressure and cooling. Concentrate with a rotor. Bradford B including CA method The concentrate is analyzed for protein by any suitable method. Also, CRBA The concentrate is therefore tested for analytical suitability.

F K-506 receptor protein is available for techniques other than isolation from mammalian target cells. It must be emphasized that it is therefore prepared and purified. For example, receptor -Proteins are produced using art-recognized recombinant DNA technology. Briefly, the cDNA encoding the receptor is purified. isolated from RNA or cDNA cloning libraries and then isolated from cells, e.g. It may also be cloned into prokaryotic cells to produce large amounts of cDNA.

The expression vector contains the FK-506 receptor cDNA, promoter and genes. Constructed to include gene control sequences, translation initiation codons, selectable markers, etc. It may then be inserted into a prokaryotic cell or a transformed eukaryotic cell. These details The cells express the receptor gene and secrete large amounts of receptor protein into the growth medium. from which it can be isolated by conventional methods.

For the purpose of CRBA according to the present invention, the protein fraction is determined by: (1) the protein being detected; statistically based on methods such as radioactivity, fluorescence polarization, chemiluminescence, etc. binds to FK-506 to a significant extent: (2) unlabeled and labeled FK-50; 6 compete with each other for specific binding sites on the receptor protein; and (3 ) Signal-to-noise ratio, i.e. total binding to non-specific binding (these terms are used below) defined) is at least 1.1, preferably less than (both about 1 ．． 2 is considered acceptable.

Gel electrophoresis and Western blot analysis were used to monitor all stages of purification. Thus, the purity of the receptor protein may be monitored and its identification performed.

Label FK-506 is required for CRBA performed according to the present invention. natural FK-506 is available from Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan) and is an aqueous parenteral It is soluble in medium solvents such as aqueous methanol. [3H]-dihydroFK- 506 converts natural FK-506 into tris(triphenyl-phosphine) rhodium. After exposure to tritium gas in the presence of a reducing agent such as chloride, normal phase and reverse phase chromatography by Amersham Corp. ), Arlington Heights, IL]. can. It was more than 98% pure according to three different TLC systems, ['' Some preparations of H]-dihydro FK-506 have 51 Ci/mmo 1 or 6 It had a specific activity of 3.2 mC1/mH. Through this process, one hydrogen atom and and one tritium atom were added to the molecule. [MeblIt-β-3 )(]Ncrosvorin A ('H-CsA) has a specific activity of 5 to 20 Ci/mmol. can be obtained from Amershas Co.

Additionally, K-506 was briefly induced by chloramine-T in the presence of NaIIJ. Reduction of tlAm can also be performed at +111, which emits γ-rays. 118 1-labeled histamine-F K-506 was obtained by the method of Long et al. It can be prepared.

For example, Abbott Laboratories s.

Inc.) [Abbott Park, IL] Suitable for use with TDX equipment Fluorescent label FK-506 may be used for fluorescence polarization detection. FK-5 06 may be labeled with a fluorophore by art-recognized methods ``1''. stomach. Suitable fluorophores include fluorescein, europium and luciferin. be caught.

Water-soluble 1,2-dioxylene that releases light energy upon cleavage by hydrolytic enzymes Chemiluminescent labels such as cetane are available from Tropix, Inc. , ) [Belford, IIA]. Also, chemiluminescence FK-506 labeling is achieved by addition of a substrate such as 1,2-dioxetane as described above. In the case of enzymes that emit light, it is also a detection method.

CRBA carried out according to the present invention can be carried out by either solution phase or solid phase methods. can also be done. The principles underlying both methods are the same. In short , a competitive equilibrium is established between a tracer amount of labeled FK-506 and a predetermined amount of the above FK-50. Unknown sample containing analyte FK-506 for binding to 6 receptor proteins occurs between After reaching equilibrium, labeled FK-5 bound to the receptor protein Measure the amount of 06. The amount of 11-sensitized receptor bound to non-tsI-identified FK-50 6, this decrease is due to the amount of unlabeled analyte present in the unknown sample. is proportional to. The decrease in label bound to the receptor and the concentration of analyte in the unknown sample The quantitative relationship between is determined by reference to a standard curve. mark To create a quasi-curve, a given amount of receptor protein is added from zero to a supersaturating concentration. Exposure to labeled FK-506 at a predetermined tracer concentration in the presence of standard FK-506. vinegar. The supersaturating concentration is ideally several orders of magnitude larger than the association constant Ka for specific binding; ('non-specific binding', N5B) indicates that non-specific binding is a primary is assumed to be the same for all Gantt concentrations. It will be done.

For standard curve analysis in solution phase, unlabeled FK-506 was prepared in aqueous alcohol (e.g. Place an aliquot of the solution (e.g., 50% ethanol in water) into a glass tube and add Evaporate the solvent with a gentle stream of N, or under cooling vacuum; The amount of FK~506 collected ranges from Ong to about 100.000 ng. in the blood For solution phase CRBA of FK-506 in For example, lower alkanols (e.g. C5-C1 linear or branched primary, secondary) or tertiary alcohol) or acetonitrile. "Amphiphilic organic solvent ” shall mean a liquid organic compound having both hydrophobic and hydrophilic properties . The precipitated sludge is removed, for example, by centrifugation, and FK-506 is The containing extracts are dried to dryness as described below. The residue to be analyzed is mixed with a small amount of water/amphiphile. in a eg 1:3 mixture of organic solvents and transferred to a reaction tube.

Each tube contains a predetermined amount of tracer in a small volume (e.g., approximately 50 μm) of stock solution. Labeled FK- (e.g., 0.5 nM, 50,000-100.OOOCPM) 506, for example [3H]-dihydro FK-506.

Each tube then receives a small amount of binding buffer, e.g. During the medium, add a solution of receptor protein and incubate until binding reaches equilibrium or steady state. These tubes are then heated to about 0°C to about 40°C, typically for about 20 to 90 minutes. Incubate at a slightly elevated temperature, such as Join 1! The composition of Li liquid is It's not important. A preferred binding buffer is 5mM 2-mercaptoethanol, 0. 20mM) containing 05% NaN, and 7.5% (v/v) fetal bovine serum) It is a buffer solution (pH 7.2). To measure non-specific binding (NSB), - The tube contains a 200-fold molar excess of non-standard material taken in a small volume, e.g. A large molar excess of unlabeled ligand, such as labeled FK-506, is included.

At the end of the reaction time, detection methods other than fluorescence polarization detect the labeled F bound to the protein. It is necessary to separate K-506 from free labeled FK-506. According to the present invention The preferred method is:

The contents of the A1 reaction mixture are diluted with a water-cold buffer, preferably at neutral pH, and the Transfer the contents to Whatman (page haLman) G F / B [Whatman filter paper (Whstman Paper), Maidstone, British Co., etc. Filter and wash the filter with water-cooled mild Ii solution. This membrane carries the labeled FK-506 compound bound to the receptor.

B. This method uses inactive proteins, e.g. Microporous filters, e.g. pre-washed with a solution of BSA or gamma globulin, e.g. For example, 0.22 μm nitrocellulose [Millipore Cor Similar to the method of A, except that the filtration is carried out on There is. This filter retains labeled FK-506 bound to the receptor. Ru.

After diluting the C1 reaction mixture with cold buffer, polyethylene glycol (molecules 115 ,000-20,000) +7) Suspensions, e.g. 30 mg/ml Suspension 1 (IE 1 ml and further preferably about 1 mg of carrier BSA or γ Add carrier protein solution containing globulin and mix the resulting suspension. Ru. Particles are collected by centrifugation; the pellet contains the receptor-bound label. Contains FK-506.

After diluting the D1 reaction mixture with cold buffer, add carbohydrates (e.g. dextran) or or coated with a carrier protein (e.g. albumin or gamma globulin). Add a suspension of charcoal particles to the tube, mix the suspension thoroughly, and then cool it down. Centrifuge to sediment the charcoal particles. The supernatant liquid binds to receptor proteins. Contains +1XaFK-506.

The E3 reaction mixture was typically divided into 100 μl aliquots in duplicate. A column of reasonable dimensions, for example 0.8X7. Ocm's LH-20 SEFADEX (Sephadex) [Pharmacia Fine Chemicals (Phar+5 acia Fine Chemicals), Piscataway, IIJ] Pour onto the molecular sieve matrix. This column is loaded with a small amount (e.g. about 0 Washing with 5 ml of buffer (e.g. phosphate buffered saline (pH 7,4)) labeled FK-506 bound to the receptor, e.g. in the first 2 ml. is eluted. LH-20 is a slightly aqueous matrix, and free FK-50 6 or CsA is delayed in such a matrix.

If a β-emitting radioactive tracer is used in Methods A and B, each frame The filter was placed in an LSC vial and scintillator combined with an aqueous organic solvent phase. system stem [e.g. pcss.

Amersham (Aa+ersham, Arlington Heights, IL) The amount of radioactivity is quantified. When using Methods C and D, the pellets are Ray 7: Suspended in In solution (e.g. PSCC) but alternatively dissolved in NaOH. diluted with scintillation solution and then counted. When using method E To do this, aliquots of void volume are diluted in scintillation solution and counted. do. In all methods, when +151 is the tracer, the label FK - place the filter, pellet or solution containing 506 into a counting tube; Count with a gamma ray counter.

The reporter molecule is AMPPD or AMPGD [l-ropisox (Tropi Chemiluminescent 1.2- By chemiluminescence, such as dioxetane, using methods A and B, When quantifying labeled FK-506, place the filter on a sheet of blotting paper. and place these filters on the 1,2-dioxetane Enzymes that decompose and produce light, such as alkaline phosphatase or galactona immerse in a solution of For example, if the filter is made of a piece of polyester film, Mylar] and then a Polaroid type 612 ) Transfer to a dark box containing instant film such as film. generate this film For example, a black-and-white RBP densitometer [Tobias Asson (Tobiag, As5oc, Inc.), IVEland, P^] to digitize the dark image. In method C, the pellet is treated with appropriate enzymes and supplements. buffer containing the factor (pH 7-12) until the maximum amount of luminescence is obtained. Suspend for 15-30 minutes, typically at 30°C, and place in a Turner 20 E or Berthol Clinilmat Instruments (Berthol) d with a luminometer such as C1iC11nilu insLuments). Read luminescence. In the method, the void volume is reduced using an appropriate hydrolytic enzyme and React for 15-30 minutes, typically at 30°C, until a large amount of luminescence is obtained. Quantify the luminescence using a luminometer. FK-506 with alkaline phosph The method for labeling with enzymes such as α- or β-galactosidase is , a suitable chemiluminescent group’! (AMPPD and AMPGD, respectively) ) is added to start light generation.

Based on fluorescence polarization (the principles underlying the analytical method are described by Robbing et al. and AbboLL Laborato. ries) 55 TDD X Instruction Manual . Fluorescence polarized CRBA carried out according to the invention involves fluorescein-FK-5 06 does not generate a polarized fluorescent signal because this molecule rotates freely. , the same molecule bound to the FK-506 receptor protein cannot rotate freely. It generates a signal even if it is closed or can rotate freely. Therefore, the receptor binds Free fluorescein-F K-506 is available for carrying out this type of analytical method. There is no need for physical separation.

Therefore, this analysis system includes standard or unknown FK-506, tjj-identified FK -506 (e.g., fluorescein-F K-506), and water-soluble receptors - It is necessary to incubate an initial sample containing protein and perform CRBA. It is essential. The polarized intensity of the signal 11 is inversely proportional to the analyte concentration, hence the low A patient sample containing a concentration of FK-506 analyte is at equilibrium 1 in the CPBA of the present invention. After this is achieved, add a high concentration of receptor-tank-bound tracer to the reaction mixture. It has a high degree of polarization.

Fluorescence polarization CRBA of FK-506 performed in accordance with the present invention, by Abbott. Laboratories (＾bboLt Laboratories) T D K System It can be easily applied to other systems. For this application, FK-506 , and wound sample into individual carts 1 and 1 of the TDK instrument. separate bars FK-50 containing buffer-surfactant solution and protein stabilizer in a vial 6.Recebutertanno solution, solution containing surfactant and protein stabilizer A metabolic reagent consisting of fluorescein-labeled FK-506 in the device (this Enter 4. Then, in a series of automated steps, the test sample is protein and fluorescein - mixed with FK-506. , incubate at 37 °C for a selected time until binding reaches equilibrium. be done. Then, transfer the sample to a glass tube and measure the fluorescence polarization signal. Ru.

When displaying CRBA data according to the present invention, the known FK-506 standard solution for, [bond+-cd+-NSB/bond,. sta+-NSBI100 vs. log[FK -506] [wherein, a bond. ,. is labeled FK bound with [$FK-506 at each concentration] - The total amount of 506, combined. , 1. , indicates the result in the absence of mark *F K-506. is the amount of labeled FK-506 combined, and NSB is the amount of Ijl quasi-FK-506 at each concentration. Draw a standard curve by plotting (representing non-specific binding at ).

Radioactivity, fluorescence polarization or chemiluminescence for unknowns and controls is then performed. [combined] value by standard calculation. . □／Join. +]100 [In the formula, binding (unjll is the quantitative value of receptor binding, binding, . . . is an appropriate value) Convert it to a standard control value]. The calculated ratio is then checked against the standard curve to determine the Estimate the concentration of FK-506 or FK-506-like molecules in a known sample.

The CRBA of the present invention can also be performed in a solid phase system. support matrix For example, the bottom of the wells of a microtiter plate, the walls of a tube, or the Cordon and inactivate the plastic beads with FK-506 receptor protein. brief exposure to sensitive proteins, such as drug-free serum or serum albumin thereby blocking the NSB site. Alifoot of labeled FK-506 solution is brought into contact with the coated surface, with gentle layering, and this solid surface is placed in an ice bath manner. Wash with cold buffer solution, e.g. PBS, at room temperature. Subsequently, FK-506, its metabolism Gently layer an aliquot of the patient sample containing the derivative or analogue. while in a cool place for an appropriate period of time, e.g. from 0 h (control) to 16 h (analyte). contact with a surface coated with receptor protein. bonds reach equilibrium Once done, remove the incubation solution and gently wash the solid surface with cold mild IIi solution. Wash.

Labeled FK-506 bound to protein is removed by detergent solution or alcohol. The precipitated proteins are removed from the solid surface by brief centrifugation. . Then radioactively labeled or chemiluminescently labeled FK- For 506, the amount of label is quantified as described above. regarding standards and unknowns. The calculation is performed as described above.

In order that those skilled in the art may more fully understand the invention, the following examples are set forth. It is listed. These examples are given for illustrative purposes only and are It should not be construed as expressing any limitations other than those stated in the claims.

Example I Binding of C5A and FK-506 to cytosolic proteins from CEM cells. 0. The binding of H-CsA to proteins in the OOOXg cytosol is determined using Method E (LH-20), which separates protein from unbound ligand. .

Total cytosol (1 mg protein/ml) was mixed with unlabeled CsA and FK-506. (10 μg/ml each) in the absence and presence of ['H]-CsA (60, 000 cpm). Marks that bind to cytoplasmic proteins Unlabeled CsA competed with labeled CsA, but FK-506 did not. (Figure 1).

Example 2 Specific binding to FK-506 in CEM cytosol (CEM cells) (1 mg protein/ml)-derived cytosol was mixed with unlabeled FK-506 (10 ug protein/ml). 3) (-FK-506 (60,000 c pm), labeled FK-506 bound to Tannokuku and Free labeled FK-506 was separated by Method E (LH-20). The result obtained The results (Figure 2) show that (, crude cytosol derived from EM lymphocytes is specific for FK-506. This indicates that it contains a specific binding protein.

Example 3 Purification of FK-506-conjugated tannokuri from CEM cytosol OEM cytosol assay Filter the sample and use a 250 μl or 500 μl loop to m's Neuiorad / (Bio-Rad BioSil) S E C125T S K column, buffer consisting of 20mM sodium phosphate (p H6,8) and a Beckman using a flow rate of 1.0 ml/min. was injected into a HPLC apparatus. Collect fractions, pool and rotary evaporate The protein was analyzed by the BCA method, and 'H-CsA (Fig. C-Cs A) in Figure 3 and ['H]-dihydro FK-506 (C-506 in Figure 3 ) was analyzed. CsA is approximately 150 kDa, 50 kDa and 1 FK-506 binds to a fraction showing a protein molecular weight of 7 kDa, whereas FK-506 binds to a fraction showing a protein molecular weight of about 8 to 10 Binds only to a single peak of kDa. 8~], Ok D FK in a fraction -506-specific binding activity was determined by CsA in either of its tannokuri fractions. (Fig. 3).

Example 4 JURKAT in the cytosol Direct binding profile of FK-506 The JURKAT cell line was treated with glutamine and antibiotics [Life Toxicity]. (Life Technology), Gaithersburg, MD] RPMI 1640 medium containing 10% FC3 [MA Bioproducts (Bi Walker Spill, MD] at 37°C and 5% CO Maintained at R. Cells (5 x 10”) were collected and pelleted, and added to culture medium three times. Wash and use immediately as a source of cytosolic proteins, or remove cell pellets. It was frozen at -70°C as a batch.

Cells for protein extraction were washed on ice with 0.02% sodium azide containing 0.5% sodium azide. Using M sodium phosphate (pH 6,8), make It was homogenized using a genizer. Completeness of cell disruption is monitored by trypan blue removal -I did. The homogenate manufactured by tII was transferred to Beckmano L5-6. Spin at 100.00Xg for 1 hour using a 5 ultracentrifuge and a 40.10-tar. The supernatant (S-100) was isolated. S-100 supernatant (cytosol ) and threw away the beret. Protein measurements were performed using Bradford (Br Cobas adford method 0 or Cobas Pius Bios) equipment [Roche, Nathalie, Japan] was used to This was done by coupling to Coosas ie blue. S The -100 supernatant was divided into aliquots and stored at -70°C.

A sample of cytosol from JURKAT lymphocytes contained [3H]-dihydroFK-5 Equilibrate with 06 or C5A (1, 1, 8 mol) and adjust the molecular weight of this solution. Bio-Rad Bio-3i 1 SEC 125 HPLC column at 1.0 ml/min. Collect fractions and paracard ・In Gold (Packard Gold) scintillation liquid, 7 inches of liquid Counts were made by Resigon analysis. The binding profile shown in Figure 4 is FK -506 mainly binds to proteins eluting in the molecular weight region of 8 to 10 kDa. but also the FK-506 binding protein. It shows that it was eluted in the 50kDa region. There is. CsA-binding proteins mainly eluted in two molecular weight regions, one of which was ca. One appeared at 15-17 kDa (cyclophilin), the other at about 51 kDa. 8~ No significant binding of CsA occurred in the 10 kDa region.

Example 5 Purification of pk-506 binding protein from JtJRKAT cytosol A, JtJR KAT S-100 cytosol was prepared as in Example 4 from 1 ml of S-100 cells. 0.1% sodium azide Madre equilibrated with 10mM sodium phosphate buffer (pH 6,8) containing Gel Bloom Column [A*1con Corp., Da Nverse, bro ^] I jumped to the top. The column is Q, 3cmX with a total bed volume of 3ml. It was a 7°Ocm Contes (Ni ontea) disposable column [Contes (K) ontea), Vinland, N. Stepwise concentration gradient (10,5o , 100, 200, 300 and 400 mM) sodium phosphate buffer. and collected in a 12 x 75 mm glass tube. Combine these fractions Analytically concentrated on a rotary evaporator for 0.02M sodium phosphate. Dialyzed against thorium buffer (pH 6,8) and protein concentration determined as above. It was measured as follows.

There is a large difference in the salinity required to elute each protein from this column matrix. This step allows the FK-506 receptor protein to become a CsA-binding protein. easily separated from proteins. As shown in the histogram in Figure 5, the bulk FK- The 506 receptor protein eluted at 50mM salt concentration, whereas most of the Cs A-binding protein eluted at approximately 300mM salt concentration. Also, this step may be undesirable. Also effective in removing bulk proteins; total loading of approximately 50 mg/ml Starting from , the pooled fraction representing the two binding proteins was only about 10 mg. ・ In addition, using Madrex Gel Blue A resin, a protein preparation of approximately 50 kDa was prepared. The product was prepared. Proteins of approximately 50 kDa do not bind to this matrix. This was used as a negative selection process.

A pool of approximately 5 Q kDa) that excludes the S-100 sample is separated by a 30 kDa barrier. Cutting filter [Centricon, Amicon (^self1con) ), Danvers, MA] and diluted with 80 mM sodium phosphate (pH 6,8). Adjust and chamber with 0.5-I MaLrexGel Blue A. Mixed at warm temperature for 15 minutes. This resin was made into Beckman's microscopic Spin and sediment in a centrifuge and retain the supernatant (containing approximately 50 kDa protein) did.

C, molecule! Sieving Madrex Bloom chromatography (B above) 7 clofilin (approximately 17 kDa), approximately 50 kDa binding protein or enhances either FK-506 receptor protein (approximately 8-12 kDa) (7) The concentrated fraction (5-I Omg/ml) was filtered through a 0.45μ filter. Ta. A sample volume of 11,500 μm was prepared using Biosil SEC125 60 0x7.5cm column, Biorad, Richmond, CA. Beckman's system gold program using Le Solvent Module (Beckman System Gold Pr ogrammable 5olvent Module)# 126HPLC Injected. The buffer was 0.02M sodium phosphate containing 0.1% sodium azide. The flow rate was 10 ml/min. Sample elution is Beckman Model #160 Absorbance Detector and Model #4 Monitoring was performed at 280 nm using a 27 integrator. Fractions are divided into 12X75mm Collect in glass tubes, test for binding, pool and rotary evaporate Concentrate with 5mM potassium phosphate, 0.5% sodium azide at pH 6.8. dialyzed against lium.

Separation using a D0 weak cation exchanger The molecular weight purified pool obtained in step C above ( 100-300 μg/ml) was filtered as above and 250 μl was added to Beckman ( Beckman) T S K CM-23W Spherogel ) column (4.6 mm x 26 cm) and 5 mM potassium phosphate at pH 6.8. um, 0.5% sodium azide at a flow rate of 0.5 ml 17 min.

These fractions were analyzed for binding, pooled, and rotary evaporated. It was concentrated using

/Clophilin size fraction (approximately 17 kDa) was subjected to weak cation exchange chromatography. When separated by , this peak corresponds to all 38-CsA binding activity (Fig. 7).

The FK-506 receptor fraction (approximately 8-12 kDa) was separated using the same column conditions. Upon separation, a single protein beak was obtained in 7-8 minutes and all 38-50 6 binding activity co-eluted with this peak (Figure 8).

The approximately 5QkDa fraction obtained in C above was chromatographed on a weak cation exchange column. When subjected to roughy, a single result matching the protein peak (12-13 minutes) was obtained. A combined peak was given (Figure 9).

E, 5DS-polyacrylamide gel electrophoresis Samples for electrophoresis were prepared by Laemli ( It was prepared and isolated as described by J. Laemlli.

In practice, the eluate is pooled and concentrated from the eluate by molecular weight removal or cation exchange. The reduced protein sample (3-5 μg) was added to sample preparation buffer (0,125M (pH 6,8), 3% SDS, 20% glycerin and 10% 2-mercapto ethanol) at l=1 and boiled for 3 minutes. Samples were run on a 10-20% gradient. Gel [Integrator Separation Sciences (Integr 5 Sciences), Boston, MA] , separated by constant current (30 mA). The gel was immobilized and Daichii (Daichii) was immobilized. ) Silver staining method [Ingredient Separation Sciences (Int. egratedSeparation　5sciences), Boston, M^] It was stained using. The results are shown in FIG. Lane l contains the molecular weight standard . Lane 2 is approximately 8-12 kD purified by molecular weight exclusion method (step C above) aJURK; Contains AT FK-506 receptor protein, lane 3 is weak The same material purified by on-exchange (Step D above). By this standard , the FK-506 receptor protein of approximately 8 to 12 kDa has been purified to homogeneity. It is clear that

Table 1 is cyclophilin, Table 2 is about 8-12 kDa FK-506 recebutartan Table 3 summarizes purification data for a binding protein of approximately 5 QkDa.

Table 1 Cyclophilin purification summary Purification process Capacity Protein binding Recovery rate (sl) (sg/ml) (cp Sushi g) (%) S-1oo 13 57 3.7 xlO'lo.

7 Tre 1 λ・Blue 3 7.4 1.36XIO’ 13 Scream Elimination Method 3 0.384.99X to’ 0.66 weak thione cross-row method 1 0,03 1 ．． 37XlG' 0.05F K-506 receptor protein purification summary purification process Capacity Protein binding Recovery rate (ml) (mg/ml) (cpra/* g) (%) S-1oo H571, OXIO31007 Torefukus Blue 3 10 15.4 XIO' 17.5 Size exclusion method 2 0.25 48J XIO' 0.4 weak cation exchange method 1. 0.02 1.25X 10'1 0.04 Table 3 Approximately 50kDa FK-506 binding protein purification summary purification process Capacity Protein Binding Recovery rate (ml) (sg/ml) (cps/sg) (%) S-10 020,04,463,OXIO' 10G Matre 1ri λ Blue 2.5 1 5.7 1. I XIO’ 43.9 Is Exclusion Method 1.5 1.24 7.7 XIO' 2.1 Matre, Ri1-Bu&-A 1.0 0.68 1.26X1 G' 0.71 Weak cation exchange method 3.2 0.0+3 7.7 XIO@0. The data in Table 2 is based on FK-5 obtained after cation exchange chromatography. 06 receptor protein is 1.250 times more purified than S-100 material. Which indicates that. The same purification scheme as shown in the data in Table 1 Therefore, cyclophilin was purified approximately 370 times compared to the S-100 material. table As shown in the data in Section 3, the approximately 50 kDa binding protein was purified by all purification methods. was purified only about 50 times, which is probably due to the starting material (S-1 This may reflect the high binding ability of the 000 cytosol).

Example 6 Scatchard Analysis of Binding Proteins Purified in Example 5 The bound protein is incubated with increasing amounts of labeled ligand, and the bound protein is incubated with increasing amounts of labeled ligand. The data were analyzed according to Scatchard. Chard pronods are shown in Figure 11, A-C.

Figure 11A shows direct binding of labeled CsA to a protein preparation of approximately 5QkDa. There is. The Kd of this interaction is approximately 54 nM, and the binding capacity is approximately 3.2 nmole/ mg protein. No cooperative binding was observed.

Figure 11B shows the direct addition of labeled 1iFK-506 to the same ~50 kDa protein preparation. Shows a bond. This interaction has a Kd of about 2 nM and a Kd of about 0.3 nanomorphs. mg protein binding capacity.

I fiber F K-506, its main receptor tongue 7 (approximately 8 to 12, D When binding to a) was analyzed, the Kd was approximately 1.3 nM, and the binding ability was approximately It was 0.5 nanomol/mg protein.

No cooperativity was observed in the interaction between FK-506 and any of its binding proteins. I couldn't.

Example 7 Receptor binding analysis of FK-506 in whole blood Binding analysis Purified JURKAT cell receptor of approximately 8-12 kDa obtained in Step C of Example 5 Binding of '1(-F　K-506 to -proteins was performed as in Example 2.

Here, receptor-bound FK-506 is obtained using the LH-20 method [see separation method E in the specification]. Separated by light. Binding buffer was 5mM 2-mercaptoethanol, 0 ．． 20mM Tris buffer (p It consisted of H7, 2) and was LX.

The receptor solution (100ul of 15μg/ml protein) was FK-506 (approximately 2 nM, 25.OOOCPM) and O ~ 70 μg/m+ non- [fiF K-506 in a 12 x 75 mm glass tube for 20 minutes at room temperature. Mixed and then placed in water. 100 μl of the cold reaction mixture was plated in binding buffer. 7. of equilibrated LH-20. Concentrate by applying to QcmXO, 8cm column. The combined and 11111 ligands were separated. receptor binding ligand Collect the first 2 ml of eluate containing the sample and place it in a scintillation vial for counting. I put it in the file. The CPM was corrected back to the total volume of the reaction mixture for at least two reactions. Expressed as the average value of repeat experiments. The standard curve thus obtained is shown in FIG. 12A.

Analysis of FK-506 in whole blood Unrecognized FK-506 was added to human whole blood at concentrations ranging from 0.5 to 10 ng/ml. . This blood 1. Qml Alico Nodo with 3ml acetonitrile (100%) , FK-506 is extracted from blood by vigorous mixing and extraction for 30 minutes. precipitated proteins were removed by centrifugation at 2.OOOXg. . The supernatant liquid was transferred to a 16 x 100 mm test tube, and the solvent was evaporated with air flow at 40 °C. I set it. The residue was dissolved in 100 μM binding buffer and the approximately 8-12 kDa protein Competitive receptor binding assays were performed as described above.

The results are shown in Figure 12B.

This analysis shows that the concentration range of FK-506 in whole blood is at least 0 to 5. On g/ml (Figure 12B). This 1 degree range is shown in Figure 1. It should be noted that within the first 10% of the standard curve shown in 2A, this This means that the useful range for the analysis of FK-506 in blood is based on the data in Figure 12B. This suggests that the range exceeds the range indicated by .

The above discussion of the invention is directed primarily to preferred embodiments and implementation thereof. . Further variations and modifications in the practical implementation of the concepts herein described. departs from the spirit and scope of the invention as specified in the following claims. It will be readily apparent to those skilled in the art that this can easily be done without any additional modifications.

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FIG. 1 FIG.2 FIG.3 fraction number FIG.4 minutes FIG. 5 Salt concentration (mM) FIG. 6A IQ 15 20 25 30 minutes FIG.6B to 15 20 25 30 minutes FIG.7 minutes FIG.8 minutes minutes Binding CsA nM Ku FIG, 12A Unlabeled FK-506 (ng/mL) Blood palpitation product (ng/mL) summary Competitive receptors for F K-506 and its biologically active metabolites, derivatives and analogs A method for targeting FK-506 action is disclosed in which the binding reagent is a target cell for FK-506 action. cells, especially in newly harvested or established cell lines, normal or purified from the soluble cytoplasm of transformed lymphocytes or recombinant DNA A specific FK~506 receptor protein synthesized by A technology. melt Liquid phase and solid phase analysis systems are disclosed.

Submission of translation of written amendment (Article 184-7, Paragraph 1 of the Patent Act) 1992 1198 Hidden

Claims (55)

[Claims] 1. (a) Homogenized and disrupted normal or transformed mammalian cells are centrifuged. separating to produce a water-soluble protein; (b) fractionating the water-soluble protein; (c) The protein fraction thus obtained was tested for FK-506 receptor activity. test; and (d) Resolve protein fractions with acceptable FK-506 receptor activity. prepared by a method characterized in that it comprises the steps of selecting for Scepter binding analysis; purified FK-506 receptor for receptor binding analysis to FK-506 Tartan protein. 2. 2. The receptor protein of claim 1, wherein said mammalian cell comprises a lymphocyte. 3. 3. The receptor protein of claim 2, wherein said lymphocytes include peripheral blood lymphocytes. 4. 4. The receptor protein of claim 3, wherein said lymphocytes include tissue culture lymphocytes. 5. The fractionation separates the water-soluble proteins. (a) an affinity chromatography column; (b) at least one molecule and (c) a cation exchange column. Receptor protein of claim 1. 6. Claim 5 further comprising passage through a hydrophobic interaction chromatography column. receptor protein. 7. The fractionation involves separating the water-soluble proteins into an affinity chromatography column. 2. The receptor protein of claim 1, comprising passing through the receptor protein. 8. 8. The method of claim 7 further comprising passage through at least one molecular weight exclusion gel column. receptor protein. 9. Claim 8 further comprising passage through a hydrophobic interaction chromatography column. receptor protein. 10. The fractionation involves transferring at least one of the soluble proteins to a molecular weight exclusion gel column. 2. The receptor protein of claim 1, comprising passing through the receptor protein. 11. 11. The receptor tank of claim 10 further comprising passing through a cation exchange column. Park. 12. Claims further comprising passage through a hydrophobic interaction chromatography column. 11 receptor proteins. 13. The fractionation involves passing the soluble protein through a cation exchange column. The receptor protein of claim 1 comprising: 14. Claims further comprising passage through a hydrophobic interaction chromatography column. 13 receptor proteins. 15. The fractionation separates the soluble proteins from a hydrophobic interaction chromatography column. 2. The receptor protein of claim 1, comprising passing the protein through a membrane. 16. The protein ranges from about 1,000 Daltons to about 200,000 Daltons. 2. The receptor protein of claim 1 having a molecular weight within. 17. The protein is within the range of about 8,000 Daltons to about 12,000 Daltons. 2. The receptor protein of claim 1 having a molecular weight of . 18. The protein ranges from about 40,000 Daltons to about 60,000 Daltons. 2. The receptor protein of claim 1 having a molecular weight within. 19. The test for acceptable FK-506 receptor activity is performed using various amounts of Place for the labeled FK-506 to bind to the FK-506 receptor protein 2. The method of claim 1 comprising a competitive binding assay in which the label FK-506 competes with a constant tracer concentration. receptor protein. 20. Receptor binding analysis of FK-506 prepared by recombinant DNA technology Purified FK-506 receptor protein for. 21. The protein ranges from about 1,000 Daltons to about 200,000 Daltons. 21. The receptor protein of claim 20 having a molecular weight within. 22. The protein is within the range of about 8,000 Daltons to about 12,000 Daltons. 21. The receptor protein of claim 20, having a molecular weight of . 23. the protein ranges from about 40,000 glutons to about 60,000 daltons 21. The receptor protein of claim 20 having a molecular weight within. 24. (a) preparing an extract of an initial liquid sample containing FK-506; (b) the FK-506 and tracer concentration of labeled FK-506 in the extract; , an aqueous solution of purified FK-506 receptor protein, and the binding is in equilibrium or steady state. (c) Labeled tracer FK not bound to the receptor Physically separate the labeled tracer FK-506 bound to the receptor from -506 death; (d) the labeled tracer specifically bound to the FK-506 receptor protein; quantifying the amount of FK-506; (e) determining the amount of FK-506 in step (d) above; The amount of labeled tracer FK-506 that specifically bound to the protein protein was quantified. The amount of FK-506 The initial liquid sample is compared to a standard curve prepared by analyzing standard solutions. The amount of FK-506 in [B[mtd]-NSB/B(o mtd)-NSB] versus log[FK] [where B(mtd) is is the amount of the standard tracer bound, B(o mtd) is the control and NSB is the non-specific binding of the sample of the labeled tracer and [FK] is the FK- 506 concentration of the standard solution] Quantitative receptor analysis method for FK-506 contained in an initial liquid sample. 25. The extract is obtained by contacting the initial liquid sample with an amphipathic organic solvent. 25. The method of claim 24, wherein the method is prepared by: 26. 2. The marking in the labeled tracer FK-506 is radioactive. Method 4. 27. the radioactivity is selected from the group consisting of alpha-, beta-, or gamma-ray emitters; 27. The method of claim 26. 28. The label in the labeled tracer FK-506 emits luminescence. 25. The method of claim 24, which is a possible compound with. 29. Possibility of the marking in the marking tracer FK-506 emitting fluorescence 25. The method of claim 24, wherein the compound is 30. The marking fabric in the labeled tracer FK-506 is capable of emitting color. 25. The method of claim 24, wherein the compound is a compound. 31. 25. The marker in the labeled tracer FK-506 is an enzyme. Method. 32. The purified FK-506 receptor protein is according to any one of claims 1 to 23. 25. The method of claim 24, prepared as described. 33. Labeled tracer FK-5 bound and unbound to said receptor The method of physically separating 06 includes filtration using a filter or membrane; From the group consisting of adsorption with charcoal, protein precipitation or filtration with molecular weight exclusion columns. 25. The method of claim 24, wherein the method is selected. 34. 25. The method of claim 24, carried out in a solid phase system. 35. The purified FK-506 receptor protein was coated onto a solid phase surface, and the The FK-506-containing extract and the marking tracer FK- of the initial liquid sample Until the binding of 506 to the nuclear receptor protein coat reaches an equilibrium or steady state. the amount of the textile tracer FK-506 contacted and bound to the receptor coat is The weight of FK-506 in the initial liquid sample is quantified and the weight of FK-506 in the initial liquid sample is determined by the amount bound to the receptor. prepared by replacing the initial liquid sample extract with a series of FK-506 standard solutions in the analysis. 35. The method of claim 34, wherein the method is quantified by comparing with a prepared standard curve. 36. (a) preparing an extract of an initial liquid sample containing FK-506; (b) The FK-506 and phosphor-textured FK-506 in the extract are purified from purified FK-506. -506 receptor protein in aqueous solution until binding reaches equilibrium or steady state. contact with; (c) Measuring the fluorescence polarization signal of the mixture thus incubated. , converting the fluorescence polarization signal into the amount of phosphor-based FK-506 bound to the receptor. and (d) a series of FK-506 standard solutions in the fluorescence polarization receptor binding assay. The initial liquid sample is compared with the fluorescence polarization standard curve obtained by analyzing the liquid. Determine the concentration of FK-506 in [B(mtd)/B(o mtd)] 100 versus log[FK] [where B(mtd)/B(o mtd) and [FK ] has the same meaning as in claim 13. Quantitative receptor analysis method for FK-506 contained in a characteristic initial liquid sample . 37. The extract is obtained by contacting the initial liquid sample with an amphipathic organic solvent. 37. The method of claim 36, wherein the method is prepared by: 38. The phosphor is selected from fluorescein, luciferin, and europium. 37. The method of claim 36, wherein the method is selected from the group consisting of: 39. The purified FK-506 receptor protein is according to any one of claims 1 to 23. 37. The method of claim 36, prepared as described. 40. (a) preparing an extract of an initial liquid sample containing FK-506; (b) Convert the FK-506 and enzyme-labeled FK-506 in the extract to purified FK-506. 506 receptor protein in aqueous solution until binding reaches equilibrium or steady state. bring into contact; (c) Enzyme-labeled FK-506 that is not bound to the receptor binds to the receptor physically separating the enzyme-labeled FK-506; (d) physically separating the enzyme-labeled FK-506; quantifying the amount of the enzyme label FK-506 differentially bound; (e) the enzyme specifically bound to the nuclear receptor protein in step (d) above; The quantified amount of standard fabric FK-506 was added to the competitive receptor in steps (a) to (d) above. Standard curve prepared by analyzing a series of FK-506 standard solutions in a binding assay Determine the amount of FK-506 in the initial liquid sample by checking with the line and [ B(mtd)]-NSB/B(o mtd)-NSB]100 vs. log[FK] [wherein the terms B(mtd), NSB, B(o mtd) and [FK] are The initial stage is characterized in that it consists of the step of plotting the following: Quantitative receptor analysis method for FK-506 contained in liquid samples. 41. The extract is obtained by contacting the initial liquid sample with an amphipathic organic solvent. 41. The method of claim 40, wherein the method is prepared by: 42. The purified FK-506 receptor protein is according to any one of claims 1 to 23. 41. The method of claim 40, prepared as described. 43. 41. The method of claim 40, wherein said quantifying comprises chemiluminescence methods. 44. The chemiluminescence method uses enzymatically degradable water-soluble chemiluminescent substances. Coupling a certain 1,2-dioxetane derivative to the enzyme-labeled tracer FK-506 decomposition with said enzyme to provide optically detectable light energy. The method of claim 43. 45. the enzyme is α- or β-galactosidase, and the chemiluminescent 45. The method of claim 44, wherein the gas substrate is AMPGD. 46. the enzyme is a phosphatase and the chemiluminescent substrate is AMPP 45. The method of claim 44, wherein D. 47. 41. The method of claim 40, carried out in solid phase. 48. (a) preparing an extract of an initial liquid sample containing FK-506; (b) said extract of said initial liquid sample and chemiluminescent compound labeled FK-50; 6 with an aqueous solution of purified FK-506 receptor protein, and the binding is at equilibrium or constant. (c) chemiluminescent material not bound to the receptor; Chemiluminescent compound label bound to receptor from chemical compound labeled FK-506 physically separate the identification FK-506; (d) quantifying the amount of the FK-506 specifically bound to the receptor; (e) the label specifically bound to the receptor obtained in step (d) above; The quantified amount of FK-506 was transferred to the competitive receptor binding in steps (a) to (d) above. The standard curve prepared by analyzing a series of FK-506 standard solutions in the analysis Check to determine the amount of FK-506 in the initial liquid sample and [B( mtd)]-NSB/B(o mtd)-NSB]100 vs. log[FK][Eq. The terms medium, B(mtd), NSB, B(o mtd) and [FK] are An initial liquid characterized by comprising a step of plotting [same meaning as in Item 13] Quantitative receptor analysis method for FK-506 contained in a sample. 49. The extract is obtained by contacting the initial liquid sample with an amphipathic organic solvent. 49. The method of claim 48, wherein the method is prepared by: 50. The purified FK-506 receptor protein is according to any one of claims 1 to 23. 49. The method of claim 48, prepared as described. 51. The quantifying includes quantifying the chemiluminescent compound labeled FK-506. The chemiluminescent compound is contacted with a specific hydrolytic enzyme and the light produced 49. The method of claim 48, comprising measuring optically detectable light energy. 52. The chemiluminescent compound is AMPGD, and the hydrolase is α - or β-galactosidase. 53. The chemiluminescent compound is AMPPD and the hydrolytic enzyme 52. The method of claim 51, wherein said sphatase is a sphatase. 54. 49. The method of claim 48, carried out in solid phase. 55. (a) Purified FK-506 receptor according to any one of claims 1 to 23. Npaku; (b) Standard fabric FK-506; and (c) FK-5 in an initial liquid sample characterized by consisting of FK-506 standard product Quantitative receptor analysis kit for 06.