JP3240135B2 - FK-506 receptor assay - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing drugs in biological fluids, and more particularly, to the immunosuppressant FK-506, and biologically active metabolites and derivatives thereof. And analog competitive receptor protein binding assays.

2. Description of the Background Art Cyclosporine, a fungal-derived cyclic undecapeptide immunosuppressant, is widely used as an anti-rejection drug for solid organ transplant patients. Cyclosporine (Cs
A) appears to be T-cell specific and induces its immunosuppressive effects by inhibiting interleukin 2 production and receptor expression ¹ . CsA, at the dosage needed to prevent rejection in the patient's organs,
Gastrointestinal and metabolic problems, hypercholesterolemia, signs and symptoms of the nervous system, and to exhibit severe toxic side effects including nephrotoxicity known ^2-6. As a result, research is actively being conducted into alternatives to immunosuppressants that have greater efficacy at lower dosages than CsA and that have fewer toxic side effects. The compound known as FK-506 is
Such a drug.

FK-506 is a registered trademark of Fujisawa Pharmaceutical Co., Ltd. of Japan.
ceutical Co.) isolated from Streptomyces tsukubaensis (822 kDa)
⁷ is a macrolide antibiotic. The structure is completely different from CsA (see Structure I), FK-506 shows similar, if not identical, anti-lymphocyte activity. However, the immunosuppressive activity of F-506 is about 100 times higher than that of CsA ⁸ , and it has been used successfully for solid organ transplant patients, especially liver transplant patients ⁹ .

The toxicity of FK-506 in human patients and experimental animals is CsA
Although reported ^lower8,10 , the dosage required for anti-rejection activity, FK-50 in such species
There are reports of 6 unwanted side effects ^2-4,10 .

The need to balance dosing requirements to prevent rejection with avoiding severe toxic side effects has resulted in FK-506.
The dosages and plasma concentrations, as in the case of CsA, carefully, must constantly monitor ^11. For this, it is necessary to monitor not only the original FK-506, but also the plasma concentration of a bioactive FK-506 metabolite produced by metabolism in the liver according to report ¹² . Therefore,
FK-506 and its bioactive metabolites, as well as accurate bioactive analogs and derivatives to be used in pharmacokinetic studies and setting dosages to patients,
There is a great need for sensitive, specific, yet rapid and inexpensive analytical methods.

Tamura (by Tamura) et ¹³ and Kyadofu (Cadoff) et ¹⁴
Describes a one- and two-step enzyme-linked immunoassay on solid phase against FK-506 using a rabbit polyclonal or mouse monoclonal antibody raised against an antigen of bovine serum albumin-FK-506 complex ( ELISA). Several problems with this method have been reported: (1) its sensitivity is highly dependent on the plasma concentration of FK-506 required before extracting the drug from the patient's plasma with an organic solvent such as benzene. (2) it is not possible to distinguish FK-506 from some of its metabolites that are immunologically active but may not be biologically active; (3) Low accuracy due to low concentration of drug in plasma and inaccurate ELISA; ¹⁴ ; and (4) a correlation between dose measured by this assay and plasma concentration ¹⁴ .

¹⁵ Zebi (Zeevi) et ¹⁵ has developed a biological assay of plasma FK-506, this analysis method is based on the proliferative response inhibition of alloreactive T cell clones by this drug. The authors reported that for all plasma samples tested, the values obtained in the above ELISA methods ¹³ , ¹⁴ for FK-506 were consistently much higher than those obtained in the bioanalytical method. . In this case, the patient's blood is bioinactive FK that is detected by ELISA but not by bioanalytical methods.
-506 metabolites. That is, since the ELISA is an immunoassay that detects any molecule having an appropriate epitope site, biologically inactive metabolites may cross-react with the anti-FK-506 antibody.

There are several problems with such FK-506 bioanalytical methods. This assay does not identify immunosuppressants. Therefore, to prevent allogeneic transplant rejection, FK-50
Steroids such as prednisone administered concurrently with 6 (or CsA) are immunosuppressive and will respond positively in any bioassay based on suppression of T cell proliferation. In addition, bioanalytical methods are not readily adaptable to normal use in hospital clinical laboratories: bioanalytical methods have low data throughput; bioanalytical methods are slow and sensitive to work; and tissue culture. Due to the biological changes between these cells, such cells become less than desirable as reagents.

Thus, there is still a great need for rapid, inexpensive, accurate, sensitive, and specific assays for FK-506 and its biologically active metabolites, derivatives and analogs. Such an assay has now been discovered and is disclosed below.

SUMMARY OF THE INVENTION FK-506 and FK-506 in a biological fluid sample
A new method for quantifying such functional activities has been discovered. The method comprises a competitive protein binding assay in which the binding reagent is a purified soluble receptor protein isolated from a target tissue for FK-506 action.

Accordingly, it is an object of the present invention to provide a purified water-soluble receptor protein for competitive binding assays specific for FK-506 and its biologically active metabolites, derivatives and analogs.

Further, using the purified receptor protein described above,
It is also an object to provide aqueous and solid phase based competitive receptor binding assays for FK-506 and its biologically active metabolites, derivatives and analogs.

It is yet another object of the present invention to provide labeled FK-506 molecules suitable for use in the competitive receptor binding assays of the present invention.

These and other objects of the present invention will become apparent from the following description of preferred embodiments thereof and from the claims.

DETAILED DESCRIPTION OF THE DRAWINGS FIG. 1 compares the binding of FK-506 and CsA to CsA binding proteins in the cytosol of CEM lymphocytes.

FIG. 2 shows FEM against cytosolic protein of CEM lymphocytes.
2 shows specific binding of K-506.

Figure 3 shows HPL on a BioRad TSK column.
FIG. 2 shows the elution profile of the FK-506 binding protein fraction fractionated from CEM lymphocyte cytosol by C molecular weight sieving chromatography.

FIG. 4 shows BioRad BioSil (BioSi
l) FK-506 from cytosolic of JURKAT lymphocytes by Beckman HPLC on SEC125 column. And CsA 2 shows the fractionation of bound proteins. Also shown are molecular weight markers.

Figure 5, Matrex Gel Blu
e) FUR-5 of JURKAT lymphocytes from A affinity column
06 And CsA Figure 3 shows the elution profile of bound protein as a function of salt concentration.

Figures 6A-B show Matrex Blue
A isolated by affinity chromatography
Figure 4 shows HPLC fractionation of pooled CaA and FK-506 bound fractions on a BioRad SEC125 molecular weight exclusion column. FIG. 6A shows the size exclusion profile of cyclophilin (about 17 kDa) and FIG. 6B is for the FK-506 receptor protein of about 8-12 kDa.

FIG. 7 shows a weak cation exchange chromatography HPLC column of the 17 kDa cyclophilin of FIG. 6A [Beckman
n) TSKCM-25W Sephalogel (Spherogel)].

FIG. 8 shows a weak cation exchange chromatography HPLC column of the FKP506 8-10 kDa binding protein of FIG. 6B [Beckman TSK CM-25W cephalogel (Spherogel)].
Show the above classification.

FIG. 9 shows the fractionation of the approximately 50 kDa protein eluted from the size exclusion column on a weak cation exchange column.

FIG. 10 shows an analytical SDS-PAGE of the purified JURKAT approximately 8-12 kDa receptor protein.

FIGS. 11A-C show an approximately 50 kDa protein preparation (FIG. 11A), FK-506 binding to the preparation (FIG. 11B), and FK-506 binding to its approximately 8-12 kDa receptor protein.
Scatchard of CsA binding to (Figure 11C)
ard) shows a plot.

FIGS. 12A-B show standard curves for receptor analysis of FK-506 in water (FIG. 12A) and human whole blood (FIG. 12B) using purified 8-12 kDa JURKAT cell receptor protein as a binding reagent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "receptor" is capable of recognizing and selectively binding certain ligands and, after binding the ligands, is the beginning of an event chain that results in a biological response It is generally accepted to mean macromolecular or glycoproteins capable of generating certain chemical or physical signals. Blecher, M. et al., "Receptors and Human Diseases," Williams and Wilkens.
ms & Wilkens), Baltimore, 1981, Chapter 1.

Accordingly, the water-soluble binding protein used as a reagent in a competitive receptor binding assay for FK-506 and its biologically active metabolites, analogs and derivatives (hereinafter collectively referred to as FK-506) is a characteristic of the natural receptor protein. Some of which are specific, saturable,
And having reversible binding is an important aspect of the present invention.

Water-soluble specific receptor proteins suitable for CRBA performed in accordance with the present invention can be grown from mammalian lymphocytes, including human or animal peripheral blood lymphocytes (PBL) ¹⁶ , primary mixed lymphocyte cultures, and organ transplant biopsies. Proliferating allogeneic T cells ^15,17 and established cell lines, such as the DQw1 specific allogeneic T cell clone DB29 ¹⁸ ,
Transformed T helper cell line (CEM) and interleukin 2 producing JURKAT human lymphocytic leukemia T cell line (JU
RKAT 77.6.8) and can be advantageously prepared from extracts of target tissues that have FK-506 action, such as ¹⁹ . Furthermore, FK-506 does not bind or inhibit peptidyl-prolyl cis-trans isomerase in the 17 kDa cyclophilin fraction, but
Do so for the isomerase activity of the approximately 8-12 kDa FK-506 binding protein ^{19 in} T cells. This enzyme
It is very sensitive to CsA and is considered to be the primary receptor for CsA action ^20-22 .

It must be emphasized that the particular cellular source of the receptor protein used in the CRBA of the present invention is not critical-that is, the requirement for this protein is that of the binding properties of the physiological receptor detailed above. It's just showing.

The cells are disrupted and soluble proteins (cytosolic proteins) may be fractionated by art-recognized methods. Typically, lymphocytes are collected, washed, and counted. Cells may be stored at −70 ° C. as a pellet until use. The cells are thawed and homogenized at ice bath temperature using a Teflon or frosted glass homogenizer. The integrity of cell destruction can be tested by trypan blue elimination. The homogenate is subjected to at least 20,000 × g, preferably at least 100,
Centrifuge at 000 × g for at least 0.5 hour. The supernatant contains both FK-506 receptor protein and cyclophilin, but is analyzed for protein content and binding activity and used immediately or stored frozen at a temperature of about -20 ° C or less.

The cytosol can be used as is for CRBA performed according to the present invention. However, preferably, FK-
506 receptor protein is purified from the cytosol,
It is concentrated before use. The FK-506 receptor protein can be partially purified by affinity chromatography. A preferred affinity column is Matrix Gel Blu
e) A [Amicon Corp., Danvers, M
A], FK-506 and CsA binding protein can be easily separated by a salt buffer concentration gradient. The cytosol can also be separated by a molecular weight exclusion method including an HPLC column. For example,
A preferred molecular sieving method is the Beckman Instrument Co. HPLC instrument and the appropriate size preparative Biorad Biosil.
A sil) SEC125 column is used (see Example 6 below). Also, FK-506 receptor protein can be obtained by cation exchange chromatography, for example, weak cation exchange Beckman TSK CM-25W spherogel (Spherog).
It can also be advantageously purified by el). A hydrophobic interaction chromatography matrix is also suitable for purifying FK-506 according to the present invention. Any sequence or combination of these fractionation systems can be used in accordance with the present invention, as long as appropriate purification is performed. For example, purification by sequences such as cytosol, affinity matrix, molecular weight exclusion gel and weak cation exchanger is particularly preferred. For the analysis of the present invention, in the case of chromatography, if a single peak represents an elution pattern of both protein and binding activity, then SDS-P
In the case of AGE, the appearance of the only major protein band indicates that the receptor protein has been purified. The fractions of the column eluate are collected, pooled, and concentrated on a rotary evaporator under cooling vacuum. The concentrate is analyzed for protein by a suitable method, including the Bradford BCA method. The concentrate is also tested for analytical suitability by CRBA.

It must be emphasized that the FK-506 receptor protein is prepared and purified by techniques other than isolation from mammalian target cells. For example, receptor proteins may be advantageously synthesized by recombinant DNA techniques ²³ which is recognized in the art. Briefly, the cDNA encoding the receptor may be isolated from a purified mRNA or cDNA cloning library and then cloned into a cell, eg, a prokaryotic cell, to produce large amounts of cDNA. The expression vector may be constructed to include the cDNA for the FK-506 receptor, promoter and gene regulatory sequences, translation initiation codons, selectable markers, etc., and then inserted into a prokaryotic or transformed eukaryotic cell. . These cells express the receptor gene and secrete large amounts of the receptor protein into the growth medium, from which they can be isolated by conventional methods.

For the purposes of the CRBA according to the invention, the protein fraction is:
(1) the protein binds to FK-506 to a statistically significant degree based on the method of detection, eg, radioactivity, fluorescence polarization, chemiluminescence, etc .; (2) unlabeled and labeled FK-506 Compete with each other for specific binding sites on the receptor protein; and (3) the signal to noise ratio, ie, the ratio of total binding to non-specific binding (as defined below) is at least 1.1. And preferably at least about 1.2 appears to be acceptable.

Gel protein electrophoresis and Western blot analysis may be used to monitor and identify the purity of the receptor protein during all stages of purification.

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 soluble in aqueous amphipathic solvents such as aqueous methanol. ^[3 H] - dihydro FK
-506 is obtained by exposing natural FK-506 to tritium gas in the presence of a reducing agent such as tris (triphenyl-phosphine) rhodium I chloride, followed by normal- and reverse-phase chromatography [Amersham (Amersham). Corp.),
Arlington Heights, IL]. According to three different TLC systems, [ ³ H] -dihydroFK- was more than 98% pure.
One preparation of 506 had a specific activity of 51 Ci / mmol or 63.2 mCi / mg. This step added one hydrogen atom and one tritium atom to the molecule.
[Mebmt-β- ³ H] cyclosporin A ( ³ H-CsA) having a specific activity of 5 to 20 Ci / mmol was obtained from Amersham.
m Co.).

K-506 also emits γ-rays by short-term reduction with chloramine-T in the presence of Na ¹²⁵ I.
It can also be labeled with ¹²⁵ I. ¹²⁵ I-labeled histamine-FK
-506 may be prepared by the method of Wong (Wong) al ^24.

For example, Abbott Laboratories
atories, Inc.) As a fluorescence polarization detection method suitable for use with the TDX device of [Abbott Park, IL], a phosphor-labeled FK-506 may be used. FK-506 may be labeled with a fluorophore by art-recognized method ²⁵ . Suitable fluorophores include fluorescein, europium and luciferin.

Chemiluminescent labels such as water-soluble 1,2-dioxetane, which release light energy upon cleavage by a hydrolase, can be obtained from Tropix, Inc. (Belford, MA). Chemiluminescence is also a detection method when the FK-506 label is an enzyme that emits light upon addition of a substrate such as 1,2-dioxetane described above.

CRBA practiced according to the present invention can be performed by either a solution phase method or a solid phase method. The principles underlying both methods are the same. Briefly, the competition equilibrium is determined by comparing the tracer amount of labeled FK-506 with a given amount of the FK-506.
Analyte FK for binding to -506 receptor protein
Occurs between unknown samples, including -506. After reaching equilibrium, the amount of labeled FK-506 bound to the receptor protein is measured. The amount of label bound to the receptor was unlabeled FK-
Although reduced in the presence of 506, this reduction is proportional to the amount of unlabeled analyte present in the unknown sample. The quantitative relationship between the loss of label bound to the receptor and the concentration of the analyte in the unknown sample is determined by reference to a standard curve. To generate a standard curve, a given amount of receptor protein was prepared from zero to supersaturated concentrations of standard FK-50.
Exposure to the indicated tracer concentration of labeled FK-506 in the presence of 6. The supersaturation concentration is ideally several orders of magnitude greater than the association constant Ka for specific binding, and this ratio ("non-specific binding", NS
B) is assumed to be the same for all ligand concentrations since non-specific binding is assumed to be a linear relationship of ligand concentration.

For standard curve analysis in solution phase, unlabeled FK-506
The amount of the added FK-506; the hydroalcoholic (e.g., 50% ethanol in water) were placed in a glass tube an aliquot of the solution, for example by mild N ₂ flow or by cooling under reduced pressure, the solvent is evaporated, Ranges from 0ng to about 100,000ng. For solution phase CRBA of FK-506 in the blood, whole blood amphiphilic organic solvents, such as lower alkanols (e.g., a first linear or branched C ₁ -C _6, secondary or tertiary (Alcohol) or acetonitrile. "Amphiphilic organic solvent" shall mean a liquid organic compound having both hydrophobic and hydrophilic properties. The precipitated protein is removed, for example, by centrifugation,
The extract containing FK-506 is evaporated to dryness as described below. The residue to be analyzed is made up of a small amount of water / amphiphilic organic solvent, for example 1:
Take in the mixture of 3 and transfer to a reaction tube. Each tube contains a small amount (eg, about 50 μl) of stock solution,
Predetermined tracer amount (for example, 0.5 nM, 50,000-100,000 CP
M) Add labeled FK-506, for example [ ³ H] -dihydroFK-506. Each tube is then filled with a small amount, for example, about 10
A solution of the receptor protein in 0 μl to 200 μl of binding buffer is added and the tubes are allowed to stand for approximately 20-90 minutes, until binding has reached equilibrium or steady state.
Incubate at a slightly higher temperature, such as 30 ° C to about 40 ° C. The composition of the binding buffer is not critical. A preferred binding buffer is 5 mM 2-mercaptoethanol, 0.05
20 mM Tris buffer (pH 7.2) containing% NaN ₃ and 7.5% (v / v) fetal calf serum. To measure non-specific binding (NSB), a set of tubes contains a small volume, eg,
A large molar excess of unlabeled ligand is included, such as a 200-fold molar excess of unlabeled FK-506 in 50 μl.

At the end of the reaction time, for detection methods other than fluorescence polarization,
The labeled FK-506 bound to the protein is replaced with the free labeled FK-506.
Need to be separated from Preferred methods according to the invention include: A. The contents of the reaction mixture are diluted with ice-cold buffer, preferably at neutral pH, and the contents are diluted with Whatman G.
Filter through a glass fiber filter such as F / B (Whatman Paper, Madestone, UK) and wash the filter with ice-cold buffer. This membrane retains the labeled FK-506 compound bound to the receptor.

B. This method is used to block non-specific sites.
A microporous filter, previously washed with a solution of an inert protein, e.g. BSA or gamma globulin, e.g. 0.22μ
mNitrocellulose [Millipore Cor
p.), Bedford, MA]
Similar to method A. This filter retains labeled FK-506 bound to the receptor.

C. After diluting the reaction mixture with cold buffer, a suspension of polyethylene glycol (molecular weight 5,000-20,000), e.g.
1 ml of the g / ml suspension and, more preferably, a solution of the carrier protein containing about 1 mg of the carrier BSA or gamma globulin are added and the resulting suspension is mixed. The particles are collected by centrifugation; the pellet contains labeled FK-506 bound to the receptor.

D. After diluting the reaction mixture with cold buffer, add a suspension of charcoal particles coated with a carbohydrate (eg, dextran) or a carrier protein (eg, albumin or gamma globulin) to the tube and allow the suspension to fully elute. And then centrifuged under cooling to precipitate the carbon particles. The supernatant is labeled FK bound to the receptor protein.
-506.

E. The reaction mixture is typically run in duplicate in 100 μl aliquots per run, using a reasonably sized column, such as 0.8 ×
7.0 cm of LH-20 Sephadex [Pharmacia Fine Chemi
cals), Piscataway, NJ]. The column is loaded with a small volume (eg, about 0.5 ml of buffer (eg, phosphate buffered saline (pH
After washing in 7.4)), labeled FK-506 bound to the receptor elutes in the void volume, for example, the first 2 ml. LH
-20 is a weakly hydrophobic matrix, free FK-506
Or CsA is delayed in such a matrix.

If a beta-emitting radioactive tracer is used in methods A and B, each filter is placed in an LSC vial and a scintillation system combining an aqueous organic solvent phase [eg PCSS, Amersham,
Arlington Heights, IL] and the amount of radioactivity is determined. When using methods C and D, suspend the pellet in a scintillation solution (eg, PSCC) or
Alternatively, dissolve in NaOH, dilute with scintillation solution and then count. When using Method E, aliquots of void volume are diluted in scintillation solution and counted. In all methods, when ¹²⁵ I is the tracer, place the filter, pellet or solution containing labeled FK-506 into a count tube,
Count with a γ-ray counter.

Using methods A and B by chemiluminescence such that the reporter molecule is a chemiluminescent 1,2-dioxetane such as AMPPD or AMPGD [Tropix, Inc., Bedford, Mass.] Label FK-50
When quantifying 6, the filters are placed on a piece of blotting paper and the filters are exposed to a solution of an enzyme that hydrolyzes 1,2-dioxetane to generate light, such as alkaline phosphatase or galactosidase. Immerse. Transfer the filter to a piece of polyester film [eg, Mylar]
It is then transferred to a dark box containing an instant film, such as a Type 612 Polaroid film. After exposing this film with the generated light, for example, a black and white RBP densitometer [Tobias, Assoc., Inc.,
Digitize the dark image using Iveyland, PA]. In Method C, the pellet is suspended in a buffer (pH 7-12) containing the appropriate enzymes and cofactors, typically at 30 ° C. for 15-30 minutes until the maximum amount of luminescence is obtained, and the mixture is turned with Turner. ) 20E or Berthold Clinilumat inst
Read the luminescence with a luminometer such as ruments). In method D, the void volume is typically increased with the appropriate hydrolase until the maximum amount of luminescence is obtained.
The reaction is performed at 30 ° C. for 15 to 30 minutes, and the luminescence is quantified using a luminometer. When FK-506 is labeled with an enzyme such as alkaline phosphatase or α-galactosidase, a suitable chemiluminescent substrate (AMPP, respectively) is used.
D and AMPGD) to start light generation.

The principles underlying fluorescence polarization-based assays are described by Robbins et al. ^{28 and} described in the Abbott Laboratories 55TDX instruction manual. Fluorescein-FK-506 is a fluorescent polarized CRBA implemented in accordance with the present invention.
Does not generate a polarized fluorescent signal because this molecule rotates freely, but the same molecule to which the FK-506 receptor protein is bound can generate a signal whether it cannot rotate or can rotate freely. . Thus, the free fluorescein-FK-506 bound receptor does not need to be physically separated to perform this type of assay.

Therefore, the standard or unknown FK
-506, labeled FK-506 (eg, fluorescein-FK-50)
It is necessary to perform CRBA by incubating 6), and the initial sample containing the water-soluble receptor protein. ²⁹ polarization intensity of the signal is inversely proportional to the analyte concentration.
Therefore, patient samples containing low concentrations of FK-506 analyte
After reaching equilibrium in the CPBA of the present invention, the reaction mixture has a high concentration of receptor protein binding tracer and a high degree of polarization.

Fluorescence polarization CRBA of FK-506 implemented according to the present invention
Is Abbott Laboratorie
s) It can be easily applied to TDX systems. For this application, FK-506 standards, controls, and patient samples were
Place in individual cartridges of the DX device. Metabolism consisting of buffer-detergent solution, FK-506 receptor protein solution containing protein stabilizer, fluorescein-labeled FK-506 in solution containing surfactant and protein stabilizer in separate vials Place the reagent pack in the device. Thereafter, in a series of automated steps, the test sample was tested for receptor protein and fluorescein-FK-50.
6 and these mixtures are incubated at 37 ° C. for a selected time until binding reaches equilibrium. Thereafter, the sample is transferred to a glass cuvette and the fluorescence polarization signal is measured.

When displaying CRBA data according to the present invention, the known FK
For the -506 standard solution, [Bind _(std) -NBS / Bind _{(0 std)} -NSB] 100 vs. log [FK
Where binding _(std) is the total amount of labeled FK-506 bound with each concentration of standard FK-506, and binding _{(0 std)} is labeled FK bound in the absence of standard FK-506 -506, where NSB represents non-specific binding at each concentration of standard FK-506].

Then, unknowns and radiation relative to the control activity, fluorescence polarization or chemiluminescence values by standard calculations, [binding _(unk) / coupling _(0)] 100 wherein binding _(unk) are the receptor-binding Quantitative value,
Binding ₍₀₎ is a suitable control value]. Then
The calculated ratio is compared with a standard curve, and the FK-
Estimate the concentration of 506 or FK-506-like molecule.

The CRBA of the present invention can also be implemented in a solid phase system. Coating a support matrix, such as the bottom of a well of a microtiter plate, the wall of a tube, or a plastic bead with FK-506 receptor protein,
NSB by short-term exposure to inactive proteins, such as drug-free serum or serum albumin
Block the site. An aliquot of the labeled FK-506 solution is brought into contact with the coated surface with gentle shaking, and the solid surface is brought to ice bath temperature at a cold buffer solution such as PBS.
Wash with. Thereafter, an aliquot of the patient sample containing FK-506, a metabolite, or derivative or analog thereof, is placed in a cool place for a suitable period of time, eg, 0 hours (control) to 16 hours (analyte), with gentle shaking. , Contact with the surface coated with the receptor protein. Once binding has reached equilibrium, the incubation solution is removed and the solid surface is gently washed with cold buffer solution.

The labeled FK-506 bound to the protein is removed from the solid surface with a detergent solution or alcohol, and the precipitated protein is removed by brief centrifugation. Thereafter, the amount of label is quantified as described above for radioactively labeled or chemiluminescently labeled FK-506. Calculations for standard and unknowns are performed as described above.

In order that those skilled in the art can more fully understand the present invention, the following examples are described. These examples are given for illustrative purposes only and should not be deemed to represent limitations other than those set forth in the appended claims.

Example 1 Binding of CsA and FK-506 to Cytosolic Protein Binding of ³ H-CsA to a protein in 100,000 × g cytosol derived from CEM cells was determined by separating bound protein from unbound ligand E (LH −20).

Whole cytosol (1 mg protein / ml) was incubated with [ ³ H] -CsA (60,000 cpm) in the absence and presence of unlabeled CsA and FK-506 (10 μg / ml each). For labeled CsA binding to cytosolic protein,
Unlabeled CsA competed, but FK-506 did not (FIG. 1).

Example 2 Specific Binding Protein to FK-506 in CEM Cytosol Cytosol from CEM cells (1 mg protein / ml) was obtained in the absence and presence of unlabeled FK-506 (10 μg / ml). ^After incubation with ³ H-FK-506 (60,000 cpm), labeled FK-506 bound to protein and free labeled FK
K-506 was separated by Method E (LH-20). The obtained results (FIG. 2) show that the crude cytosol derived from CEM lymphocytes was F
This indicates that the protein contains a binding protein specific to K-506.

Example 3 Purification of FK-506 Binding Protein from CEM Cytosol CEM cytosol sample was filtered and 250 μl or 500 μl
Using a 7.5 × 300 mm Bio-Rad BioSil SEC 125 TSK column, 20 mM
The solution was injected into a Beckman HPLC apparatus using a buffer consisting of sodium phosphate (pH 6.8) and a flow rate of 1.0 ml / min. Fractions were collected, pooled, concentrated on a rotary evaporator, analyzed for protein by the BCA method, and analyzed for ³ H-CsA (C-CsA in FIG. 3) and [ ³ H]-
The binding to dihydro FK-506 (C-506 in FIG. 3) was analyzed. CsA binds to fractions showing protein molecular weights of about 150 kDa, 50 kDa and 17 kDa, whereas FK-506 binds to about 8-10 kDa.
Only binds to a single Da peak. The binding activity specific for FK-506 in the 8-10 kDa fraction was substantially greater than for CsA in any of the protein fractions (FIG. 3).

Example 4 Direct Binding Profile of FK-506 in the JURKAT Cytosol The JURKAT cell line was engineered with RPMI 1640 medium containing 10% FCS supplemented with L-glutamine and antibiotics [Life Technologies, Geysersburg, MD]. Maintained at 37 ° C. and 5% CO _{2 in} [MA Bioproducts Walkersville, MD]. Collect cells (5 × 10 ⁹ ), pellet them, and add 3
Washed twice and used immediately as a source of cytosolic protein or frozen at -70 ° C as a cell pellet. Cells for protein extraction were homogenized on ice with a Teflon or frosted glass homogenizer using 0.02 M sodium phosphate (pH 6.8) containing 0.5% sodium azide. Cell disruption integrity was monitored by trypan blue exclusion. The crude homogenate was spun at 100,00 × g for 1 hour using a Beckman L5-65 ultracentrifuge and a 40.1 rotor, and the supernatant (S-
100) was isolated. The S-100 supernatant (cytosol) was removed and the pellet was discarded. The protein was measured using the method ³⁰ of Bradford or on a Cobas Bios instrument [Roch
e) Company, Natalie, NJ]
e) By binding to blue. S-1
The supernatant was divided into aliquots and stored at -70 ° C.

The cytosolic sample derived from JURKAT lymphocytes was [ ³ H]-
The solution was equilibrated with dihydro FK-506 or CsA (11.8 mol) and the solution was adjusted to a molecular weight adjusted Bio-Rad (Bio-Ra
d) 1.0-well on Bio-Sil SEC125 HPLC column.
It was passed at ml / min. Fractions were collected and counted in Packard Gold scintillation fluid by liquid scintillation analysis. The binding profile shown in FIG. 4 shows that FK-506 binds primarily to proteins that elute in the 8-10 kDa molecular weight region, while FK-506 binding proteins also elute in the 50 kDa region. The CsA binding protein eluted primarily in two molecular weight regions, one of which was approximately 15-17 kDa (cyclophilin),
The other appeared at about 51 kDa; in the 8-10 kDa region, no significant binding of CsA occurred.

Example 5 Purification of Fk-506 binding protein from JURKAT cytosol A. JURKAT S-100 cytosol was prepared as in Example 4.

B. Affinity purification by Matrex Gel Blue A.

Matrex Gel Blue A equilibrated with 1 ml of S-100 cytosol (40-55 mg / ml, from Example 4) with 10 mM sodium phosphate buffer (pH 6.8) containing 0.1% sodium azide Loaded on a column (Amicon Corp., Danvers, MA). The column has a total bed capacity
It was a 3 ml 0.8 cm x 7.0 cm disposable Kontes disposable column [Kontes, Vinland, NJ].
Sodium phosphate buffer with a step concentration gradient (10, 50, 100, 200, 300 and 400 mM) consisting of 3 ml each was passed through the column and collected in a 12 x 75 mm glass tube. These fractions were analyzed for binding, concentrated on a rotary evaporator, and added to 0.02 M sodium phosphate buffer (pH 6.
Dialyzed against 8) and the protein concentration was measured as described above.

This step allows the FK-506 receptor protein to be easily separated from the CsA binding protein due to the large difference in salt concentration required to elute each protein from the column matrix. As shown in the histogram in FIG. 5, bulk FK-506 receptor protein eluted at 50 mM salt concentration, whereas most CsA binding proteins eluted at approximately 300 mM salt concentration. This step is also effective in removing undesired bulk proteins; starting with a total loading of about 50 mg / ml, the pool fraction showing two bound proteins was only about 10 mg.

Also, using Matrex Gel Blue A resin,
A protein preparation of approximately 50 kDa was prepared. The approximately 50 kDa protein did not bind to this matrix and was used as a negative selection step. Size to exclude S-100 sample (about 50k
Da) pool with a 30 kDa blocking filter [Centricon, Amicon, Danvers, M
A], adjusted to 80 mM sodium phosphate (pH 6.8), and mixed with 0.5 ml of Matrex Gel Blue A at room temperature for 15 minutes. The resin was spun down in a Beckman microcentrifuge and the supernatant (containing about 50 kDa protein) was retained.

C. Molecular weight sieving Obtained from Matrix Blue A chromatography (above B), cyclophilin (approx.
A concentrated fraction (5-10 mg / ml) enriched for either 17 kDa), about 50 kDa binding protein or FK-506 receptor protein (about 8-12 kDa) was filtered through a 0.45μ filter. A sample volume of 500 μl was applied to Biosil SEC 12
Beckman System Gold programmable solvent module (Beckman) using a 5 × 600 × 7.5 cm column [Biorad, Richmond, CA]
odule) injected on a # 126 HPLC. The buffer consisted of 0.02 M sodium phosphate (ph 6.8) containing 0.1% sodium azide and the flow rate was 1.0 ml / min. Sample elution was monitored at 280 nm using a Beckman model # 160 absorbance detector and a model # 427 integrator. Fractions were collected in 12 x 75 mm glass tubes, tested for binding, pooled, concentrated on a rotary evaporator, and dialyzed at pH 6.8 against 5 mM potassium phosphate, 0.5% sodium azide.

D. Separation by Weak Cation Exchanger The pool (100-300 μg / ml) of the molecular weight purified obtained in C above was filtered as above, and 250 μl was Beckman TSK CM-2SW
It was injected into a Spherogel column (4.6 mm x 26 cm) and collected using 5 mM potassium phosphate, pH 6.8, 0.5% sodium azide at a flow rate of 0.5 ml / min. These fractions were analyzed for binding, pooled, and concentrated using a rotary evaporator.

Separation of the cyclophilin-sized fraction (approximately 17 kDa) by weak cation exchange chromatography eluted only one protein peak (in 9-10 minutes), which peak corresponds to all ³ H-CsA binding activity ( (FIG. 7).

Separation of the FK-506 receptor fraction (approximately 8-12 kDa) by the same column conditions resulted in a single protein peak in 7-8 minutes and all ³ H-506 binding activity eluted with this peak ( (FIG. 8).

The approximately 50 kDa fraction obtained in C above gave a single binding peak consistent with the protein peak (12-13 minutes) when chromatographed on a weak cation exchange column (FIG. 9).

E. SDS-polyacrylamide gel electrophoresis Electrophoresis samples were prepared and separated as described by Laemlli ³¹ . Briefly, protein samples (3-5 μg) pooled and concentrated from the eluate by molecular weight removal or cation exchange were combined with a sample preparation buffer (0.
1: 1 with 125 M Tris (pH 6.8), 3% SDS, 20% glycerin and 10% 2-mercaptoethanol.
Boil for a minute. Samples were run on a 10-20% gradient gel [Integrated Separation Sciences (Inte
grated Separation Sciences), Boston, Mass.] and separated at a constant current (30 mA). The gel was immobilized and stained with the Daichii silver stain [Integrated Separatio
n Sciences), Boston, MA]. The results are shown in FIG. Lane 1 contains molecular weight standards. Lane 2 contains the approximately 8-12 kDa JURKAT FK-506 receptor protein purified by molecular weight exclusion (step C above), and lane 3 is the same material purified by weak cation exchange (step D above). By this criterion, it is clear that the FK-506 receptor protein of about 8-12 kDa has been purified to homogeneity.

Table 1 shows cyclophilin, and Table 2 shows FK-506 of about 8 to 12 kDa.
Receptor proteins, Table 3, summarizes the purification data for the binding protein of approximately 50 kDa.

The data in Table 2 shows that the FK-506 receptor protein obtained after cation exchange chromatography was 1,250-fold more purified than the S-100 material.
As shown in the data in Table 1, by the same purification scheme, cyclophilin was found to be about 370 compared to the S-100 material.
Two-fold purification. As shown in the data in Table 3,
Approximately 50 kDa bound protein is only about 50
It was only purified twice, which probably reflects the high binding capacity of the starting material (S-100 cytosol).

Example 6 Scatchard Analysis of Bound Protein Bound protein purified in Example 5 was incubated with increasing amounts of labeled ligand and binding data was analyzed according to Scatchard ³² . Scatchard plots are shown in FIGS.

FIG. 11A shows the direct binding of labeled CsA to a protein preparation of approximately 50 kDa. The Kd for this interaction was about 64 nM and the binding capacity was about 3.2 nmol / mg protein. No cooperative binding was observed.

FIG. 11B shows labeled FK- to the same approximately 50 kDa protein preparation.
506 shows direct binding of 506. This interaction is about 2 nM
It provided a binding capacity of Kd and about 0.3 nmol / mg protein.

Analysis of the binding of labeled FK-506 to its major receptor protein (about 8-12 kDa) revealed a Kd of about 1.3 nM and a binding capacity of about 0.5 nmol / mg protein.

No cooperativity was observed in the interaction of FK-506 with any of its binding proteins.

Example 7 Analysis of receptor binding of FK-506 in whole blood Binding analysis Purified JURKA of about 8 to 12 kDa obtained in step C of Example 5.
Binding of ³ H-FK-506 to the T cell receptor protein was performed as in Example 2. Here, receptor-bound FK-
506 was separated by the LH-20 method [see separation method E in the specification]. Binding buffer consisted of 5 mM 2-mercaptoethanol, 20 mM Tris buffer containing 0.05% NaN ₃ and 7.5% FCS (pH7.2). Receptor solution (15μg / m
l protein (100 μl) is 50 μl of ³ H-FK-506 (about 2 n
M, 25,000 CPM) and 0-70 μg / ml unlabeled FK-506
And in a 12 x 75 mm glass tube at room temperature for 20 minutes, then placed on ice. 100 μl of the cold reaction mixture
Bound and free ligand were separated by running on a 7.0 cm x 0.8 cm column of LH-20 equilibrated in binding buffer. The first 2 ml of eluate containing the receptor binding ligand was collected and placed in scintillation vials for counting. CPM was corrected for the total volume of the reaction mixture and expressed as the average of at least two replicates. The standard curve thus obtained is shown in FIG. 12A.

Analysis of FK-506 in whole blood Unlabeled FK-506 was added to human whole blood in a concentration range of 0.5 to 10 ng / ml. FK-506 was extracted from the blood by extracting a 1.0 ml aliquot of this blood with 3 ml of acetonitrile (100%) for 30 minutes with vigorous mixing; precipitated proteins were removed by centrifugation at 2,000 xg. did. Transfer the supernatant to a 16 x 100 mm test tube and remove the solvent
Evaporated with a stream of ° C. The residue was dissolved in 100 μl of binding buffer and a competitive receptor binding assay using approximately 8-12 kDa protein was performed as described above. The results are shown in FIG. 12B.

This analysis appears to be useful when the concentration range of FK-506 in whole blood is at least 0-5.0 ng / ml (FIG. 12B). It should be noted that this concentration range is within the first 10% of the standard curve shown in FIG. 12A, which indicates that a useful range for the analysis of FK-506 in blood is
This suggests that the range shown by the data in FIG. 12B was exceeded.

The above discussion of the invention is primarily directed to preferred embodiments and their implementation. Further changes and modifications in the practice of the concepts described herein may be readily made without departing from the spirit and scope of the invention as specified in the following claims. It will be readily apparent to one skilled in the art.

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Claims (9)

(57) [Claims] The present invention relates to the following steps: 1. Specific binding to cyclosporin and FK-506, having a molecular weight of about 50 kDa, and the following steps: (a) centrifuging the disrupted human T cells to obtain a water-soluble protein-containing cytosol; And (b) fractionating the cytosol with an affinity matrix, a molecular weight exclusion gel, and a weak cation exchanger, and (c) selecting a fraction of a purified immunosuppressant-binding protein. Drug binding protein. 2. A quantitative protein binding assay for the analytes cyclosporin and / or FK-506 contained in a tissue sample, the method comprising: (a) extracting an extract of the tissue sample containing the analyte; (B) contacting the analyte and a tracer amount of labeled cyclosporin or labeled FK-506 in the extract simultaneously or sequentially with the binding protein of claim 1; (c) binding to the protein Physically separating labeled cyclosporin or labeled FK-506 bound to protein from unlabeled cyclosporin or labeled FK-506; (d) determining the amount of labeled cyclosporin or labeled FK-506 specifically bound to the bound protein. (E) quantifying the quantified amount of labeled cyclosporin or labeled FK-506 specifically bound to the binding protein in step (d) above; Comprising the step of determining the amount of against the standard curve generated by analyzing a series of standard solutions of the analyte the analyte of the tissue sample by protein binding analysis). 3. The method of claim 2, wherein said label is selected from the group consisting of radioactivity, chemiluminescent compounds, fluorescent compounds, coloring compounds and enzymes. 4. The method of claim 2, wherein the contact of the labeled cyclosporine or labeled FK-506 and the analyte with the binding protein occurs in a solution phase or on a solid support. 5. The method of claim 2, wherein said label is a chemiluminescent compound or an enzyme capable of producing light from a chemiluminescent compound, and said quantification comprises a chemiluminescence method. 6. The quantification of an enzymatically degradable water-soluble chemiluminescent 1,2-dioxetane derivative substrate with cyclosporine or an enzyme covalently linked to FK-506 to produce optically detectable light energy. The method of claim 5 comprising: 7. A quantitative fluorescent polarization protein binding method for determining the analytes cyclosporin and / or FK-506 contained in a liquid sample, comprising: (a) an analyte-containing extract of the liquid sample. And (b) the analyte and the fluorophore-labeled cyclosporin or fluorophore-labeled FK- in the extract until the binding reaches equilibrium.
Contacting 506 with the aqueous solution of the binding protein of claim 1; (c) measuring the fluorescence polarization signal of the mixture thus incubated, and then binding the fluorescence polarization signal to the protein, the fluorophore-labeled cyclosporin Or converted to the amount of fluorophore-labeled FK-506; and (d) comparing the liquid with a fluorescence polarization standard curve obtained by analyzing a series of standard solutions of the analyte in the fluorescence polarization protein binding assay Determining the concentration of the analyte in the sample. 8. A kit for quantitative binding protein analysis for the analyte cyclosporin or FK-506 in a liquid sample, comprising: (a) one or more purified binding proteins according to claim 1; and (B) A kit comprising an analyte standard in a separation container. 9. The kit according to claim 8, further comprising labeled cyclosporin and / or labeled FK-506.