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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
  • G01N33/9493—Immunosupressants

Definitions

• the invention relates to assays for drugs in biological fluids, more particularly to a competitive receptor protein binding assay for the
• Cyclosporine appears to be T-cell specific and induces its immunosuppressive effects by impairing interleukin-2 production and receptor expression. 1 In the dosages required to prevent organ rejection in patients, CsA is known to exhibit serious toxic side effects, inducing
• FK-506 is a such a drug.
• FK-506 is a 822 kDa macrolide antibiotic isolated from the fungus Streptomyces tsukubaensis by the
• FK-506 exhibits similar, if not identical, antilymphocytic activities.
• the immunosuppressive activity of FK-506 is, however, about 100-fold greater than that of CsA, 8 and it is being used successfully in solid organ transplant patients, particularly in liver transplant patients.
• 9 Although the toxicity of FK-506 in human patients and experimental animals has been reported to be less than that of CsA 8 ' 10 there are reports of undesirable side effects of FK-506 in those species at the dosages required for anti-rejection activity. 2-4,10
• Tamura et al. 13 and Cadoff et al. 14 have disclosed solid state, one-step and two-step enzyme-linked immunoassays (ELISA) for FK-506 that employ rabbit polyclonal or mouse monoclonal antibodies raised against a bovine serum albumin-FK-506 complex antigen.
• ELISA enzyme-linked immunoassays
• steroids such as prednisone that are given concurrently with FK-506 (or CsA) to prevent allograft rejections, are given concurrently with FK-506 (or CsA) to prevent allograft rejections.
• This method comprises a competitive protein binding assay wherein the binding reagent is a purified soluble receptor protein isolated from a target tissue of FK-506 action.
• FIGURE 1 compares the binding of FK-506 and CsA to CsA binding proteins in CEM lymphocyte cytosol.
• FIGURE 2 shows specific binding of FK-506 to a CEM lymphocyte cytosolic protein.
• FIGURE 3 shows the elution profile of FK-506-binding protein fractions fractionated from CEM lymphocyte cytosol by HPLC molecular weight sieving chromatography on a BioRad TSK column.
• FIGURE 4 shows the fractionation by Beckman HPLC on a
• lymphocyte cytosol lymphocyte cytosol. Molecular weight markers are also shown.
• FIGURE 5 shows the elution profile of JURKAT lymphocyte FK-506 and CsA binding proteins from a
• Matrex Gel Blue A affinity column as a function of salt concentration.
• FIGURES 6A-B show the HPLC fractionation on a BioRad SEC125 molecular weight exclusion column of pooled CsA and FK-506 binding fractions isolated by Matrex Blue A affinity chromatography.
• Fig . 6A shows the size exclusion profile of cyclophilin (about 17 kDa), and 6B that for the about 8-12 kDa FK-506 receptor protein.
• FIGURE 7 shows the fractionation of 17 kDa cyclophilin from Fig. 6A on a weak cation exchange chromatography HPLC column (Beckman TSK CM-25W Spherogel).
• FIGURE 8 shows the fractionation of the FK-506 8-10 kDa binding protein from Fig. 6B on a weak cation exchange chromatography HPLC column (Beckman TSK CM-25W
• FIGURE 9 shows the fractionation of about 50 kDa proteins eluted from a size exclusion column on a weak cation exchange column.
• FIGURE 10 shows analytical SDS-PAGE of the purified JURKAT about 8-12 kDa receptor protein.
• FIGURE 11A-C show Scatchard plots for CsA binding for the about 50 kDa protein preparation (Fig. 11A), FK-506 binding to the same preparation (Fig. 11B), and FK-506 binding to its about 8-12 kDa receptor protein (Fig. 11C).
• FIGURE 12A-B show standard curves for the receptor assay of FK-506 in water (Fig. 12A) and in whole human blood (Fig. 12B) using the purified 8-12 kDa JURKAT cell receptor protein as binding reagent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
• receptor refers to a macromolecular protein or glycoprotein capable of recognizing and selectively binding with some ligand, and which, after binding the ligand, is capable of generating some chemical or physical signal that initiates the chain of events leading to the biological response.
• water-soluble binding protein used as a reagent in the competitive receptor binding assay
• FK-506 metabolites, analogues and derivatives
• FK-506 has several of the characteristics of a natural receptor protein, i.e., specific, saturable and reversible binding.
• Water-soluble, specific receptor proteins suitable for CRBAs carried out in accordance with this invention may be advantageously prepared from extracts of target tissues of FK-506 action, such as mammalian
• lymphocytes including human or animal peripheral blood lymphocytes (PBL), 16 primary mixed lymphocytes cultures, proliferating alloreactive T-cells propagated from organ transplant biopsies, 15 ' 17 and established cell lines such as the DQwl-specific alloreactive T-cell clone DB29, 18 a transformed T-helper cell line (CEM) and the interleukin-2-producing JURKAT human lymphocytic leukemia T-cell line (JURKAT 77.6.8). 19 As will be shown below, and has been reported elsewhere, 19 FK-506 and CsA do not share binding proteins in common.
• PBL peripheral blood lymphocytes
• CEM transformed T-helper cell line
• JURKAT 77.6.8 interleukin-2-producing JURKAT human lymphocytic leukemia T-cell line
• FK-506 does not bind to, and inhibit the peptidyl-prolyl cis-trans isomerase of the 17 kDa cyclophilin fraction, although it does do so to the isomerase activity of the JURKAT T-cell about 8-12 kDa FK-506 binding protein. 19 .
• This enzyme which is highly sensitive to CsA is thought to be a primary receptor for CsA action. 20-22
• the particular cellular source of the receptor protein used in the CRBA of the invention is not critical - the protein need only exhibit the binding properties of a
• Cells may be disrupted and the soluble proteins (cytosolic proteins) fractionated by art-recognized methods. Typically, lymphocytes are collected, washed and counted. Cells may be stored as a pellet at -70°C until used. Cells are thawed and homogenized with a Teflon or ground glass homogenizer at ice bath
• Homogenates are centrifuged in the cold under reduced pressure at least 20,000 x g, preferably at least
• the supernatant fluid which contains both the FK-506 receptor protein and cyclophilin is analyzed for protein content and binding activity and is either used immediately or stored frozen at temperatures at or below about -20°C.
• Cytosols can be used as such for the CRBA carried out in accordance with the invention.
• FK-506 receptor protein is purified from the cytosol and concentrated prior to use.
• FK-506 receptor protein can be partially purified by affinity
• a preferred affinity column is Matrix Gel Blue A (Amicon Corp., Danvers, MA) on which FK-506 and CsA binding proteins are readily separable by salt buffer gradients.
• Cytosol can also be fractionated by molecular weight exclusion methods, including HPLC columns. For example, a preferred molecular sieve method uses a Beckman Instrument Co. HPLC instrument and a preparative Biorad Biosil SEC 125 column of appropriate dimension (see, Example 6, below).
• FK-506 receptor protein can also be advantageously purified by cation exchange chromatography, e.g., by the weak cation exchange Beckman TSK CM-25W Spherogel.
• Hydrophobic interaction chromatographic matrices are also suitable for purifying the FK-506 according to this invention. Any sequences or combinations of these fractionation systems can be used according to this invention as long as appropriate purifications are obtained. For example, purification by the sequence: cytosol, affinity matrix, molecular weight exclusion gel and weak cation exchanger is particularly useful.
• a receptor protein is deemed to be purified if, upon chromatography, a single peak describes the elution pattern of both protein and binding activity, and if, upon SDS-PAGE, only one major protein band appears. Fractions of column eluate are collected, pooled, and concentrated in the cold by rotary
• Concentrates are assayed for protein by any suitable method, including the Bradford BCA method. Concentrates are also tested for assay suitability by the CRBA.
• FK-506 receptor protein may be prepared and purified by techniques other than by isolation from mammalian target cells.
• the receptor protein may advantageously be synthesized by art-recognized recombinant DNA
• cDNA coding for the receptor may be isolated from purified mRNA or from a cDNA cloning library, then cloned into cells, e.g.,
• An expression vector may be constructed
• cDNA for a FK-506 receptor containing the cDNA for a FK-506 receptor, promoter and gene regulation sequences, a translation start codon, selectable markers, etc., and then inserted into prokaryotic or transformed eukaryotic cells. These cells will express the receptor gene and secrete the receptor protein in large quantities into the growth medium, from which it can be isolated by conventional methods.
• a protein fraction is deemed acceptable if: (1) the protein binds FK-506 to a statistically
• the signal-to-noise ratio i.e., the ratio of total binding to nonspecific binding (as these terms are defined below) is at least 1.1, preferably at least about 1.2.
• Gel electrophoresis and Western blot analyses may be used to monitor the purity of, and identify the receptor protein throughout the stages of purification.
• Labeled FK-506 is required for the CRBA carried out in accordance with this invention.
• Native FK-506 is available from Fujisawa Pharmaceutical Co., Osaka, Japan and is soluble in aqueous amphipathic solvents, e.g., aqueous methanol.
• [ 3 H]-dihydro FK-506 can be prepared by exposure of native FK-506 to tritium gas in the presence of a reducing agent such as tris
• K-506 can also be labeled with ⁇ -ray emitting 125 I by brief reduction by chloramine-T in the presence of Na 125 I.
• 125 I-labeled histamine-FK-506 can be prepared by the method of Wong, et al. 24
• fluorophore-labeled FK-506 may be used. FK-506 may be labeled with a fluorophore by art-recognized methods. 25 Suitable fluorophores include fluorescein, europium and luciferin.
• Chemiluminescent labels such as the water-soluble 1,2-dioxetane derivatives that release light energy upon cleavage with a hydrolytic enzyme 26,27 can be obtained from Tropix, Inc., Bedford, MA.
• Chemiluminescence may also be the detection method when the FK-506 label is an enzyme that releases light upon addition of a substrate such as the aforementioned 1,2-dioxetanes.
• the CRBA carried out in accordance with this invention can be performed by either solution phase or solid phase methods.
• the principle underlying both methods is the same. Briefly, a competition
• equilibrium is set up between a tracer amount of labeled FK-506 and unknown samples containing analyte FK-506 for binding to a fixed amount of the FK-506 receptor protein described above. Following attainment of equilibrium, the amount of labeled FK-506 bound to the receptor protein is determined. The amount of label bound to the receptor will be reduced in the presence of unlabeled FK-506, and this reduction is proportional to the amount of unlabeled analyte present in the unknown sample. The quantitative relationship between the reduction of receptor-bound label and the concentration of analyte in the unknown sample is determined by reference to a standard curve.
• a fixed amount of receptor protein is exposed to a fixed tracer amount of labeled FK-506 in the presence of zero-to-supersaturating concentrations of standard FK-506.
• the supersaturating concentration is, ideally, several orders of magnitude greater than the association constant, Ka, of specific binding, and this fraction ("nonspecific binding," NSB) is assumed to be the same for all ligand
• aliquots of an aqueous alcoholic (e.g., 50% ethanol in water) solution of unlabeled FK-506 are added to glass tubes, and the solvent evaporated, e.g., by a gentle stream of N 2 or in the cold under reduced pressure; the amount of FK-506 delivered ranges between zero and about 100,000 ng.
• whole blood is extracted with an amphipathic organic solvent, such as a lower alkanol (e.g., C 1 to C 6 straight or branched chain, primary, secondary or tertiary alcohol) or acetonitrile.
• Amphipathic organic solvent is intended to mean a liquid organic compound having both hydrophilic and hydrophobic properties.
• the precipitated proteins are removed by, e.g., centrifugation, and the extract containing FK-506 taken to dryness as described below. The residue to be analyzed is taken up in a small volume of a
• a solution of receptor protein in a small volume e.g., about
• binding buffer incubated until equilibrium or steady state binding is reached, typically about 20 to 90 mins . , at a slightly elevated temperature such as about 30°C to about 40°C.
• the composition of the binding buffer is not critical.
• a preferred binding buffer is 20 mM Tris Buffer, pH 7.2, containing 5 mM 2-mercaptoethanol, 0.05% NaN 3 and 7.5% (v/v) fetal calf serum.
• NBS non-specific binding
• one set of tubes contains a large molar excess of unlabeled ligand, such as a 200-fold molar excess of unlabeled FK-506 delivered in a small volume, e.g., 50 ⁇ l.
• the contents of the reaction mixture are diluted with ice-cold buffer, preferably at neutral pH, the contents filtered through a glass fiber filter such as Whatman GF/B (Whatman Paper, Maidstone, England), and the filter washed with ice-cold buffer.
• the membrane retains the receptor-bound labeled FK-506 compound.
• B. This method is analogous to that of A, except that filtration is carried out on a microporous filter such as 0.22 ⁇ m nitrocellulose (Millipore Corp., Bedford, MA) prewashed with a solution of an inert protein, e.g., BSA or ⁇ -globulin, to block
• an inert protein e.g., BSA or ⁇ -globulin
• the filter retains the
• a suspension of polyethyleneglycol e.g., 1 ml of a 30 mg/ml suspension, plus a solution of a carrier protein, preferably delivering about 1 mg of carrier BSA or ⁇ -globulin, are added, and the resulting suspension of polyethyleneglycol (MW 5,000 to 20,000), e.g., 1 ml of a 30 mg/ml suspension, plus a solution of a carrier protein, preferably delivering about 1 mg of carrier BSA or ⁇ -globulin, are added, and the resulting
• the supernatant fluid will contain the receptor protein-bound labeled FK-506.
• reaction mixture typically a 100 ⁇ l aliquot run in duplicate, is poured onto a column of convenient dimension, such as 0.8 x 7.0 cm of a molecular sieve matrix such as LH-20 Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ).
• washing the column with a small volume e.g., about 0.5 ml of a buffer (e.g., phosphate-buffered saline, pH 7.4), will elute in the void volume, e.g., the first 2 ml, the receptor-bound labeled FK-506.
• a buffer e.g., phosphate-buffered saline, pH 7.4
• LH-20 is a weakly hydrophobic matrix, and free FK-506 or CsA will be retarded in such a matrix.
• each filter is placed in a LSC vial, an aqueous-organic solvent phase combining scintillation system (e.g., PCSS, Amersham, Arlington Heights, IL) is added, and the amount of radioactivity quantified.
• an aqueous-organic solvent phase combining scintillation system e.g., PCSS, Amersham, Arlington Heights, IL
• the pellet is suspended in scintillant solution (e.g., PCSS) or dissolved in NaOH and diluted with scintillant
• chemiluminescence when the reporter molecule is a chemiluminescent 1,2-dioxetane such as AMPPD or AMPGD (Tropix, Inc., Bedford, MA) with methods A and B filters are placed on a sheet of blotting paper, and the filters soaked with a solution of the enzyme, e.g., alkaline phosphatase or galactosidase, that hydrolyses the 1,2-dioxetane and produces light. Filters are transferred to a piece of polyester film (e.g.. Mylar), and then to a black box containing instant film, such as Type 612 Polaroid film. After exposure of the film to the emitted light, the dark image is digitized using e.g., a black and white RBP Densitometer, Tobias,
• Method C the pellet is suspended in a buffer (pH 7-12) containing the
• Method D the void volume is reacted with an
• FK-506 is labeled with the enzyme, such as alkaline phosphatase or ⁇ - or ⁇ -galactosidase except that the appropriate chemiluminescent substrate (AMPPD and AMPGD,
• fluorescein-FK- 506 will not produce a polarized fluorescence signal as this molecule rotates freely, whereas the same molecule bound by a FK-506 receptor protein will produce a signal as it is not free or as free, to rotate. Thus, receptor-bound and free fluorescein-FK-506 do not need to be physically separated in order to carry out this type of assay.
• the assay system thus involves carrying out a CRBA with incubation of an initial sample containing
• FK-506, labeled FK-506 e.g., fluorescein-FK-506
• water-soluble receptor e.g., fluorescein-FK-506
• the polarization intensity of the signal is inversely related to analyte concentration.
• concentration of FK-506 analyte will, after equilibrium has been reached in the CPBA of this invention, have a high concentration of receptor protein bound tracer in the reaction mixture, and polarization will be high.
• the fluorescence polarization CRBA for FK-506 carried out in accordance with this invention is readily adaptable to the Abbott Laboratories TDX
• a Metabolite Reagent Pack containing, in separate vials, a buffer-surfactant solution, a solution of FK-506 receptor protein containing a protein stabilizer, fluorophore-labeled- FK-506 in a solution containing a surfactant and protein stabilizer, is placed in the instrument.
• test samples are mixed with receptor protein and
• Bound (std) is the total amount of labeled FK-506 bound at each concentration of standard FK-506,
• Bound (o std) is the amount of labeled FK-506 bound in the absence of standard FK-506 , and NSB represents
• Bound (unk) /Bound (o) 100 wherein Bound (unk) is the quantitative value of receptor- bound and Bound(o) is the appropriate control value, by standard calculations. The calculated ratio is then referenced to the standard curve for estimation of the concentration of FK-506 or FK-506-like molecules in the unknown samples.
• the CRBA of the invention can also be carried out in a solid state system.
• a supporting matrix e.g., the bottom of wells of a microtitre plate or the walls of a tube or plastic beads is coated with FK-506 receptor protein, and NSB sites are blocked by brief exposure to an inert protein, e.g., drug-free serum or serum albumin.
• An aliquot of a solution of labeled FK-506 is contacted with the coated surface with gentle shaking, and the solid surface washed with cold buffer solution, e.g., PBS at ice-bath temperatures.
• an aliquot of a patient sample containing FK-506 , its metabolites, or derivatives or analogues is contacted with a receptor protein-coated surface with gentle shaking for a suitable period, e.g., 0 hrs. (control) to 16 hrs (analyte) in the cold.
• a suitable period e.g., 0 hrs. (control) to 16 hrs (analyte) in the cold.
• the incubation fluid is removed, and the solid surface washed gently with cold buffer solution.
• Protein-bound labeled FK-506 is removed from the solid surface by a surfactant solution or an alcohol, and the precipitated proteins removed by brief
• Binding of 3 H-CsA to proteins in a 100,000 x g cytosol derived from CEM cells was determined using Method E (LH-20) for separating protein bound from unbound ligands.
• CEM cytosol samples were filtered and injected using either a 250 ⁇ l or 500 ⁇ l loop with a Beckman
• HPLC instrument using a 7.5 x 300 mm Bio-Rad BioSil SEC 125 TSK column, a buffer consisting of 20 mM sodium phosphate, pH 6.8, and a flow rate of 1.0 ml/min.
• the JURKAT cell line was maintained in RPMI 1640 medium (MA Bioproducts, Walkersville, MD) with 10% FCS supplemented with L-glutamine and antibiotics (Life Technologies, Gaithersburg, MD) at 37°C and 5% CO 2 .
• Cells (5 x 10 9 ) were collected, pelleted and washed with medium three times, and either used immediately as a source for cytosolic proteins or were frozen as a cell pellet at -70°C.
• Cells for extraction of proteins were homogenized with either a Teflon or ground glass homogenizer, using 0.02 M sodium phosphate pH 6.8 with 0.5% sodium azide on ice. Completeness of cellular disruption was monitored with trypan blue exclusion. The crude homogenates were spun at 100,00 x g for
• lymphocytes was equilibrated with [ 3 H]-dihydro FK-506 or CsA(11.8 mol), and the solution passed through a molecular weight calibrated Bio-Rad Bio-Sil SEC 125 HPLC column at 1.0 ml/min. Fractions were collected and counted by liquid scintillation spectrometry in Packard Gold scintillation fluid.
• the binding profile shown in Fig. 4 demonstrates that FK-506 was bound primarily to a protein eluting in the 8-10 kDa
• CsA binding proteins eluted primarily in two molecular weight ranges, one at about 15-17 kDa (cyclophilin) and the other at about 51 kDa; no significant binding of CsA occurred in the 8-10 kDa range.
• JURKAT S-100 cytosol was prepared as in Example 4.
• the about 50 kDa protein preparation was also separated using Matrex Gel Blue A resin. As the about 50 kDa proteins did not bind to this matrix, it was used as a negative selection step. Pools of size excluded S-100 samples (about 50 kDa) were concentrated with a 30 kDa cut-off filter
• Lane 1 contains the molecular weight standards. Lane 2 contains the about 8-12 kDa JURKAT FK-506 receptor protein purified through molecule weight exclusion (step C, above), and Lane 3 the same material purified through weak cation exchange (Step D, above). It is clear that, by this criterion, the FK-506 receptor protein of about 8-12 kDa has been purified to homogeneity. Table 1 summarizes the purification data for cyclophilin. Table 2 that for the about 8-12 kDa FK-506 receptor protein, and Table 3 that for the about 50 kDa binding protein.
• Example 5 The binding proteins purified in Example 5 were incubated with increasing amounts of labeled ligand and the binding data analyzed according to Scatchard. 32 Scatchard plots are shown in Figs. 11A-C.
• Fig. 11A represents the direct binding of labeled CsA to the about 50 kDa protein preparation.
• the Kd of this interaction was about 64 nM, with a binding capacity of about 3.2 nmoles/mg protein. Cooperative binding was not observed.
• Fig. 11B represents the direct binding of labeled FK-506 to the same about 50 kDa protein preparation. This interaction yielded a Kd of about 2 nM and a binding capacity of about 0.3 nmoles/mg protein.
• the Kd was about 1.3 nM and the binding capacity was about 0.5 nmoles/mg protein.
• the Binding Buffer consisted of 20 mM Tris buffer, pH 7.2,
• Unlabeled FK-506 was added to whole human blood in the concentration range of 0.5 to 10 ng/ml. FK-506 was extracted from blood by extracting 1.0 ml aliquots of the blood with 3 ml of acetonitrile (100%) by vigorous mixing for 30 seconds; precipitated proteins were removed by centrifugation at 2000 x g. Supernatant fluids were transferred to 16 x 100 mm test tubes, and the solvent evaporated by a stream of air at 40°C.
• the assay appears to be useful over the
• concentration range of at least 0 to 5.0 ng/ml FK-506 in whole blood (Fig. 12B). It should be noted that this concentration range falls within the first 10% of the standard curve shown in Fig. 12A, suggesting that the useful range for assays of FK-506 in blood will extend beyond that shown by the data in Fig. 12B.

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