Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
  • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/963—Methods of stopping an enzyme reaction or stabilizing the test materials
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/826—Additives, e.g. buffers, diluents, preservatives
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/2525—Stabilizing or preserving

Definitions

• the invention relates to a method for stabilizing endogenous, physiologically active peptides in human whole blood, serum or plasma samples before and / or during the determination of the concentration of these peptides according to immunological or physical determination methods.
• Peptides have important biological functions as hormones and biomodulators. Because of their high biological effectiveness, their half-life in the organism is very limited, and there are different biological functions for the various physiologically active peptides
• a blood sample must first be taken in the blood, from which a serum sample or plasma sample can then be obtained in a known manner, which is then used in the determination method for determining the concentration of the respective biomolecule.
• concentration of different biomolecules can also be measured by means of physical methods, such as chromatographic methods, spectroscopic methods or by separation such as dialysis or ultrafiltration, biomolecules in the blood become due to the small amounts in which they occur and due to the clinical examinations required high determination accuracy usually measured by means of various immunodiagnostic methods.
• the serum sample or plasma sample is incubated together with the other substances required for the respective immunodiagnostic method (labeled or unlabeled, suspended or immobilized antibodies, labeled tracer molecules, buffers) for specific times.
• Physiologically active endogenous peptides that have a limited lifespan or are potential candidates for proteolytic degradation in blood, serum or plasma samples are, for example, the adrenocorticotropic hormone (corticotropin, ACTH), the angiotensins, the atrial peptides, including the atrial natriuretic peptide (ANP), bradykinin, calcitonin, calcitonin precursor and the calcitonin gene-related peptide (calcitonin gene-related peptide), cholecystokinin, glucagon, interleukins, insulin, catacalcin (PDN-21) , the luteinizing hormone releasing hormone (LHRH) and the parathyroid hormone or parathyroid hormone (PTH).
• ACTH adrenocorticotropic hormone
• ACTH corticotropin
• angiotensins the atrial peptides, including the atrial natriuretic peptide (ANP)
• the focus is on the stabilization of the unstable peptides ACTH (adrenocorticotropic hormoma) and ANP (atrial natriuretic peptide).
• the invention has for its object to provide a method by which endogenous, physiologically active peptides in human whole blood and in particular human serum and plasma samples can be stabilized in such a way that their concentration before and during the determination cannot be determined by the measurement results falsifying degradation can be changed.
• a method for stabilizing endogenous, physiologically active peptides in human whole blood, serum or plasma samples before and / or during the determination of the concentration of these peptides by immunodiagnostic or physical determination methods which is characterized in that a stabilizer combination is added to the samples, which consists of the protease inhibitors amastatin and leupeptin and ethylenediaminetetraacetic acid (EDTA) or contains the compounds mentioned, for example in the form of a solution in a suitable buffer.
• a stabilizer combination is added to the samples, which consists of the protease inhibitors amastatin and leupeptin and ethylenediaminetetraacetic acid (EDTA) or contains the compounds mentioned, for example in the form of a solution in a suitable buffer.
• the components of the stabilizer combination of the respective sample in particular serum or plasma sample, normally have to be added in such amounts that the respective sample contains minimum concentrations of 4 mM EDTA, 25 ⁇ M amastatin and 50 ⁇ M leupeptin.
• the method according to the invention has a certain similarity to the method of the applicant, which is described in its German patent 38 33 935.
• the method described in the cited patent does not concern the stabilization of any endogenous peptide to be measured, but rather the stabilization of a specific, artificially produced oligopeptide tracer, which in a typical immunoassay, in which the substance to be determined and a tracer are mixed with each other by a deficit amount of antibody compete, is added.
• a combination of leupeptin and amastatin a degradation of this oligopeptide tracer which falsifies the measurement results can be avoided while the determination is being carried out.
• reaction mixture was each analyzed chromatographically by means of reversed-phase HPLC, the result of the chromatography being continuously analyzed using a radio
• the resulting degradation products deliver more radioactive peaks.
• the percentage breakdown of the starting substances results from the peak integral of the product or products
• the normal breakdown of the starting peptides is measured in a control experiment, and the measurement is then repeated under identical conditions in the presence of different potential proteolysis inhibitors.
• Inhibitor solution mixed in 15-fold concentration (controls were mixed with 5 ⁇ l water). The reaction was then started by adding 50 ⁇ l of freshly obtained serum or EDTA plasma. After the addition, the Batches incubated at 22 "C (ACTH in serum for 18 h, ACTH in plasma for 25 h, ANP in serum for 1 h, ANP in plasma for 3 h). The reactions were stopped by freezing the samples. Immediately before the chromatographic HPLC analysis, the samples were thawed and mixed with 1 ml of the solvent A described below, sterile filtered through a 0.2 ⁇ m filter and using a ⁇ -Bondapack C18 column (0.4 ⁇ 30 cm) from Waters via HPLC separated.
• the substances were eluted using a gradient consisting of a mobile phase A (LMA) made of acetonitrile: water: trifluoroacetic acid in a volume ratio of 5: 95: 0.1 and a mobile phase B (LMB) made of acetonitrile: water: trifluoroacetic acid was generated in the volume ratio 90: 10: 0.1 as follows:
• LMA mobile phase A
• LMB mobile phase B
• the inhibitors used in the tests were commercial products. The following are listed together with their respective sources of supply (in brackets): EDTA (Fluka 03610), DPFP (Serva 77205), PMSF (Merck 7349), CCPS (Sigma C-4503), NEM (Serva 11331), Bestatin (Novabiochem A02341), Amastatin (Biomol 50360), Pepstatin (Serva 52682 ), Elastatinal (Sigma E-0881), leupeptin (Biomol 12136), phosphoramidon (Novabiochem A01239), benzamidine (Sigma B-6505), trasylol or aprotinin (Sigma A-6012), heparin (Serva 63036), soybean trypsin Inhibitor (Sigma T-900), Antithrombin III (Sigma A-7388).
• hACTH (Bachem PACT100), hANP (Novabiochem 05-23-0300).
• Table 2 shows that a mixture of amastatin, leupeptin and EDTA not only stabilizes ACTH in the serum or plasma, but also stabilizes the very differently constructed cyclic peptide ANP in a similarly effective manner.
• the concentrations of the individual inhibitors required for adequate protection of the peptides in the presence of the other inhibitors were varied several times. It turned out that for an effective As protection, the minimum concentrations present in the sample for EDTA are about 4 mM, about 25 ⁇ M for amastatin and 50 ⁇ M for leupeptin.
• a kit for carrying out an immunoradiometric determination method for ACTH contains the following components typical of a kit of the present type:
• a The tracer in the form of 125 I anti-ACTH (34-39) antibodies (mouse, monoclonal), a bottle of 10 ml, colored red, concentrate, which is to be reconstituted with 23 ml tracer buffer before use.
• b The tracer reconstitution buffer, a 23 ml bottle, ready for use.
• c Coated tubes (test tubes), coated with immobilized anti-ACTH (25-21) antibody (mouse, monoclonal), twice 50 pieces, ready for use.
• d Wash solution, 2 bottles of 11 ml, concentrate. In front
• each bottle with distilled water to a volume of 550 ml, e. ACTH zero standard (human serum), 1 bottle, 10 ml ready to use.
• ACTH zero standard human serum
• 1 bottle 10 ml ready to use.
• 1 to 6 ACTH standards intact human ACTH (1-39), in 6 vials, lyophilized.
• the concentrations are 5; 15; 50; 150; 500; 1500 pg / ml.
• the standards must be reconstituted with 1 ml of zero serum each. i, ii. Control sera I and II (human serum), 2 bottles, lyophilized.
• the kit also contains a buffer A, which contains the protease inhibitors for inhibiting ACTH degradation in lyophilized form and is to be dissolved in 11 ml of a buffer B, which is also added, before use.
• the first step of the method is to transfer the solution obtained by mixing the lyophilized buffer A with the liquid buffer B into the respective test tubes for the standards and the patient sera, i.e. the stabilizing buffer is introduced and the ACTH-containing components are stabilized by buffer A from the beginning during the test.
• the measurement is then carried out in a manner known per se by incubating the sera or standards in the coated test tubes, then adding a washing solution and carrying out a solid / liquid separation and, if necessary, repeating this step, after which the markings on the solid phase-bound ACTH molecules of the labeled antibody in a suitable buffer is added in a further test step. After the incubation, a solid / liquid separation and washing, the bound radioactivity is measured and the hACTH concentration in the respective sample is determined from this, taking into account the values obtained for the standard.

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• 1991
  • 1991-08-13 US US08/066,156 patent/US5541116A/en not_active Expired - Lifetime
  • 1991-09-30 DE DE4132587A patent/DE4132587C1/de not_active Expired - Fee Related
• 1992
  • 1992-08-13 EP EP92917643A patent/EP0559853B1/de not_active Expired - Lifetime
  • 1992-08-13 CA CA002096336A patent/CA2096336A1/en not_active Abandoned
  • 1992-08-13 DE DE59207385T patent/DE59207385D1/de not_active Expired - Lifetime
  • 1992-08-13 WO PCT/EP1992/001855 patent/WO1993007489A1/de not_active Ceased
  • 1992-08-13 AT AT92917643T patent/ATE144327T1/de active IP Right Revival
  • 1992-08-13 JP JP50656693A patent/JP3273154B2/ja not_active Expired - Lifetime

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