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1. WO2013015717 - METHOD FOR DETERMINING THE ACTIVITY OF A PROTEOLYTIC ENZYME (VARIANTS), DEVICE FOR THE IMPLEMENTATION OF SAME AND DIAGNOSTIC METHOD

Publication Number WO/2013/015717
Publication Date 31.01.2013
International Application No. PCT/RU2012/000570
International Filing Date 16.07.2012
IPC
C12Q 1/56 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
56involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
G01N 21/76 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
76Chemiluminescence; Bioluminescence
G01N 21/64 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
G01N 33/48 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
CPC
C12Q 1/37
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
34involving hydrolase
37involving peptidase or proteinase
C12Q 1/56
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
56involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
G01N 2021/6439
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
6439with indicators, stains, dyes, tags, labels, marks
G01N 21/6408
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
6408with measurement of decay time, time resolved fluorescence
G01N 21/6456
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
21Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
63optically excited
64Fluorescence; Phosphorescence
645Specially adapted constructive features of fluorimeters
6456Spatial resolved fluorescence measurements; Imaging
G01N 2333/95
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
2333Assays involving biological materials from specific organisms or of a specific nature
90Enzymes; Proenzymes
914Hydrolases (3)
948acting on peptide bonds (3.4)
95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
Applicants
  • ОБЩЕСТВО С ОГРАНИЧЕННОЙ ОТВЕТСТВЕННОСТЬЮ «ГЕМАТОЛОГИЧЕСКАЯ КОРПОРАЦИЯ» OBSCHESTVO S OGRANICHENNOY OTVETSTVENNOSTYU «GEMATOLOGICHESKAYA KORPORATSIYA» [RU]/[RU] (AllExceptUS)
  • АТАУЛЛАХАНОВ, Фазоил Иноятович ATAULLAKHANOV, Fazoil Inoyatovich [RU]/[RU] (UsOnly)
  • ДАШКЕВИЧ, Наталья Михайловна DASHKEVICH, Nataljya Mikhajlovna [RU]/[RU] (UsOnly)
  • ОВАНЕСОВ, Михаил Владимирович OVANESOV, Mikhail Vladimirovich [RU]/[RU] (UsOnly)
  • САРБАШ, Василий Иванович SARBASH, Vasilii Ivanovich [RU]/[RU] (UsOnly)
  • ПАНТЕЛЕЕВ, Михаил Александрович PANTELEEV, Mikhail Aleksandrovich [RU]/[RU] (UsOnly)
  • КАРАМЗИН, Сергей Сергеевич KARAMZIN, Sergey Sergeevich [RU]/[RU] (UsOnly)
  • КОНДРАТОВИЧ, Андрей Юрьевич KONDRATOVICH, Andrey Yurjevich [RU]/[RU] (UsOnly)
Inventors
  • АТАУЛЛАХАНОВ, Фазоил Иноятович ATAULLAKHANOV, Fazoil Inoyatovich
  • ДАШКЕВИЧ, Наталья Михайловна DASHKEVICH, Nataljya Mikhajlovna
  • ОВАНЕСОВ, Михаил Владимирович OVANESOV, Mikhail Vladimirovich
  • САРБАШ, Василий Иванович SARBASH, Vasilii Ivanovich
  • ПАНТЕЛЕЕВ, Михаил Александрович PANTELEEV, Mikhail Aleksandrovich
  • КАРАМЗИН, Сергей Сергеевич KARAMZIN, Sergey Sergeevich
  • КОНДРАТОВИЧ, Андрей Юрьевич KONDRATOVICH, Andrey Yurjevich
Priority Data
201113129326.07.2011RU
Publication Language Russian (RU)
Filing Language Russian (RU)
Designated States
Title
(EN) METHOD FOR DETERMINING THE ACTIVITY OF A PROTEOLYTIC ENZYME (VARIANTS), DEVICE FOR THE IMPLEMENTATION OF SAME AND DIAGNOSTIC METHOD
(FR) PROCÉDÉ POUR DÉTERMINER LA RÉPARTITION SPATIALE ET TEMPORELLE DE L'ACTIVITÉ D'UN FERMENT PROTÉOLYTIQUE DANS UN SYSTÈME HÉTÉROGÈNE (ET VARIANTES), DISPOSITIF POUR METTRE EN œUVRE LEDIT PROCÉDÉ ET PROCÉDÉ DE DIAGNOSTIC DU SYSTÈME DE PERTURBATION DE L'HOMÉOSTASIE SUR LA BASE DE LA MODIFICATION DE LA RÉPARTITION SPATIALE ET TEMPORELLE DE L'ACTIVITÉ D'UN FERMENT PROTÉOLYTIQUE DANS UN SYSTÈME HÉTÉROGÈNE
(RU) СПОСОБ ОПРЕДЕЛЕНИЯ АКТИВНОСТИ ПРОТЕОЛИТИЧЕСКОГО ФЕРМЕНТА (ВАРИАНТЫ), УСТРОЙСТВО ДЛЯ ЕГО РЕАЛИЗАЦИИ И СПОСОБ ДИАГНОСТИКИ
Abstract
(EN)
The invention relates to the field of biotechnology. The method for determining the spatial and temporal distribution of the activity of a proteolytic enzyme in an in vitro heterogeneous system, such as blood or blood plasma, involves the introduction of a luminescent, fluorogenic or chromogenic substrate into a sample with the subsequent release of a detectable tag as the proteolytic enzyme cleaves the substrate, and the recording of the optical characteristics of the sample, which makes it possible to assess the spatial and temporal distribution of the activity of the enzyme. The device for the implementation of the above method comprises an in vitro system, a means for illuminating the sample, a recording means and a control means. A method for diagnosing homeostatic imbalances according to a change in the spatial and temporal distribution of the activity of a proteolytic enzyme in a blood sample is also proposed.
(FR)
L'invention concerne les domaines de la médecine et de la biologie et peut s'utiliser notamment à des fins de diagnostic et de recherche pour déterminer les caractéristiques d'un système de coagulation de sang et de ses composants. Le but visé par la présente invention consiste en un examen de la dynamique spatiale des facteurs de coagulation dans le cadre du processus de croissance d'un caillot de fibrine et l'évaluation du rôle des facteurs de coagulation à des phases différentes du processus de coagulation du sang dans un système hétérogène, ce qui permet de diagnostiquer des maladies isolées et d'évaluer l'activité des préparations influant sur les paramètres de coagulation du sang. L'objectif est réalisé en ce que l'on crée un procédé de détermination de la répartition spatiale et temporelle de l'activité du ferment protéolytique dans un système hétérogène dans lesquels on assure l'existence d'un système in vitro qui comporte un échantillon du milieu examiné sélectionné dan le groupe constitué du plasma du sang, du sang entier, de l'eau, du lymphe, de la solution colloïde, de la solution cristalloïde ou du gel, et un ferment protéolytique réparti dans l'échantillon ou son précurseur; on ajoute au système in vitro un substrat fluorogène puis on assure la libération du marqueur lors de la libération d'un marqueur au moment du clivage du substrat par le ferment protéolytique; on éclaire l'échantillon du milieu étudie à des moments voulus pour exciter un signal de fluorescence du marqueur, on enregistre en effectuant l'éclairage à des moments voulus la distribution spatiale su signal de fluorescence du marqueur dans l'échantillon, on obtient sur la base de l'ensemble des répartitions spatiales du marqueur dans l'échantillon fluorescent, on obtient à partir le l'ensemble des distributions spatiales du signal du marqueur fluorescent à des moments temporels voulus la répartition de l'activité de l'agent du ferment protéolytique par la résolution du problème inverse « réaction - diffusion - convection » en tenant compte du degré de liaison du marqueur aux composants du milieu examiné. En tant que substrat, on peut ajouter un substrat chromogène ou luminescent. L'invention porte également sur un dispositif pour la mise en oeuvre du procédé de l'invention.
(RU)
Изобретение относится к области биотехнологии. Способ определения пространственно-временного распределения активности протеолитического фермента в гетерогенной системе in vitro, такой как кровь или плазма крови включает введение в образец люминесцентного, флуорогенного или хромогенного субстрата с последующим высвобождением детектируемой метки при расщеплении протеолитическим ферментом, и регистрирование оптических характеристик образца, что позволяет оценить пространственно-временное распределение активности фермента. Устройство для реализации заявленного способа содержит систему in vitro, средство для освещения образца, средство регистрации и средство управления. Предложен также способ диагностики нарушений системы гемостаза по изменению пространственно-временного распределения активности протеолитического фермента в образце крови.
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