DK163189B - METHOD AND REAGENT OF DETERMINATION OF PLASMAS FIBRINOLYTIC CONDITIONS - Google Patents

METHOD AND REAGENT OF DETERMINATION OF PLASMAS FIBRINOLYTIC CONDITIONS Download PDF

Info

Publication number
DK163189B
DK163189B DK030686A DK30686A DK163189B DK 163189 B DK163189 B DK 163189B DK 030686 A DK030686 A DK 030686A DK 30686 A DK30686 A DK 30686A DK 163189 B DK163189 B DK 163189B
Authority
DK
Denmark
Prior art keywords
fibrin
plasma
reagent
thrombin
fibrinogen
Prior art date
Application number
DK030686A
Other languages
Danish (da)
Other versions
DK30686D0 (en
DK163189C (en
DK30686A (en
Inventor
Knut Bartl
Helmut Lill
Original Assignee
Boehringer Mannheim Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Publication of DK30686D0 publication Critical patent/DK30686D0/en
Publication of DK30686A publication Critical patent/DK30686A/en
Publication of DK163189B publication Critical patent/DK163189B/en
Application granted granted Critical
Publication of DK163189C publication Critical patent/DK163189C/en

Links

Classifications

    • 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
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • 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
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
    • C12Q2337/10—Anilides
    • C12Q2337/12—Para-Nitroanilides p-NA
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3153—Streptokinase
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/4609—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from reptiles
    • G01N2333/4613—Snake venom
    • G01N2333/462—Snake venom from Agkistrodon sp., e.g. acutase, ACTE
    • G01N2333/4623—Snake venom from Agkistrodon sp., e.g. acutase, ACTE from Agkistrodon rhodostama (Malayan pit viper); Arvin (R); Batroboxin; Ancrod
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9723—Urokinase
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9726—Tissue plasminogen activator
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Neurosurgery (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Drying Of Semiconductors (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Control Of Indicators Other Than Cathode Ray Tubes (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)
  • Discharge Heating (AREA)
  • Measuring Oxygen Concentration In Cells (AREA)
  • Electrotherapy Devices (AREA)
  • Control Of Stepping Motors (AREA)

Abstract

1. Process for the determination of the fibrinolytic state of plasma, characterised in that to a native plasma sample one a) adds an amount of fibrin sufficient for a turbidity formation or produces this in situ and measures the time-dependent turbidity change or the time-dependent formation of fission products or b) adds a chromogenic plasmin substrate and an amount of fibrin, fibrinogen fission products or of an enzyme producing fibrin in situ insufficient for the turbidity formation and measures the time-dependent colour formation.

Description

DK 163189 BDK 163189 B

iin

Fibrinolyse er den modsatte reaktion af blodkoagulering. Ved 5 blodkoagulering opstår der udfra fibrinogen under indflydelse af enzymet thrombin fibrinmonomer, som under indflydelse af kalciumioner og andre faktorer polymeriserer til et uopløseligt skelet af fibrin. Det fremkomne fibrinmolekyle bevirker, at den koagulerede blodmasse stivner. Da denne fibrindannelses-jO proces stadig forløber i karsystemet, er et modsat rettet system, det fibrinolytiske system, ligeledes stadig aktivt for at forhindre, at der uden særlig anledning sker dannelse af thromber. Dette fibrinolytiske system fører under indflydelse af plasmi-nogenaktivator til dannelse af plasminud fra plasminogen og 15 plasmin forårsager opløsning af fibrin under dannelse af fibrin-spalteprodukter. Bestemmelsen af et plasmas fibrinolytiske tilstand er derfor af stor betydning til vurdering af thrombosefa-rer.Fibrinolysis is the opposite reaction of blood coagulation. By blood coagulation, fibrinogen is produced under the influence of the enzyme thrombin fibrin monomer, which under the influence of calcium ions and other factors polymerizes to an insoluble skeleton of fibrin. The resulting fibrin molecule causes the coagulated blood mass to harden. As this fibrin formation process is still ongoing in the vascular system, an oppositely directed system, the fibrinolytic system, is also still active to prevent the formation of thrombi without particular cause. This fibrinolytic system, under the influence of plasmin protein activator, leads to plasminogen formation from plasminogen and 15 plasmin causes dissolution of fibrin to form fibrin cleavage products. The determination of the fibrinolytic state of a plasma is therefore of great importance in the assessment of thrombosis spheres.

20 Der kendes allerede prøvemetoder, der måler blods (plasmas) spontane fibrinolytiske aktivitet eller den fibrinolytiske reaktion på stimulanter, der frigør plasminogenaktivator fra karvæggen. Det er her nødvendigt at udkoble indvirkningen af plasmainhibitorer, hvilket imidlertid i praksis for det meste kun 25 lykkes utilstrækkeligt og samtidigt også betyder en kunstig forandring af prøven.Test methods are already known that measure the spontaneous fibrinolytic activity of the blood (plasma) or the fibrinolytic response to stimulants that release plasminogen activator from the vessel wall. Here, it is necessary to disable the effect of plasma inhibitors, which, however, in practice, for the most part, only 25 succeeds insufficiently and at the same time also means an artificial change of the sample.

Ved bestemmelsen af euglobulin-lyse-tid går man ud fra Citratplasma. Til dette sættes der eddikesyre og der fracentrifugeres. 30 Bundfaldet opløses i puffer, og der tilsættes thrombin·Der inkuberes ved 37°C, og tiden indtil opløsning af den koagulerede masse måles. Måletiden bliver herved ganske vist væsentligt for-. kortet, men man bestemmer herved en in vitro fibrinolyseaktivi- tet, der ikke nødvendigvis er korreleret med aktiviteten in vivo. 35In the determination of euglobulin lysis time, one assumes citrate plasma. To this is added acetic acid and centrifuged. The precipitate is dissolved in buffer and thrombin is added. · Incubate at 37 ° C and measure the time until dissolution of the coagulated mass. The meal time is hereby considerably reduced. but in this case an in vitro fibrinolysis activity is not determined which is not necessarily correlated with the activity in vivo. 35

Man har derfor også allerede udviklet metoder, der gennemføres uden eliminering af plasmasinhibitorer. En sådan metode er f.eks.Therefore, methods have already been developed that are implemented without the elimination of plasma inhibitors. One such method is e.g.

DK 163189 BDK 163189 B

2 bestemmelsen af helblod^-lyse-tiden.2 the determination of the whole blood ^ light time.

5 Således sættes der ved bestemmelsen af helblod-lyse-tiden thrombin til helblod, og den dannede koagulerede masse inkuberes ved 37°C, og tiden indtil dens opløsning måles. Denne tid udgør ved normale værdier ikke under 24 timer. Metoden er derfor 10 tidskrævende, kan ikke automatiseres og egner sig- kun til grov vurdering af den fibrinolytiske tilstand. En unormal lav fibri-nolytisk aktivitet kan endog slet ikke bestemmes.Thus, in determining the whole blood lysis time, thrombin is added to whole blood and the resulting coagulated mass is incubated at 37 ° C and the time until its solution is measured. At normal values this time does not exceed 24 hours. The method is therefore 10 time-consuming, cannot be automated and is suitable only for rough assessment of the fibrinolytic state. An abnormally low fibrinolytic activity cannot even be determined at all.

Der opnås noget kortere tider, når der arbejdes med fortyndet 15 blod, men for øvrigt er de nævnte ulemper der stadig.Somewhat shorter times are obtained when working with diluted blood 15, but otherwise the mentioned disadvantages are still there.

Den såkaldte fibrinpladeprøve, der arbejder med citratplasma eller gensuspenderet euglobulinbundfaid, skulle give nøjagtige resultater.Hovedulempen ligger her ved den lange inkubations-20 tid på 18 timer ved 37°C.The so-called fibrin plate sample, which works with citrate plasma or resuspended euglobulin bundle fluid, should give accurate results. The main disadvantage here lies at the long incubation time of 18 hours at 37 ° C.

Det er således formålet med den foreliggende opfindelse at tilvejebringe en global fibrinolyseprøve, som undgår ulemperne ved de ovennævnte kendte metoder og navnlig på forholdsvis 25 kort tid giver resultater, der på pålidelig måde genspejler tilstanden in vivo, lader sig automatisere og kan udnyttes fotometrisk .It is thus the object of the present invention to provide a global fibrinolysis assay which avoids the disadvantages of the above known methods and in particular, in a relatively short time, produces results that reliably reflect the in vivo state, are automated and can be utilized photometrically.

Dette opnås ifølge opfindelsen ved hjælp af en fremgangsmåde 30 til bestemmelse af plasmas fibrinolytiske tilstand, hvilken fremgangsmåde er ejendommelig ved, at man til en naturlig plasmaprøve « a) tilsætter en til uklarhedsdannelse tilstrækkelig mængde 35 fibrin eller tilvejebringer dette in situ og måler uklarheden eller de dannede fibrin-spalteprodukter, ellerThis is achieved according to the invention by a method 30 for determining the fibrinolytic state of the plasma, which is characterized by adding to the natural plasma sample a) a sufficient amount of 35 fibrin or providing it in situ and measuring the cloudiness or the formed fibrin cleavage products, or

DK 163189BDK 163189B

3 b) tilsætter et farvedannende plasminsubstrat og en til uklar-g hedsdannelse ikke tilstrækkelig mængde af fibrin, fibrino- gen-spalteprodukter eller et enzym, der danner fibrin in situ og måler den dannede farve.3 b) does not add a sufficient amount of fibrin, fibrinogen cleavage products or an enzyme which produces fibrin in situ to measure the color formed, and a color-forming plasma substrate and a fuzzy formation.

Med uklarhedsmåling forstås her indenfor den foreliggende op-10 findelses rammer målingen af uklarhedens formindskelse eller forøgelse pr. tidsenhed, målingen af tiden indtil opnåelse af uklarhedsmaksimum eller måling af tiden indtil opnåelse af en bestemt nedsættelse eller forøgelse af uklarheden, regnet i forhold til uklarhedsmaksimummet. Der kan også anvendes en 15 kombination af disse målemuligheder. Særligt foretrækkes det at måle tiden indtil opnåelse _ af en defineret uklarhedsnedsættelse eller tiden indtil opnåelse af uklarhedsmaksimum.By ambiguity measurement here is understood within the scope of the present invention, the measurement of the reduction of obscurity or increase per minute. unit of time, the measurement of time until the cloudiness peak is reached, or the measurement of time until a certain reduction or increase in cloudiness, calculated in relation to the cloudiness peak. A combination of these measurement options can also be used. In particular, it is preferred to measure the time until attainment of a defined cloud reduction or the time until attainment of cloudiness.

Farvemålingen kan foregå ved at følge farvedannelsen kinetisk, 20 idet ekstinktionsændringen pr. tidsenhed fastsættes. Ligeledes kan man bestemme den efter en i forvejen angivet tid opnåede ekstinktion eller farvedannelsesreaktionen kan efter en i forvejen angivet tid afbrydes, og derefter kan man på et vilkårligt senere tidspunkt måle den dannede farve.The color measurement can be carried out by following the color formation kinetically. time unit is determined. Likewise, one can determine the extinction obtained at a predetermined time or the color formation reaction can be interrupted after a predetermined time, and then at any later time the color formed can be measured.

25 Målingen af de dannede fibrin-spalteprodukter kan foregå ved hjælp af i handelen almindeligt tilgængelige prøvereagenser eller prøvemetoder. Eksempler er stafylokok-dumping-test (fremstillet af Boehringer Mannheim GmbH) eller immunologiske 30 metoder f.eks. ved hjælp af antistofbelagte latexpartikler (fremstillet af Wellcome Corp.).The measurement of the fibrin cleavage products formed can be carried out by commercially available test reagents or test methods. Examples are staphylococcal dumping tests (manufactured by Boehringer Mannheim GmbH) or immunological methods e.g. using antibody coated latex particles (manufactured by Wellcome Corp.).

. Afbrydelsen af farvedannelsesreaktionen kan ske ved tilsætning af egnede inhibitorer, fortrinsvis plasmininhibitorer eller 35 syrer som f.eks. eddikesyre eller citronsyre. Fortrinsvis måles tiden indtil opnåelsen af en bestemt ekstinktion.. The color formation reaction may be interrupted by the addition of suitable inhibitors, preferably plasmin inhibitors or acids such as e.g. acetic or citric acid. Preferably, the time until the attainment of a particular extinction is measured.

44

DK 163189 BDK 163189 B

Den ovenfor definerede fremgangsmåde ifølge opfindelsen giver 5 en bestemmelse af plasmas hyperfibrinolytiske aktivitet. Her ved udløser allerede i plasmaen tilstedeværende eller fremkomne plasminogenaktivatorer reaktionen. Denne udførelsesform for fremgangsmåden ifølge opfindelsen egner sig navnlig til at følge en fibrinolyseterapi. Man kan dermed fastslå, hvorledes det 10 naturlige plasmas fibrinolytiske tilstand ændrer sig i løbet af terapien.The method defined above according to the invention provides a determination of the hyperfibrinolytic activity of the plasma. Hereby plasminogen activators already present or produced in the plasma trigger the reaction. This embodiment of the method according to the invention is particularly suitable for following a fibrinolysis therapy. It is thus possible to ascertain how the fibrinolytic state of the natural plasma changes over the course of therapy.

Ifølge en yderligere udføreIsesform for fremgangsmåden ifølge opfindelsen tilsættes der yderligere en bestemt mængde plasmi-15 nogenaktivator, idet der fortrinsvis benyttes terapeutisk an vendte koncentrationer. Ved denne udførelsesform for fremgangsmåden bestemmer man plasmaets mulige reaktivitet i nærværelse af definerede mængder plasminogenaktivator. Dette er særlig vigtigt for indledningen af en fibrinolyseterapi. Man kan der-20 ved fastslå, hvorledes plasmaets fibrinolytiske tilstand ændrer sig, når der tilsættes de til.en fibrinolyseterapi påtænkte terapeutika, såsom t-PA (EPA) , urokinase eller streptokinase.According to a further embodiment of the method according to the invention, a further amount of plasma activator is further added, preferably using therapeutically used concentrations. In this embodiment of the method, the potential reactivity of the plasma is determined in the presence of defined amounts of plasminogen activator. This is particularly important for the initiation of a fibrinolysis therapy. One can then ascertain how the fibrinolytic state of the plasma changes when added to the fibrinolysis therapy intended therapeutics such as t-PA (EPA), urokinase or streptokinase.

Ifølge en foretrukket udførelsesform tilvejebringes fibrinet 25 in situ ved tilsætning af thrombin eller et thrombinlignende enzym, såsom batroxobin eller arvin.According to a preferred embodiment, the fibrin 25 is provided in situ by the addition of thrombin or a thrombin-like enzyme such as batroxobin or arvin.

Som fibrinogenspalteprodukter anvendes fortrinsvis sådanne, der opnås ved behandling af fibrinogen med bromcyan (I.H.Ver-30 heijen, Thromb. Haemostas 48, 266-269 (1982). Fortrinsvis anvendes en koncentration fra 10 til 150 μ g/ml.Preferably, as fibrinogen cleavage products, those obtained by treating fibrinogen with bromocyan (I.H.V.-Verheijen, Thromb. Haemostas 48, 266-269 (1982)) are used.

. Fibrinmonomerer fremstilles ved behandling af fibrinogen med batroxobin, inaktivering af batroxobin med diisopropylfluor-35 fosfat, fjernelse og efterfølgende opløsning af fibrinklumper i 1 til 3 molær -urinstofopløsning. Fortrinsvis anvendes koncentrationer på 1 til 100 μ g/ml fibrinmonomer.. Fibrin monomers are prepared by treating fibrinogen with batroxobin, inactivating batroxobin with diisopropyl fluorophosphate, removing and subsequently dissolving fibrin lumps in 1 to 3 molar urea solution. Preferably, concentrations of 1 to 100 µg / ml fibrin monomer are used.

55

DK 163189 BDK 163189 B

Som farvedannende plasminsubstrat kan anvendes ethvert plas-5 minsusbstrat, hvorfra der under indvirkning af plasmin fra spaltes en til farvedannelse egnet gruppe. Som farvedannende grupper kommer her både sådanne på tale, der kan måles direkte fotometrisk som f.eks. nitroanilin,. dinitroanilin og derivater deraf, såvel som sådanne forbindelser, der ved reaktion med en 10 yderligere komponent bevirker en farvedannelse. Eksempler her på er:Any plasmid substrate may be used as a dye-forming plasma substrate from which a group suitable for dye formation is cleaved under the action of plasmin. As color-forming groups, both here come into question which can be measured directly photometrically such as e.g. nitroaniline ,. dinitroaniline and derivatives thereof, as well as such compounds which, upon reaction with an additional component, effect coloration. Examples here are:

Ved en udførelsesform for fremgangsmåden ifølge opfindelsen under tilsætning af en plasminogenaktivator' kan man som plas-15 minogenaktivator anvende EPA (Extrinsic Plasminogen-Aktivator t-PA), urokinase eller streptokinase. Et foretrukket kromogent plasminsubstrat er Tos-Gly-Pro-Lys-p-nitroanilin.In one embodiment of the method of the invention with the addition of a plasminogen activator, one may use as a plasminogen activator EPA (Extrinsic Plasminogen Activator t-PA), urokinase or streptokinase. A preferred chromogenic plasma substrate is Tos-Gly-Pro-Lys-β-nitroaniline.

Fremgangsmåden ifølge opfindelsen kan gennemføres ved en vil-20 kårlig temperatur mellem ca. 20 og 40°C.The process according to the invention can be carried out at any temperature between approx. 20 and 40 ° C.

En yderligere genstand for opfindelsen er et reagens til bestemmelse af plasmas fibrinolytiske tilstand, som er egnet til gennemføring af fremgangsmåden ifølge opfindelsen. Et sådant 25 reagens er ejendommelig ved, at det indeholder plasminogenakti vator, thrombin eller et thrombinlignende enzym og puffer.A further object of the invention is a reagent for determining the fibrinolytic state of the plasma suitable for carrying out the process of the invention. Such a reagent is characterized in that it contains plasminogen activator, thrombin or a thrombin-like enzyme and buffer.

I stedet for thrombin eller thrombinlignende enzym kan reagenset også indeholde fibrinspalteprodukter eller fibrinmomo-30 mer.Instead of thrombin or thrombin-like enzyme, the reagent may also contain fibrin cleavage products or fibrin monomers.

Dersom reagenset ifølge opfindelsen er bestemt±il varianten . b) ved fremgangsmåden ifølge opfindelsen, dvs. farvemålingen, indeholder det yderligere et farvegivende plasminsubstrat.If the reagent of the invention is determined ± in the variant. b) by the method according to the invention, i. the color measurement, it further contains a color-giving plasma substrate.

3535

Ifølge en foretrukket udførelsesform indeholder reagenset i-følge opfindelsen desuden mindst et stof fra gruppen polyethy- 6In accordance with a preferred embodiment, the reagent of the invention further contains at least one substance of the polyethylene group.

DK 163189 BDK 163189 B

lenglokol, ikke-ionisk, overfaIdeaktivt middel og okseserum-5 albumin.elongol, nonionic, surfactant and bovine serum albumin.

I en første udførelsesform for reagenset ifølge opfindelsen indeholder dette EPA, batroxobin, trispuffer pH 7 til 8, poly-ethylenglykol og ikke-ionisk-overfladektivt middel.In a first embodiment of the reagent according to the invention, this contains EPA, batroxobin, tris buffer pH 7 to 8, polyethylene glycol and nonionic surfactant.

1010

Til farvemåling indeholder dette reagens hensigtsmæssigt i stedet for batroxobin fibrinogen-spalteprodukter fremstillet ved bromcyanbehandling af fibrinogen eller i stedet fibrinmo-nomer og desuden Tos-Gly-Pro-Lys-p-nitroanilin, 15For color measurement, this reagent conveniently contains instead of batroxobin fibrinogen cleavage products prepared by bromocyanine treatment of fibrinogen or instead fibrin monomers and in addition Tos-Gly-Pro-Lys-p-nitroaniline,

For de enkelte bestanddele af reagenset ifølge opfindelsen gælder tilsvarende de ovennævnte udførelsesformer i sammenhæng med den nærmere forklaring af fremgangsmåden.Similarly, for the individual components of the reagent of the invention, the above embodiments apply in the context of the further explanation of the process.

20 De følgende eksempler belyser opfindelsen yderligere i forbindelse med den vedføjede tegning.The following examples further illustrate the invention in connection with the accompanying drawings.

På tegningen viser: 25 fig.l en justeringskurve for en uklarhedsprøve med EPA som plas-minogenaktivator.In the drawing: Fig. 25 shows an adjustment curve for a turbidity test with EPA as a plasminogen activator.

Eksempel 1 30Example 1 30

Uklarhedsprøve med EPA som plasminogenaktivatorFuzzy test with EPA as plasminogen activator

Reagenssammensætning: * Tris/HCL, 0,1 mol/1, pH 7,5Reagent composition: * Tris / HCL, 0.1 mol / l, pH 7.5

Polyethylenglykol 6000, 2 vægt% 35 l p\ overfladeaktivt middel ("Tween"v '80) 0,1 vægt% okseserumalbumin (RSA) , 1 vægt% batroxobin 0,02 BU/ml EPA (0,01 - 10 ng/ml prøveportion) 7Polyethylene glycol 6000, 2 wt% 35 lp \ surfactant ("Tween" v '80) 0.1 wt% bovine serum albumin (RSA), 1 wt% batroxobin 0.02 BU / ml EPA (0.01 - 10 ng / ml sample portion) ) 7

DK 163189 BDK 163189 B

Prøveportion: 50 μΐ prøve (plasma) 5 1000 μΐ reagensSample portion: 50 μΐ sample (plasma) 5 1000 μΐ reagent

Reagens og prøve blandes ved 25°C, og uklarheden følges straks med et fotometer ved λ = 334 nmReagent and sample are mixed at 25 ° C and the cloudiness is immediately followed by a photometer at λ = 334 nm

Ekstinktionsændringen registreres ved hjælp af en skriver (pa-10 pirhastighed 0,1 cm/min).The extinction change is recorded with the help of a printer (p-10 pir speed 0.1 cm / min).

Fremstillingen af en justeringskurve sker enten ved at fastlægge tiden t^ indtil der nås uklarhedsmaksimum (fig. 1, kurve 1) eller den tid t„ indtil der - udgående fra uklarhedsmaksimum -15 ^ opnås en ekstinktionsnedsættelse på 100 mE (fig. 1, kurve 2). Eksempel 2 20 Uklarhedsprøve med streptokinase eller urokinase som plasmino-genaktivator.An adjustment curve is produced either by determining the time t ^ until the cloudiness peak is reached (Fig. 1, curve 1) or the time t «until - based on the cloudiness maximum -15 ^, an extinction reduction of 100 mE is obtained (Figure 1, curve 2). EXAMPLE 2 Fuzziness test with streptokinase or urokinase as plasminogen activator.

Reagenssammensætning, prøveportion og prøvegennemførelse svarer til eksempel 1, idet der i stedet for EPA tilsættes strep-25 tokinase eller urokinase i forskellige koncentrationer.Reagent composition, sample portion, and assay are similar to Example 1, with strep-tokinase or urokinase added at various concentrations instead of EPA.

Der opnås derved følgende måleværdier; a) streptokinase koncentration iU/ml prøveportion 0 0,05 0,10 0,15 0,20 0,25 t^ [min] (jvf. eksempel 1) 55 40 36,5 17,0 15,0 10,5 t- [min] 35 ^ (jvf. eksempel 1) 35 27 23 15 14 8The following measurement values are thus obtained; a) streptokinase concentration in IU / ml sample portion 0 0.05 0.10 0.15 0.20 0.25 t ^ [min] (cf. Example 1) 55 40 36.5 17.0 15.0 10.5 t - [min] 35 ^ (cf. Example 1) 35 27 23 15 14 8

DK 163189 BDK 163189 B

b) Urokinase 5 koncentration ng/ml prøveportion 0 10 50 100 200 300 400 t^ [min] 65 50 23,5 16f5 12,0 9,5 8,5 10 t2 [min] °° 55 13 7,5 4,0 · 3,5 3,0b) Urokinase 5 concentration ng / ml sample portion 0 10 50 100 200 300 400 t ^ [min] 65 50 23.5 16f5 12.0 9.5 8.5 10 t2 [min] ° 55 55 13 7.5 4, 0 · 3.5 3.0

Eksempel 3 15Example 3 15

Farveprøve med EPA som plasminogenaktivatorColor sample with EPA as plasminogen activator

Reagens sammensætning: 20 Tris/HCL 0,1 mol/1, pH 7,5Reagent composition: Tris / HCL 0.1 mol / l, pH 7.5

Polyeth^lenglyko1 6000, 2 vægt% "Tween” 80, 0,1 vægt% RSA, 1 vægt% EPA: 0,01 til 10 ng/ml prøveportion 25 BirCN-fragmenter af fibrin, 75 μg/mlPolyethylene glycol 1.6000, 2 wt% Tween 80, 0.1 wt% RSA, 1 wt% EPA: 0.01 to 10 ng / ml sample portion 25 BirCN fragments of fibrin, 75 µg / ml

Plasminsubstrat Tos-Gly-Pro-Lys-pNA, 0,15 mmol/1Plasma substrate Tos-Gly-Pro-Lys pNA, 0.15 mmol / l

Prøveportion: 50 μΐ prøve (plasma) 1000 jjI reagens 30 oSample portion: 50 µl sample (plasma) 1000 µl reagent 30 o

Reagens og prøve blandes ved 25 C, og farvedannelsen følges med et fotometer ved \ = 405 nm. Ekstinktionsændringen registreres ved hjælp af en skriver (papirhastighed 0,1 cm/min).Reagent and sample are mixed at 25 ° C and the color formation is monitored with a photometer at \ = 405 nm. The extinction change is recorded using a printer (paper speed 0.1 cm / min).

**

Der måles den tid t^ indtil der gående ud fra grundlinien (re- gensblindværdi) - er opnået en ekstinktionsforøgelse på 100 mE. 35The time t ^ is measured until starting from the baseline (reg. Blind value) - an extinction increase of 100 mE is obtained. 35

Claims (11)

1. Fremgangsmåde til bestemmelse af plasmas fibrinolytiske tilstand, kendetegnet ved, at man til en naturlig plasmaprøve a) tilsætter en til uklarhedsdannelse tilstrækkelig mængde fi- *1 2 brin eller tilvejebringer denne in situ og måler uklarheden eller de dannede fibrin-spalteprodukter, eller b) tilsætter et farvedannende plasminsubstrat og en til uklarhedsdannelse ikke tilstrækkelig mængde fibrin, fibrinogen- 20 spalteprodukter eller et enzym, der danner fibrin in situ, og måler den dannede farve.A method for determining the fibrinolytic state of the plasma, characterized in that a sufficient amount of fibrin is added to the natural plasma sample (a) or provides it in situ and provides the cloudiness or the fibrin cleavage products formed, or b ) does not add a sufficient amount of fibrin, fibrinogen cleavage products, or an in situ fibrin enzyme to measure the color produced, a color-forming plasma substrate and a cloud-forming agent. 2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at man yderligere tilsætter en bestemt mængde plasminogenaktiva- 25 tor.Process according to claim 1, characterized in that a certain amount of plasminogen activator is further added. 3. Fremgangsmåde ifølge krav 1 eller 2, kendetegnet ved, at man tilvejebringer fibrin in situ ved tilsætning af thrombin eller et thrombinlignende enzym såsom batroxobin el- 30 , ler arvin. Fremgangsmåde ifølge krav 2 eller 3, kendetegnet ved, at man som plasminogenaktivator tilsætter EPA, urokinase eller streptokinase. 35 2 Fremgangsmåde ifølge et af de foregående krav, kendetegnet ved, at man som fibrinogen-spalteprodukter anven- DK 163189 B der sådanne, der vindes ved behandling af fibrinogen med bromcyan .Process according to claim 1 or 2, characterized in that fibrin is provided in situ by the addition of thrombin or a thrombin-like enzyme such as batroxobin or arvin. Process according to claim 2 or 3, characterized in that EPA, urokinase or streptokinase are added as a plasminogen activator. A method according to any one of the preceding claims, characterized in that as fibrinogen cleavage products, such as are obtained by the treatment of fibrinogen with bromocyanone, are used. 6. Fremgangsmåde ifølge et af de foregående krav, kende-5 tegnet ved, at man som farvedannende pi asmi nsubstrat anvender Tos-Gly-Pro-Lys-p-nitroani 1 in.Process according to one of the preceding claims, characterized in that Tos-Gly-Pro-Lys-β-nitroani 1 in is used as a color forming pie as a substrate. 7. Reagens til bestemmelse af plasmas fibrinolytiske tilstand ved fremgangsmåden ifølge krav 1-6, kendetegnet 10 ved, at det indeholder plasminogenaktivator, thrombin eller thrombinlignende enzym og puffer.A reagent for determining the fibrinolytic state of the plasma by the method of claims 1-6, characterized in that it contains plasminogen activator, thrombin or thrombin-like enzyme and buffer. 8. Reagens til bestemmelse af plasmas fibrinolytiske tilstand ved fremgangsmåden ifølge krav 1-6, kendetegnet 15. ved, at det indeholder plasminogenaktivator og fibrinogen- spalteprodukter eller fibrinmonomer.Reagent for determining the fibrinolytic state of plasma by the method of claims 1-6, characterized in that it contains plasminogen activator and fibrinogen cleavage products or fibrin monomer. 9. Reagens ifølge krav 7 eller 8, kendetegnet ved, at det yderligere indeholder et farvedannende plasminsubstrat. 20Reagent according to claim 7 or 8, characterized in that it further contains a color-forming plasma substrate. 20 10. Reagens ifølge krav 7-9, kendetegnet ved, at det indeholder polyethylenglykol, et ikke-ionisk overfladeaktivt middel og/eller okseserumalbumin.Reagent according to claims 7-9, characterized in that it contains polyethylene glycol, a nonionic surfactant and / or bovine serum albumin. 11. Reagens ifølge et af kravene 7 - 10, kendetegnet ved, at det indeholder EPA, batroxobin, trispuffer pH 7 til 8, polyethylenglykol og ikke-ionisk overfladeaktivt middel.Reagent according to one of claims 7 to 10, characterized in that it contains EPA, batroxobin, tris buffer pH 7 to 8, polyethylene glycol and nonionic surfactant. 12. Reagens ifølge krav 11, kendetegnet ved, at det 30 indeholder EPA, trispuffer pH 7 til 8, polyethylenglykol, ikke-ionisk overfladeaktivt middel og ved bromcyanbehandling af fibrinogen fremstillede fibrinogen-spalteprodukter eller fibrin-monomer og Tos-Gly-Pro-Lys-p-nitroani1 in.Reagent according to Claim 11, characterized in that it contains EPA, tris buffer pH 7 to 8, polyethylene glycol, nonionic surfactant and by bromocyanine treatment of fibrinogen produced fibrinogen cleavage products or fibrin monomer and Tos-Gly-Pro-Lys -p-nitroani1 in. 13. Anvendelse af thrombin eller thrombinlignende enzym sammen med puffer som reagens til bestemmelse af plasmas fibrinolyti-ske tilstand.13. Use of thrombin or thrombin-like enzyme together with buffer as reagent to determine plasma fibrinolytic state.
DK030686A 1985-01-29 1986-01-21 METHOD AND REAGENT OF DETERMINATION OF PLASMAS FIBRINOLYTIC CONDITIONS DK163189C (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3502878 1985-01-29
DE19853502878 DE3502878A1 (en) 1985-01-29 1985-01-29 METHOD FOR DETERMINING THE FIBRINOLYTIC STATE OF PLASMA

Publications (4)

Publication Number Publication Date
DK30686D0 DK30686D0 (en) 1986-01-21
DK30686A DK30686A (en) 1986-07-30
DK163189B true DK163189B (en) 1992-02-03
DK163189C DK163189C (en) 1992-06-22

Family

ID=6261029

Family Applications (1)

Application Number Title Priority Date Filing Date
DK030686A DK163189C (en) 1985-01-29 1986-01-21 METHOD AND REAGENT OF DETERMINATION OF PLASMAS FIBRINOLYTIC CONDITIONS

Country Status (8)

Country Link
EP (1) EP0189910B1 (en)
JP (1) JPS61178000A (en)
AT (1) ATE45771T1 (en)
CA (1) CA1276097C (en)
DE (2) DE3502878A1 (en)
DK (1) DK163189C (en)
ES (1) ES8701841A1 (en)
NO (1) NO167588C (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU193126B (en) * 1985-08-15 1987-08-28 Valeria Duschanek Method for forecasting quality of flesh of the livestocks and for selecting livestocks on the basis of this
DE3705744A1 (en) * 1987-02-23 1988-09-01 Behringwerke Ag METHOD FOR THE FUNCTIONAL DETERMINATION OF PROTEIN C-INHIBITOR
DE3838529A1 (en) * 1988-11-14 1990-05-17 Behringwerke Ag GLOBALTEST FOR DETECTING THE MAIN COMPONENTS OF THE FIBRINOLYSIS SYSTEM
DE3900493A1 (en) * 1989-01-10 1990-07-12 Thomae Gmbh Dr K METHOD FOR DETERMINING OVERALL FIBRINOLYTIC ACTIVITY IN PLASMA
CA2100882A1 (en) * 1991-11-25 1993-05-26 Rosa M. F. Denis Method for measuring fibrinolytic capacity within whole human plasma
ES2090835T3 (en) * 1992-02-17 1996-10-16 Akzo Nobel Nv CALIBRATOR AND ITS USE IN AN IMMUNOLOGICAL TEST.
US5708591A (en) * 1995-02-14 1998-01-13 Akzo Nobel N.V. Method and apparatus for predicting the presence of congenital and acquired imbalances and therapeutic conditions
US6429017B1 (en) 1999-02-04 2002-08-06 Biomerieux Method for predicting the presence of haemostatic dysfunction in a patient sample
US6898532B1 (en) 1995-06-07 2005-05-24 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US6321164B1 (en) 1995-06-07 2001-11-20 Akzo Nobel N.V. Method and apparatus for predicting the presence of an abnormal level of one or more proteins in the clotting cascade
US6502040B2 (en) 1997-12-31 2002-12-31 Biomerieux, Inc. Method for presenting thrombosis and hemostasis assay data
ATE282208T1 (en) 1999-02-04 2004-11-15 Bio Merieux Inc METHOD AND APPARATUS FOR PREDICTING HEMOSTATIC FUNCTION IN PATIENT SAMPLES
US7179612B2 (en) 2000-06-09 2007-02-20 Biomerieux, Inc. Method for detecting a lipoprotein-acute phase protein complex and predicting an increased risk of system failure or mortality

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2431342C3 (en) * 1973-07-27 1978-10-26 Behringwerke Ag, 3550 Marburg Method for measuring the plasminogen content by determining the formation of fibrin in a sample
DE2525804B2 (en) * 1975-06-10 1980-04-03 Behringwerke Ag, 3550 Marburg Stable clumping factor, use of the same for the detection of fibrinogen and fibrin breakdown products and production of the same
JPS59210900A (en) * 1983-05-14 1984-11-29 Mihama Hisaharu Measurement of activity of plasmin in blood
DE3330699A1 (en) * 1983-08-25 1985-03-07 Boehringer Mannheim Gmbh, 6800 Mannheim METHOD FOR THE SIMULTANEOUS DETERMINATION OF FIBRINOGEN AND FIBRINOGEN Fission Products in the Plasma

Also Published As

Publication number Publication date
EP0189910B1 (en) 1989-08-23
EP0189910A2 (en) 1986-08-06
NO167588B (en) 1991-08-12
ATE45771T1 (en) 1989-09-15
DK30686D0 (en) 1986-01-21
DK163189C (en) 1992-06-22
NO860089L (en) 1986-07-30
DK30686A (en) 1986-07-30
DE3665196D1 (en) 1989-09-28
EP0189910A3 (en) 1986-12-10
JPS61178000A (en) 1986-08-09
DE3502878A1 (en) 1986-07-31
CA1276097C (en) 1990-11-13
ES8701841A1 (en) 1986-12-16
ES551389A0 (en) 1986-12-16
NO167588C (en) 1991-11-20

Similar Documents

Publication Publication Date Title
EP0420332B1 (en) Method for determining the endogenous thrombin potential of plasma and blood, and also a kit for use in said method
US9428790B2 (en) Simultaneous measurement of thrombin generation and clot strength in plasma and whole blood
DK163189B (en) METHOD AND REAGENT OF DETERMINATION OF PLASMAS FIBRINOLYTIC CONDITIONS
JP4718833B2 (en) Diagnostic tests to determine the concentration of transient proteolytic activity in complex biological media
DK171086B1 (en) Method for photometric determination of protein C and / or protein S activity
Myrmel et al. Characteristics of the association between prothrombin fragment 2 and α-thrombin
Tans et al. Autoactivation of human plasma prekallikrein.
HU176068B (en) Process and reagent for the determination of biologically active heparine in blood
TWI443193B (en) Protein assay
EP2529221B1 (en) Thrombin generation determination method
JPH0258920B2 (en)
Eisen Kinin formation and fibrinolysis in human plasma
Pochron et al. A fluorescent substrate assay for plasminogen
US5188940A (en) Method of determining the total fibrinolytic activity in the plasma
Lauritsen Urokinase inhibitor in human plasma
US3778352A (en) Plasminogen assay system
Donaldson C′ l activation in hereditary angioneurotic edema plasma: role of urokinase and inhibitors
US5529905A (en) Method of assaying plasma proteins with prothrombin fragments
Stafford The fibrinolytic mechanism in haemostasis: a review
CA1313488C (en) Assay
Heimburger et al. Blood coagulation and fibrinolysis
US4210420A (en) Detection of fibrin monomer and composition therefor
Gjønnæss Cold promoted activation of factor VII
Valeri et al. Reaction of antithrombin with proteases. Evidence for a specific reaction with papain
CA2598757A1 (en) Method for determining the total coagulation activity of a blood or plasma sample