NO309902B1 - Method of determining fibrinogen - Google Patents
Method of determining fibrinogen Download PDFInfo
- Publication number
- NO309902B1 NO309902B1 NO931773A NO931773A NO309902B1 NO 309902 B1 NO309902 B1 NO 309902B1 NO 931773 A NO931773 A NO 931773A NO 931773 A NO931773 A NO 931773A NO 309902 B1 NO309902 B1 NO 309902B1
- Authority
- NO
- Norway
- Prior art keywords
- fibrinogen
- thrombin
- meizothrombin
- heparin
- carried out
- Prior art date
Links
- 108010049003 Fibrinogen Proteins 0.000 title claims abstract description 43
- 102000008946 Fibrinogen Human genes 0.000 title claims abstract description 43
- 229940012952 fibrinogen Drugs 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 20
- 108090000190 Thrombin Proteins 0.000 claims abstract description 36
- 229960004072 thrombin Drugs 0.000 claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 13
- 102000009123 Fibrin Human genes 0.000 claims abstract description 11
- 108010073385 Fibrin Proteins 0.000 claims abstract description 11
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229950003499 fibrin Drugs 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 108010011227 meizothrombin Proteins 0.000 claims description 26
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 22
- 229960002897 heparin Drugs 0.000 claims description 22
- 229920000669 heparin Polymers 0.000 claims description 22
- 230000015271 coagulation Effects 0.000 claims description 17
- 238000005345 coagulation Methods 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 11
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 230000007017 scission Effects 0.000 claims description 7
- 229940122388 Thrombin inhibitor Drugs 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000003868 thrombin inhibitor Substances 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000000337 buffer salt Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 230000002345 thrombinlike Effects 0.000 abstract description 5
- 108010094028 Prothrombin Proteins 0.000 abstract description 3
- 102100027378 Prothrombin Human genes 0.000 abstract description 3
- 229940039716 prothrombin Drugs 0.000 abstract description 3
- 239000000282 fibrinogen degradation product Substances 0.000 abstract 1
- 239000012634 fragment Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 4
- 102000004411 Antithrombin III Human genes 0.000 description 4
- 108090000935 Antithrombin III Proteins 0.000 description 4
- 229960005348 antithrombin iii Drugs 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 102000003839 Human Proteins Human genes 0.000 description 3
- 108090000144 Human Proteins Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 239000003998 snake venom Substances 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 229960002319 barbital Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108010027612 Batroxobin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 101000651439 Homo sapiens Prothrombin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 241000360071 Pituophis catenifer Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000008841 allosteric interaction Effects 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 229960002210 batroxobin Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 208000015294 blood coagulation disease Diseases 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 230000009852 coagulant defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 108010085662 ecarin Proteins 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229940039715 human prothrombin Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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
-
- 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)
- G01N2333/974—Thrombin
Abstract
Description
Oppfinnelsen vedrører en fremgangsmåte ved bestemmelse av fibrinogen ved hjelp av enzymatisk omvandling med trombin, meizotrombin, meizotrombin (des. Fl) eller blandinger derav, og påfølgende påvisning av det dannede fibrin hhv. fibrinogenspaltningsprodukter. Dessuten omfatter oppfinnelsen et reagens for bestemmelse av fibrinogeninnholdet i en eventuelt ufortynnet og eventuelt også heparinholdig plasmaprøve. The invention relates to a method for the determination of fibrinogen by means of enzymatic conversion with thrombin, meizothrombin, meizothrombin (des. Fl) or mixtures thereof, and subsequent detection of the formed fibrin or fibrinogen cleavage products. In addition, the invention includes a reagent for determining the fibrinogen content in a possibly undiluted and possibly also heparin-containing plasma sample.
Bestemmelsen av fibrinogen i plasma er innenfor rammen av undersøkelser av koaguleringsforstyrrelser av stor betyd-ning. For bestemmelse av fibrinogenet er det kjent såvel immunologiske som funksjonelle metoder, såsom f.eks. koaguleringstester. Ved koaguleringstestene bestemmes fibrinogeninnholdet ved tidsmåling av koagulatdanne 1 sen. The determination of fibrinogen in plasma is within the framework of investigations of coagulation disorders of great importance. Both immunological and functional methods are known for determining the fibrinogen, such as e.g. coagulation tests. In the coagulation tests, the fibrinogen content is determined by measuring the time of coagulate formation for 1 second.
En vanlig bestemmelsesmetode er beskrevet av Clauss (Acta Haemat. 17, 237-246, 1957). Oksalatplasma fortynnes i en buffer med pH 7,4, og en konsentrert trombinoppløsning tilsettes. Derefter bestemmes tiden inntil koagulerings-sluttpunktet. Koaguleringstiden av plasmaprøven som er omsatt med trombin, er imidlertid proporsjonalt med fibrinogeninnholdet bare, når det velges et tilstrekkelig stort overskudd av trombin til plasma, for å koble ut plasmatiske forstyrrelsesfaktorer. Som forstyrrelses-faktor regnes antitrombin III (AT III), som fremfor alt sammen med heparin hemmer trombinvirkningen. Heparin er en vanlig antikoagulant og anvendes som tilsetning til blodprøver eller som terapeutikum. Et heparininnhold i en plasmaprøve kan føre til forlengede koaguleringstider og således til et falskt resultat. A common method of determination is described by Clauss (Acta Haemat. 17, 237-246, 1957). Oxalate plasma is diluted in a buffer with pH 7.4, and a concentrated thrombin solution is added. The time until the coagulation endpoint is then determined. However, the coagulation time of the plasma sample reacted with thrombin is proportional to the fibrinogen content only, when a sufficiently large excess of thrombin is selected for plasma, to exclude plasmatic disturbance factors. Antithrombin III (AT III), which above all together with heparin inhibits thrombin action, is considered to be an interfering factor. Heparin is a common anticoagulant and is used as an additive to blood tests or as a therapeutic. A heparin content in a plasma sample can lead to prolonged coagulation times and thus to a false result.
Virkningen av antitrombin III i plasma blir her sterkt forminsket, takket være den høye trombinkonsentrasjon og fortynning av plasmaet og dermed forminskningen av konsentrasjonen av AT III hhv. heparin. Når plasma ikke blir fortynnet, er antitrombinvirkningen flere ganger sterkere og koaguleringstiden forlenget. The effect of antithrombin III in plasma is greatly reduced here, thanks to the high thrombin concentration and dilution of the plasma and thus the reduction of the concentration of AT III or heparin. When plasma is not diluted, the antithrombin effect is several times stronger and the coagulation time is prolonged.
En plasmafortynning og det dermed forbundne ytterligere fremgangsmåtetrinn betyr ved rutineundersøkelser en be-traktelig belastning. Ved siden av øket arbeidsbyrde og ytterligere håndtering med potensielt infeksjonsfremkal-lende plasma, er enhver fortynning en prinsipiell feilkilde. Dessuten må fortynninger ofte gjennomføres i samsvar med fibrinogeninnholdet, noe som vanskeliggjør arbeidet med analyseautomater. A plasma dilution and the associated further method step means a considerable burden in routine examinations. In addition to increased workload and further handling of potentially infection-causing plasma, any dilution is a fundamental source of error. In addition, dilutions often have to be carried out in accordance with the fibrinogen content, which makes work with automatic analyzers difficult.
Vanligvis anvendes for plasmafortynning veronalbuffer, fordi denne buffer letter dannelsen av målbare fibrin-koagulater, noe som forbedrer bestemmelsene i plasma med lite fibrinogeninnhold. Veronal er imidlertid samtidig også et bedøvelsesmiddel som kan føre til avhengighet, hvorfor omgangen med denne substans helst skal unngås. Generally, veronal buffer is used for plasma dilution, because this buffer facilitates the formation of measurable fibrin coagulates, which improves determinations in plasma with a low fibrinogen content. However, Veronal is also an anesthetic that can lead to addiction, which is why dealing with this substance should preferably be avoided.
Ytterligere forsøk med å utelukke forstyrrelsesfaktoren antitrombin III hhv. heparin, medfører nøytralisasjon hhv. fjerning av heparinet. I Clinical Chemistry 29, 614-617 (1983) er det beskrevet en fremgangsmåte hvor heparinet i en plasmaprøve nøytraliseres ved tilsetning av plybren. Plasma fortynnes i en buffer med pH 7,4, og overføres efter tilsetning av trombin til et fotometer. Derefter bestemmes reaksjonshastigheten nefelometrisk. Further attempts to rule out the interfering factor antithrombin III or heparin, leads to neutralization or removal of the heparin. In Clinical Chemistry 29, 614-617 (1983) a method is described in which the heparin in a plasma sample is neutralized by adding plybren. Plasma is diluted in a buffer with pH 7.4, and after addition of thrombin is transferred to a photometer. The reaction rate is then determined nephelometrically.
Ved anvendelse av slangegiftenzymer med en trombinlignende virksomhet (Batroxobin) i stedet for trombin, kan fibrinogen i plasma bestemmes uavhengig av de nevnte forstyrrelsesfaktorer. Disse slangegiftenzymer inhiberes nemlig ikke ved AT III hhv. heparin. By using snake venom enzymes with a thrombin-like activity (Batroxobin) instead of thrombin, fibrinogen in plasma can be determined independently of the aforementioned interfering factors. These snake venom enzymes are not inhibited by AT III or heparin.
Anvendelsen av proteiner som ikke regnes som naturlige koaguleringsenzymer såsom f.eks. slangegiftenzymer, er problematiske ved fremgangsmåter for bestemmelse av humane proteiner. Reaksjonene mellom et ikke-pattedyr-enzym og et humant protein er ofte uspesifikke. Ifølge prin-sippet for biokjemisk analyse, å sammenligne og å under-søke bare likt med likt, prøver man ved bestemmelsen av humane proteiner likeledes å anvende bare enzymer fra pattedyr og fremfor alt humane enzymer som reaksjons-partnere. The use of proteins that are not considered natural coagulation enzymes such as e.g. snake venom enzymes, are problematic in methods for the determination of human proteins. The reactions between a non-mammalian enzyme and a human protein are often non-specific. According to the principle of biochemical analysis, to compare and to examine only like with like, when determining human proteins, one also tries to use only enzymes from mammals and above all human enzymes as reaction partners.
Trombin kan fremstilles ved aktivering av protrombin. Som forstadium oppstår under aktiveringen meizotrombin (MT) og meizotrombin (desFl). De tokjedede trombin-mellomtrinn viser imidlertid i motsetning til protrombin en proteaseaktivitet som ligner trombinets trombinlignende aktivitet. De ar derfor i stand til likeledes å spalte substrater som kan omsettes av trombin. En trombinlignende aktivitet er således en proteaseaktivitet som er i stand til å spalte trombinsubstrater hvis reaksjons-kinetikk og reaksjonsparametre, såsom pH- eller ione-styrkeoptima, inhibitorer, effektorer, allosteriske vek-selvirkninger osv., imidlertid ikke nødvendigvis må ligne trombinets. Thrombin can be produced by activation of prothrombin. As a precursor, meizothrombin (MT) and meizothrombin (desFl) occur during activation. However, unlike prothrombin, the two-chain thrombin intermediates show a protease activity similar to thrombin's thrombin-like activity. They are therefore also capable of cleaving substrates that can be converted by thrombin. A thrombin-like activity is thus a protease activity capable of cleaving thrombin substrates whose reaction kinetics and reaction parameters, such as pH or ionic strength optima, inhibitors, effectors, allosteric interactions, etc., however, do not necessarily have to resemble those of thrombin.
Aktiviteten av de bovine enzymer meizotrombin, meizotrombin (desFl) og trombin blir sammenlignet i J. of Biol. Chemistry 265, 10693-10701 (1990) ved en pH på 7,4. Meizotrombin viser bare 2 % av trombinets "fibrinogen clotting activity". Denne ubetydelige aktivitet kan imidlertid også føres tilbake til et 10 til 15 % MT(desFl)-innhold i MT-sammensetningen. For MT(desFl) ble aktiviteten funnet å være 14 % av trombinets. The activity of the bovine enzymes meizothrombin, meizothrombin (desFl) and thrombin is compared in J. of Biol. Chemistry 265, 10693-10701 (1990) at a pH of 7.4. Meizothrombin shows only 2% of thrombin's "fibrinogen clotting activity". However, this negligible activity can also be traced back to a 10 to 15% MT(desFl) content in the MT composition. For MT(desFl), the activity was found to be 14% of that of thrombin.
Testen for enzymatisk omvandling av fibrinogen i fibrin gjennomføres vanligvis ved en pH-verdi mellom 7,4 og 8,0. Dette pH-område anvendes generelt ved trombinets enzymatiske reaksjoner, det tilsvarer nemlig det fysiologiske pH-område. Den optimale pH-verdi for trombinaktivitet ligger tett opptil ved pH 8. The test for enzymatic conversion of fibrinogen to fibrin is usually carried out at a pH value between 7.4 and 8.0. This pH range is generally used for thrombin's enzymatic reactions, namely it corresponds to the physiological pH range. The optimal pH value for thrombin activity is close to pH 8.
Oppfinnelsens oppgave består i å tilveiebringe en fremgangsmåte ved bestemmelse av fibrinogen ved hjelp av en koaguleringstest hvor ulempene hos de kjente fremgangsmåter unngås, og som kan gjennomføres spesielt også på ufortynnede plasmaprøver uavhengig av AT III- hhv. heparininnholdet. The task of the invention consists in providing a method for determining fibrinogen by means of a coagulation test where the disadvantages of the known methods are avoided, and which can be carried out in particular also on undiluted plasma samples independently of AT III or the heparin content.
Denne oppgave løses ifølge oppfinnelsen ved en fremgangsmåte for bestemmelse av fibrinogen, for det meste uavhengig av trombininhibitorer, ved enzymatisk omvandling med trombin, meizotrombin, meizotrombin (des. Fl) eller blandinger derav og påfølgende påvisning av det dannede fibrin hhv. av fibrinogenspaltningsproduktene, karakterisert ved at den enzymatiske omvandling av fibrinogenet gjennomføres ved en pH mellom 4 og 7,3 og påvisningen av det dannede fibrin foretas ved bestemmelse av koaguleringstiden hhv. fibrinogenspaltningsproduktene. Derefter påvises det dannede fibrinkoagulat. Påvisningen kan foretas fotometrisk eller turbidimetrisk via bestemmelse av koaguleringstiden. Konsentrasjonen hhv. blandingsforhol-dene av de anvendte enzymer skal velges slik at en lineær sammenheng sikres over et størst mulig område av fibrinogeninnholdet i en prøve. This task is solved according to the invention by a method for determining fibrinogen, mostly independent of thrombin inhibitors, by enzymatic conversion with thrombin, meizothrombin, meizothrombin (des. Fl) or mixtures thereof and subsequent detection of the formed fibrin or of the fibrinogen cleavage products, characterized in that the enzymatic conversion of the fibrinogen is carried out at a pH between 4 and 7.3 and the detection of the formed fibrin is carried out by determining the coagulation time or the fibrinogen cleavage products. The fibrin clot formed is then detected. The detection can be carried out photometrically or turbidimetrically via determination of the coagulation time. The concentration or the mixing ratios of the enzymes used must be chosen so that a linear relationship is ensured over the largest possible range of the fibrinogen content in a sample.
Også genteknisk fremstilte proteiner som på grunn av sin struktur viser en aktivitet som ligner trombinets og i sammenligning med trombin har en mindre affinitet til antitrombin III, er egnet for fremgangsmåten ifølge oppfinnelsen . Also genetically engineered proteins which, due to their structure, show an activity similar to that of thrombin and, in comparison with thrombin, have a lower affinity to antithrombin III, are suitable for the method according to the invention.
Gjennomføringen av bestemmelsesfremgangsmåten i det an-gitte pH-område gjør denne så langt uavhengig av de nevnte forstyrrelsesfaktorer AT III og heparin at fibrinogenbestemmelsen kan gjennomføres på enkel måte med en ufortynnet og eventuelt også heparinholdig plasmaprøve. Det ytterligere fremgangsmåtetrinn med fortynning av plasma kan utelates ved fremgangsmåten ifølge oppfinnelsen, hvorved testresultatet forbedres ved at en eventuell feilkilde utkobles. Carrying out the determination procedure in the indicated pH range makes it so far independent of the aforementioned interfering factors AT III and heparin that the fibrinogen determination can be carried out in a simple way with an undiluted and possibly also heparin-containing plasma sample. The further method step of diluting the plasma can be omitted in the method according to the invention, whereby the test result is improved by switching off a possible source of error.
Oppfinnelsen beror på den erkjennelse at inhibisjonen av trombin ved AT III/heparin ved en pH på 4 til 7,3 kan neglisjeres, hvorved trombinaktiviteten til tross for suboptimale betingelser er tilstrekkelig for enzymatisk omvandling av fibrinogen. En noe forminsket aktivitet av trombin har til og med den fordel at koagulerings tidene ikke blir for korte og lettere kan bestemmes. The invention is based on the recognition that the inhibition of thrombin by AT III/heparin at a pH of 4 to 7.3 can be neglected, whereby the thrombin activity is sufficient despite suboptimal conditions for the enzymatic conversion of fibrinogen. A somewhat reduced activity of thrombin even has the advantage that the coagulation times are not too short and can be determined more easily.
Ved bruk av trombin, meizotrombin, meizotrombin (des Fl) eller blandinger derav har det overraskende vist seg at den trombinaktige aktivitet i det nevnte pH-område, fortrinnsvis ved en pH på 5 til 7 eller mest foretrukket ved en pH på ca. 6, er optimal for bestemmelse av fibrinogen uavhengig av trombininhibitorer. When using thrombin, meizothrombin, meizothrombin (des Fl) or mixtures thereof, it has surprisingly been shown that the thrombin-like activity in the aforementioned pH range, preferably at a pH of 5 to 7 or most preferably at a pH of approx. 6, is optimal for the determination of fibrinogen independent of thrombin inhibitors.
En enzymatisk omvandling av fibrinogenet gjennomføres fortrinnsvis i en buffer med en buffersaltmolaritet på mellom 0,02 til 0,5 M, mest foretrukket 0,1 til 0,25 M. Ionestyrken kan eventuelt økes ved tilsetning av salter, såsom natriumklorid. Det har vist seg at trombin fremfor alt ved lave ionestyrker er ufølsomme i forhold til AT III-inhibisjonen. For gjennomføring av fremgangsmåten ifølge oppfinnelsen er imidlertid også mulig å anvende et miljø som i forhold til plasmaprøven har en lik eller høyere bufferkapasitet, for å holde en bestemt pH under bestemmelsesmetoden. An enzymatic conversion of the fibrinogen is preferably carried out in a buffer with a buffer salt molarity of between 0.02 to 0.5 M, most preferably 0.1 to 0.25 M. The ionic strength can optionally be increased by adding salts, such as sodium chloride. It has been shown that thrombin above all at low ionic strengths is insensitive to AT III inhibition. For carrying out the method according to the invention, however, it is also possible to use an environment which, in relation to the plasma sample, has an equal or higher buffer capacity, in order to maintain a specific pH during the determination method.
Fordelaktig skjer fibrinogenbestemmelsen med en ufortynnet og eventuelt også heparinholdig plasmaprøve hvor heparin altså ikke foreligger i nøytralisert form. Advantageously, the fibrinogen determination takes place with an undiluted and possibly also heparin-containing plasma sample where heparin is therefore not present in a neutralized form.
Oppfinnelsen vedrører også et reagens for bestemmelse av f ibrinogeninnholdet for det meste uavhengig av trombininhibitorer av en eventuelt ufortynnet og eventuelt også heparinholdig plasmaprøve karakterisert ved at det inneholder trombin, meizotrombin, meizotrombin (des Fl) eller blandiger derav for enzymatisk omvandling av fibrinogen og påfølgende påvisning av det dannede fibrin via koaguleringstiden hhv. fibrinogenspaltningsproduktene i en buffer med en pH mellom 4 og 7,3. Med reagenset oppnås en enkel bestemmelse av fibrinogen uten å måtte ta om-stendelige fortynninger av plasmaprøver eller nøytralise-ring hhv. fjerning av heparin fra plasmaprøver med på kj øpet. The invention also relates to a reagent for determining the fibrinogen content, mostly independent of thrombin inhibitors, of an optionally undiluted and optionally also heparin-containing plasma sample characterized in that it contains thrombin, meizothrombin, meizothrombin (des Fl) or mixtures thereof for enzymatic conversion of fibrinogen and subsequent detection of the formed fibrin via the coagulation time or the fibrinogen cleavage products in a buffer with a pH between 4 and 7.3. With the reagent, a simple determination of fibrinogen is achieved without having to take extensive dilutions of plasma samples or neutralization or removal of heparin from plasma samples included in the purchase.
Oppfinnelsen skal forklares nærmere ved hjelp av de et-terfølgende eksempler. The invention shall be explained in more detail by means of the following examples.
Bestemmelse av fibrinogen ved pH 6 og pH 8: Determination of fibrinogen at pH 6 and pH 8:
Til sammenligning ble meizotrombin og trombin ved pH 6 og pH 8 anvendt, og koaguleringstidene ble bestemt avhengig av fibrinogeninnholdet i plasmaprøver. Meizotrombin ble fremstilt ved aktivering av humant protrombin med immobi-lisert "Ecarin" (firma Pentapharm) efter forskrift fra Stocker und Mviller (Thromb. Haemostasis 65 (6) , Abstract 855 (1991)) . Plasmaholdige prøver med forskjellig fibrinogeninnhold ble anvendt for testene. 50 fil plasmaprøve ble blandet med 100 fil buffer (0,2 M tris HCl-buffer, pH 8 eller 0,2 M fosfat-buffer, pH 6), og 150 /il meizotrombin (15 NIH-analog E/ml) eller trombin (10 NIH E/ml, "Thrombin Reagent", firma Immuno), og koaguleringstiden ved 37°C ble bestemt med et Schnitger-Gross-koagulometer (firma Amelung). Bestemmelsen ble utført såvel i nærvær som i fravær av heparin (1 E/ml). For comparison, meizothrombin and thrombin at pH 6 and pH 8 were used, and the coagulation times were determined depending on the fibrinogen content of plasma samples. Meizothrombin was produced by activation of human prothrombin with immobilized "Ecarin" (company Pentapharm) according to instructions from Stocker und Mviller (Thromb. Haemostasis 65 (6), Abstract 855 (1991)). Plasma-containing samples with different fibrinogen content were used for the tests. 50 µl plasma sample was mixed with 100 µl buffer (0.2 M tris HCl buffer, pH 8 or 0.2 M phosphate buffer, pH 6), and 150 µl meizothrombin (15 NIH analog U/ml) or thrombin (10 NIH U/ml, "Thrombin Reagent", company Immuno), and the clotting time at 37°C was determined with a Schnitger-Gross coagulometer (company Amelung). The determination was carried out both in the presence and in the absence of heparin (1 U/ml).
Fra tabellen fremgår at referansekurven for fibrinogen ved pH 6 er uavhengig av heparininnholdet i prøven. På grunn av heparininnholdet i prøven, blir imidlertid koaguleringstidene ved pH 8 forlenget; dvs. såvel for meizotrombin som for trombin som koaguleringsenzym. The table shows that the reference curve for fibrinogen at pH 6 is independent of the heparin content in the sample. However, due to the heparin content of the sample, the coagulation times at pH 8 are prolonged; i.e. both for meizothrombin and for thrombin as a coagulation enzyme.
Meizotrombin Meizothrombin
Trombin Thrombin
Claims (4)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0099992A AT399055B (en) | 1992-05-15 | 1992-05-15 | METHOD FOR DETERMINING FIBRINOGENS |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| NO931773D0 NO931773D0 (en) | 1993-05-14 |
| NO931773L NO931773L (en) | 1993-11-16 |
| NO309902B1 true NO309902B1 (en) | 2001-04-17 |
Family
ID=3504619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO931773A NO309902B1 (en) | 1992-05-15 | 1993-05-14 | Method of determining fibrinogen |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0570354B1 (en) |
| JP (1) | JPH0646898A (en) |
| AT (2) | AT399055B (en) |
| CA (1) | CA2096215A1 (en) |
| CZ (1) | CZ87893A3 (en) |
| DE (1) | DE59308942D1 (en) |
| DK (1) | DK0570354T3 (en) |
| ES (1) | ES2123043T3 (en) |
| FI (1) | FI932196A (en) |
| HU (1) | HU216866B (en) |
| MX (1) | MX9302827A (en) |
| NO (1) | NO309902B1 (en) |
| SK (1) | SK48693A3 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2462721C2 (en) * | 2008-05-22 | 2012-09-27 | Этикон, Инк. | Method for thrombin and fibrinogen activity and functionality test |
| US9896716B2 (en) | 2008-05-22 | 2018-02-20 | Ethicon, Inc. | Protein assay |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2994557B2 (en) | 1994-09-02 | 1999-12-27 | 株式会社アズウェル | Fibrinogen measuring method and its measuring reagent |
| JP4399615B2 (en) * | 1999-09-27 | 2010-01-20 | シスメックス株式会社 | Stabilizing means and composition for thrombin |
| US6448024B1 (en) * | 2000-10-03 | 2002-09-10 | Roche Diagnostics Corporation | Method, reagent, cartridge, and device for determining fibrinogen |
| FR3028204B1 (en) | 2014-11-10 | 2017-04-21 | Sidel Participations | "THERMOPLASTIC CONTAINER MANUFACTURING FACILITY INCORPORATING A GRINDING DEVICE AND SUCH A GRINDING DEVICE" |
| CN110257475B (en) * | 2019-06-28 | 2023-05-02 | 深圳市国赛生物技术有限公司 | Fibrinogen detection reagent, preparation method thereof and detection reagent product |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
1992
- 1992-05-15 AT AT0099992A patent/AT399055B/en not_active IP Right Cessation
-
1993
- 1993-05-12 DE DE59308942T patent/DE59308942D1/en not_active Expired - Fee Related
- 1993-05-12 CZ CZ93878A patent/CZ87893A3/en unknown
- 1993-05-12 DK DK93890097T patent/DK0570354T3/en active
- 1993-05-12 ES ES93890097T patent/ES2123043T3/en not_active Expired - Lifetime
- 1993-05-12 AT AT93890097T patent/ATE170569T1/en not_active IP Right Cessation
- 1993-05-12 EP EP93890097A patent/EP0570354B1/en not_active Expired - Lifetime
- 1993-05-13 CA CA002096215A patent/CA2096215A1/en not_active Abandoned
- 1993-05-14 FI FI932196A patent/FI932196A/en unknown
- 1993-05-14 NO NO931773A patent/NO309902B1/en not_active IP Right Cessation
- 1993-05-14 MX MX9302827A patent/MX9302827A/en unknown
- 1993-05-14 HU HU9301401A patent/HU216866B/en not_active IP Right Cessation
- 1993-05-14 JP JP5112801A patent/JPH0646898A/en active Pending
- 1993-05-14 SK SK486-93A patent/SK48693A3/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2462721C2 (en) * | 2008-05-22 | 2012-09-27 | Этикон, Инк. | Method for thrombin and fibrinogen activity and functionality test |
| US9896716B2 (en) | 2008-05-22 | 2018-02-20 | Ethicon, Inc. | Protein assay |
Also Published As
| Publication number | Publication date |
|---|---|
| HU9301401D0 (en) | 1993-09-28 |
| CA2096215A1 (en) | 1993-11-16 |
| ATE170569T1 (en) | 1998-09-15 |
| DE59308942D1 (en) | 1998-10-08 |
| HUT64146A (en) | 1993-11-29 |
| ATA99992A (en) | 1994-07-15 |
| NO931773L (en) | 1993-11-16 |
| FI932196A (en) | 1993-11-16 |
| ES2123043T3 (en) | 1999-01-01 |
| SK48693A3 (en) | 1993-12-08 |
| NO931773D0 (en) | 1993-05-14 |
| HU216866B (en) | 1999-09-28 |
| JPH0646898A (en) | 1994-02-22 |
| DK0570354T3 (en) | 1999-05-31 |
| EP0570354A1 (en) | 1993-11-18 |
| FI932196A0 (en) | 1993-05-14 |
| EP0570354B1 (en) | 1998-09-02 |
| CZ87893A3 (en) | 1994-02-16 |
| MX9302827A (en) | 1994-05-31 |
| AT399055B (en) | 1995-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Soria et al. | A new type of congenital dysfibrinogenaemia with defective fibrin lysis—Dusard syndrome: possible relation to thrombosis | |
| US4692406A (en) | Process and a reagent for the simultaneous determination of fibrinogen and fibrinogen fission products in plasma | |
| US5292664A (en) | Functional test and reagent for determining fibrinogen | |
| DE59813326D1 (en) | Method for determining the anticoagulant potential of a sample and method for determining the glycosylation of thrombomodulin | |
| US5766869A (en) | Factor V ratio blood test for susceptibility to thromboembolism | |
| CA2155503C (en) | A method for detecting defects in protein c/protein s mediated blood clotting | |
| US20100227346A1 (en) | Activation Mixture | |
| NO309902B1 (en) | Method of determining fibrinogen | |
| US5529905A (en) | Method of assaying plasma proteins with prothrombin fragments | |
| US5753510A (en) | Calibrator for use in test methods for detecting a defective coagulation factor V | |
| US5476771A (en) | Test for quantitative thrombin time | |
| EP0440775B1 (en) | Factor ix chromogenic assay | |
| AU654962B2 (en) | Factor VIII:Ca chromogenic assay | |
| AU729843B2 (en) | Method for the functional detection of disorders in the protein C system | |
| Becker et al. | Development of a photometric assay for activated partial thromboplastin time and its application to the cobas (R) biocentrifugal analyzer | |
| WO1993025220A1 (en) | Test for quantitative thrombin time | |
| Grannis et al. | The spectrophotometry determination of thrombin activity in plasma | |
| Giddings et al. | Laboratory support in the diagnosis of coagulation disorders | |
| van Putten et al. | Automated determination of heparin with chromogenic substrates | |
| Laugen et al. | Hereditary dysfibrinogenemia characterized by slow fibrinopeptide release and competitive inhibition of thrombin | |
| JPH06331628A (en) | Measuring method for anti-phospholipid antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MM1K | Lapsed by not paying the annual fees |