CA2096215A1 - Method of assaying fibrinogen and a reagent therefor - Google Patents
Method of assaying fibrinogen and a reagent thereforInfo
- Publication number
- CA2096215A1 CA2096215A1 CA002096215A CA2096215A CA2096215A1 CA 2096215 A1 CA2096215 A1 CA 2096215A1 CA 002096215 A CA002096215 A CA 002096215A CA 2096215 A CA2096215 A CA 2096215A CA 2096215 A1 CA2096215 A1 CA 2096215A1
- Authority
- CA
- Canada
- Prior art keywords
- fibrinogen
- thrombin
- assaying
- set forth
- plasma sample
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Neurosurgery (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Materials For Medical Uses (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Compounds Of Unknown Constitution (AREA)
- Measuring Pulse, Heart Rate, Blood Pressure Or Blood Flow (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
There is disclosed a method of assaying fibrinogen by enzymatic conversion with thrombin, prothrombin fragments having a thrombin-like activity or mixtures thereof with subsequent detection of fibrin or fibrinogen cleavage products formed, in which the enzymatic conversion of fibrinogen is effected at a pH
ranging from 4 to 7.3.
There is disclosed a method of assaying fibrinogen by enzymatic conversion with thrombin, prothrombin fragments having a thrombin-like activity or mixtures thereof with subsequent detection of fibrin or fibrinogen cleavage products formed, in which the enzymatic conversion of fibrinogen is effected at a pH
ranging from 4 to 7.3.
Description
2~
The invention relates to a method of assaying fibrinogen by enzymatic conversion with thrombin, prothrombin fragments having a thrombin-like activi-ty or mixtures thereof and subsequent detection of the fibrin or fibrinogen cleavage products formed.
Furthermore, the invention relates to a reagent for assaying the fibrinogen content of an optionally undiluted and optionally also heparin-containing ~lasma sample.
The assaying of fibrinogen in plasma is very important within the field of the investigation of coagulation defects. For the assaying of fibrinogen, both immunological and functional methods, such as, e.g., coagulation tests, have been kno~n. In the coagulation tests, the fibrinogen content is determined by measuring the time of clot formation.
A common assaying method has been described by Clauss (Acta HaematO 17, 237-246, 1957). Oxalate plasma is diluted in a buffer of pH 7.~, and a concentrated thrombin solution is admixed. Subsequen-tly, the time until the end of coagulation is determined. Yet, the coagulation time of the plasma sample which has been reacted with -thrombin is only proportionate to the fibrinogen cont~nt, if a sufficiently large excess of thrombin relative to plasma is chosen, so as to eliminate plasmatic interfering factors. Antithrombin III (AT III) which effectively inhibits the action of thrombin, primarily together with heparin, is considered an interfering fac-tor. Heparin is a commonly used anticoagulant and i5 used as addi-tive when taking blood or as a therapeutic agent. A heparin content in a plasma sample may lead -to extended coagulation times and thus to falsified results.
The action of antithrombin III in the plasma here is greatly reduced, on account of the high thrombin concentration and on account of the dilution of the plasma, and thus on account of the reduction oX the concentrations of ATIII and heparin, respectively. If plasma is not dilutPd, the antithrombin effect is stronger by a multiple and the coagula-tion time is extended.
A plasma dilution and the additional method step involved therein is a substantial burden in routine check-ups. Beside increased operational expenditures and the additional handling of potentially infectious plasma, each dilution in principle constitutes a source of error. Furthermore, dilutions often have to be carried out according to the fibrinogen content, and this makes working with analysis automats more difficult.
Usually veronal buffer is used to dilute the plasma, because this buffer facilitates the formation of measureable fibrin clots, which improves assaying particularly with plasmas having low fibrinogen contents. Yet, veronal is also a narcotic having the character of an addic-tive druy, and -therefore use of this substance should be avoided, if possible.
Further attempts to eliminate the interferîng factors antithrombin III or heparin, respectively, involve the neutralization or the removal of -the heparin. In Clinical Chemistry 29, 61A-617 (1983), a method is described according to which heparin is-neutralized in a plasma sample by adding polybrene.
Plasma is diluted in a buffer having a pH of 7.4, and after the addition of thrombin, it is transferred into a photometer. Subsequently, the reaction speed is determined nephelometrically.
By using snake venom enzymes having a thrombin-like activity (batroxobin) instead of thrombin, fibrinogen may be assayed in plasma independently of the interfering factors mentioned, the reason for this being that these snake venom enzymes are not inhibited by AT III or heparin.
The use of proteins which are not considered to be natural coagulation enzymes, such as snake venom enzymes~ e.g~, in methods for assaying human proteins does have its problems. The reactions hetween a non-mammal enzyme and a human protein often are unspecific.
In accordance with the principle of biochemical analytics, to compare and to assay only s things that are alike, one aims at using only mammalian and, above all, human enzymes as the reaction par-tners for assaying human proteins.
Thrombin may be produced by ac-tiva-tion of prothrombin. As precursors, Meizothrombin (MT) and Meizothombin (desF1) form during activation. Yet, contrary to prothrombin, the double-chained -thrombin in-termediates have a protease activity similar to-that of thrombin ("thrombin-like activity"). Therefore, they can also cleave substrates that can be reacted by thrombin. Thus, a "thrombin-like activity" is a protease activity which is capable of cleaving thrombin suhstrates, whose reaction kinetics and reac-tion param~ters, such as pH or ionic strength optima, inhibitors, effectors, allosteric interaction ancl so on, need not necessarily be equal to those of thrombin.
The activities of the bovine enzyme meizothrombin, meizothrombin (desFl~ and thrombin are compared in J. of Biol. Chemistry 265, 10693-:L0701 (1990) at a pH of 7.4. Meizothrombin exhibits only 2~
of the "fibrinogen clotting activity" of thrombin. This slight activity may, however! also be due to a 10 to 15 ~ content of MT(desFl) in the MT composition. For MT(desF'1) an activity of 1~ of that of thromb:in was found.
The test for enzymatically converting fibrinogen into fibrin is usually carried out at a pH in the range ~ J
of from 7.4 to 8Ø This pH range is generally used in enzymatic reactions of thrombin, since it corresponds to the physiological pH range. The optimum pH for thrombin activity is close to pH 8.
The invention has as its object to provide a method of assaying fibrinogen by means of a coagulation test, by which the disadvantages of the known methods described above are avoided and which may particularly also be carried out in undiluted plasma samples, independetly of their AT III or heparin contents.
According to the invention, this object is achieved by a method of assaying fibrinogen largely independently of thrombin inhibitors by enzymatic conversion with thrombin, prothrombin fragments having a "thrombin-like activity" or mixtures thereof, and subsequent detection of the fibrin or fibrinogen cleavage products formed, which is characterised in that the enzymatic conversion of the fibrinogen is carried out at a pH in the range of from 4 to 7.3, and the detection of the fibrin formed is effected by determining the coagulation time or the fibrinogen cleavage products. Subsequently, the fibrin clot formed is detec-ted. The detection may be effected by determining the coagulation time photometrically or turbidimetrically. Among the prothrombin fragments having a "thrombin-like act:ivity" there are particularly MT, MT(desFl) or mixtures thereof. The I~J~Ç;J~ t~
concentrations and the mixing ratios of the enzymes used are to be chosen such that a linear relation is guaranteed over as broad a range of the fibrinogen content in a sample as possible.
Also proteins produced by genetic engineering, which have a thrombin-like activity on account of their structure and which have a slighter affinity to antithrombin III as compared to thrombin, are suited for -the method according to the invention.
Carrying out the assaying method in ths pH range indicated makes the latter independent of the above-mentioned interfering factors AT III and heparin to such an ex-tent that the fibrinogen assay may be efected in a simple manner also from an undiluted and, optionally also heparin-containing, plasma sample. The additional me-thod step of diluting plasma can be obviated in the me-thod according to the invention, thereby improving the assay result by eliminating a possible source of error.
The invention is based on the finding that the inhibition of thrombin by AT III/heparin at a pH of from 4 to 7.3 may be neglected, wherein -the thrombin activity suffices for an enzymatic conversion of fibrinogen, daspite suboptimal conditions. A somewhat reduced activi-ty of thrombin even has the advantage tha-t the coagulation times are not too short and can be recorded more easily.
When using thrombin, prothrombin fragments having a "thrombin-like activity" or mi~tures thereof, it has surprisingly been found that the thrombin-like activity in the pH range mentioned, preferably at a p~ of from 5 to 7, or, most preferred, at a pH of appro~imately 6, is best suited for assaying fibrinogen independently of thrombin inhibitors.
The enzymatic conversion of fibrinogen is preferably carried out in a buffer having a molarity of buffer salts in a range of from 0.02 to 0.5 M, most preferred of 0.1 to 0.25 M, buffer. If desired, the ionic strength may also be increased by the acldition of salts, such as sodium chloride. It has been found that, particularly at low ionic strengths, thrombin i5 insensitive to AT III inhibition. For carrying out the method according to the invention, however, also an environment having an equal or a higher buffer capacity compared to the plasma sample is practicable to maintain a certain pH during the assaying procedure.
Advantageously, fibrinogen assaying is ef~scted from an undiluted and, possibly also heparin-containing, plasma sample, i.e. a sample, in which heparin is present and not neutralized.
The invention also relates to a reagent for assaying the fibrinogen content largely independently of thrombin inhibitors o* a possibly undiluted and possibly also heparin-containing plasma sample, s~
containing thrombin, prothrombin fragments having a "thrombin-like activity" or mixtures -thereof, for enzymatically converting fibrinogen in a buffer having a pH in -the range of from A -to 7.3 and subsequ~nt detection of the fibrin formad by determining -the coagulation time or the fibrinogen cleavage products.
This reagent serves for the simple assaying of fi~rinogen, without having to put up with a cumbersome dilu-tion of plasma samples or neutralization or removal of heparin from the plasma samples.
I'he invention will now be e~plained in more de-tail by way of -the following examples.
Assaying of fibrinogen a-t pH 6 and pH 8:
For reasons of comparison, meizothrombin and thrombin were used at pH 6 and pH 8, and the coagulation times were determined in dependence on the fibrinogen content in plasma samples. Meizothrombin was produced by activating human prothrombin with immobilized ecarin (Pentapharm) according to the protocol of S-tocker and Muller (Thromb. Haemostasis 65 (6), Abstract 855 (1991)). Plasma-containing samples having different fibrinogen contents were used for the tests.
50 ~1 plasma sample were admixed with 100 ~1 buffer (0.2 M Tris HCl buffer, pH 8, or 0.2 M phosphate buffer, pH 6) and 150 ~1 meizothrombin (15 NIH
analogous U/ml) or thrombin (10 NIH U/ml, Thrombin Reagent, Immuno), and -the coagulation ~imes at 37C
were determined by means of a Schnitger-Gross coagulometer (Amelung). The determining method was carried out both in the presence and in the absence of heparin (1 U/ml).
From the Tables it becomes apparP.nt that at pH 6, the course of the calibration curves for fibrinogen is independent of the heparin content in the sample..
However, by a content of heparin in the sample the coagulation times are extended at pH 8, both, for meizothrombin and for thrombin as coagulation enzymes.
Meizothrombin pH 8 pH 6 Fibrinogen without withwithout with content (mg/ml) hep. hep. hep. hep.
310 8.0 10.630.~ 30.1 238 11.1 15.150.7 49.1 94 26.0 35.5166.4 157.5 62.5 35.6 >250 >250 >250 Thrombin pH 8 pH 6 Fibrinogen without withwithout with content (mg/ml) hep. hep. hep. hep.
373 11.6 >20018.6 17.3 310 16.1 >20024.1 24.1 238 25.6 >20028.4 30.1 100 50.0 >20085.9 89.8
The invention relates to a method of assaying fibrinogen by enzymatic conversion with thrombin, prothrombin fragments having a thrombin-like activi-ty or mixtures thereof and subsequent detection of the fibrin or fibrinogen cleavage products formed.
Furthermore, the invention relates to a reagent for assaying the fibrinogen content of an optionally undiluted and optionally also heparin-containing ~lasma sample.
The assaying of fibrinogen in plasma is very important within the field of the investigation of coagulation defects. For the assaying of fibrinogen, both immunological and functional methods, such as, e.g., coagulation tests, have been kno~n. In the coagulation tests, the fibrinogen content is determined by measuring the time of clot formation.
A common assaying method has been described by Clauss (Acta HaematO 17, 237-246, 1957). Oxalate plasma is diluted in a buffer of pH 7.~, and a concentrated thrombin solution is admixed. Subsequen-tly, the time until the end of coagulation is determined. Yet, the coagulation time of the plasma sample which has been reacted with -thrombin is only proportionate to the fibrinogen cont~nt, if a sufficiently large excess of thrombin relative to plasma is chosen, so as to eliminate plasmatic interfering factors. Antithrombin III (AT III) which effectively inhibits the action of thrombin, primarily together with heparin, is considered an interfering fac-tor. Heparin is a commonly used anticoagulant and i5 used as addi-tive when taking blood or as a therapeutic agent. A heparin content in a plasma sample may lead -to extended coagulation times and thus to falsified results.
The action of antithrombin III in the plasma here is greatly reduced, on account of the high thrombin concentration and on account of the dilution of the plasma, and thus on account of the reduction oX the concentrations of ATIII and heparin, respectively. If plasma is not dilutPd, the antithrombin effect is stronger by a multiple and the coagula-tion time is extended.
A plasma dilution and the additional method step involved therein is a substantial burden in routine check-ups. Beside increased operational expenditures and the additional handling of potentially infectious plasma, each dilution in principle constitutes a source of error. Furthermore, dilutions often have to be carried out according to the fibrinogen content, and this makes working with analysis automats more difficult.
Usually veronal buffer is used to dilute the plasma, because this buffer facilitates the formation of measureable fibrin clots, which improves assaying particularly with plasmas having low fibrinogen contents. Yet, veronal is also a narcotic having the character of an addic-tive druy, and -therefore use of this substance should be avoided, if possible.
Further attempts to eliminate the interferîng factors antithrombin III or heparin, respectively, involve the neutralization or the removal of -the heparin. In Clinical Chemistry 29, 61A-617 (1983), a method is described according to which heparin is-neutralized in a plasma sample by adding polybrene.
Plasma is diluted in a buffer having a pH of 7.4, and after the addition of thrombin, it is transferred into a photometer. Subsequently, the reaction speed is determined nephelometrically.
By using snake venom enzymes having a thrombin-like activity (batroxobin) instead of thrombin, fibrinogen may be assayed in plasma independently of the interfering factors mentioned, the reason for this being that these snake venom enzymes are not inhibited by AT III or heparin.
The use of proteins which are not considered to be natural coagulation enzymes, such as snake venom enzymes~ e.g~, in methods for assaying human proteins does have its problems. The reactions hetween a non-mammal enzyme and a human protein often are unspecific.
In accordance with the principle of biochemical analytics, to compare and to assay only s things that are alike, one aims at using only mammalian and, above all, human enzymes as the reaction par-tners for assaying human proteins.
Thrombin may be produced by ac-tiva-tion of prothrombin. As precursors, Meizothrombin (MT) and Meizothombin (desF1) form during activation. Yet, contrary to prothrombin, the double-chained -thrombin in-termediates have a protease activity similar to-that of thrombin ("thrombin-like activity"). Therefore, they can also cleave substrates that can be reacted by thrombin. Thus, a "thrombin-like activity" is a protease activity which is capable of cleaving thrombin suhstrates, whose reaction kinetics and reac-tion param~ters, such as pH or ionic strength optima, inhibitors, effectors, allosteric interaction ancl so on, need not necessarily be equal to those of thrombin.
The activities of the bovine enzyme meizothrombin, meizothrombin (desFl~ and thrombin are compared in J. of Biol. Chemistry 265, 10693-:L0701 (1990) at a pH of 7.4. Meizothrombin exhibits only 2~
of the "fibrinogen clotting activity" of thrombin. This slight activity may, however! also be due to a 10 to 15 ~ content of MT(desFl) in the MT composition. For MT(desF'1) an activity of 1~ of that of thromb:in was found.
The test for enzymatically converting fibrinogen into fibrin is usually carried out at a pH in the range ~ J
of from 7.4 to 8Ø This pH range is generally used in enzymatic reactions of thrombin, since it corresponds to the physiological pH range. The optimum pH for thrombin activity is close to pH 8.
The invention has as its object to provide a method of assaying fibrinogen by means of a coagulation test, by which the disadvantages of the known methods described above are avoided and which may particularly also be carried out in undiluted plasma samples, independetly of their AT III or heparin contents.
According to the invention, this object is achieved by a method of assaying fibrinogen largely independently of thrombin inhibitors by enzymatic conversion with thrombin, prothrombin fragments having a "thrombin-like activity" or mixtures thereof, and subsequent detection of the fibrin or fibrinogen cleavage products formed, which is characterised in that the enzymatic conversion of the fibrinogen is carried out at a pH in the range of from 4 to 7.3, and the detection of the fibrin formed is effected by determining the coagulation time or the fibrinogen cleavage products. Subsequently, the fibrin clot formed is detec-ted. The detection may be effected by determining the coagulation time photometrically or turbidimetrically. Among the prothrombin fragments having a "thrombin-like act:ivity" there are particularly MT, MT(desFl) or mixtures thereof. The I~J~Ç;J~ t~
concentrations and the mixing ratios of the enzymes used are to be chosen such that a linear relation is guaranteed over as broad a range of the fibrinogen content in a sample as possible.
Also proteins produced by genetic engineering, which have a thrombin-like activity on account of their structure and which have a slighter affinity to antithrombin III as compared to thrombin, are suited for -the method according to the invention.
Carrying out the assaying method in ths pH range indicated makes the latter independent of the above-mentioned interfering factors AT III and heparin to such an ex-tent that the fibrinogen assay may be efected in a simple manner also from an undiluted and, optionally also heparin-containing, plasma sample. The additional me-thod step of diluting plasma can be obviated in the me-thod according to the invention, thereby improving the assay result by eliminating a possible source of error.
The invention is based on the finding that the inhibition of thrombin by AT III/heparin at a pH of from 4 to 7.3 may be neglected, wherein -the thrombin activity suffices for an enzymatic conversion of fibrinogen, daspite suboptimal conditions. A somewhat reduced activi-ty of thrombin even has the advantage tha-t the coagulation times are not too short and can be recorded more easily.
When using thrombin, prothrombin fragments having a "thrombin-like activity" or mi~tures thereof, it has surprisingly been found that the thrombin-like activity in the pH range mentioned, preferably at a p~ of from 5 to 7, or, most preferred, at a pH of appro~imately 6, is best suited for assaying fibrinogen independently of thrombin inhibitors.
The enzymatic conversion of fibrinogen is preferably carried out in a buffer having a molarity of buffer salts in a range of from 0.02 to 0.5 M, most preferred of 0.1 to 0.25 M, buffer. If desired, the ionic strength may also be increased by the acldition of salts, such as sodium chloride. It has been found that, particularly at low ionic strengths, thrombin i5 insensitive to AT III inhibition. For carrying out the method according to the invention, however, also an environment having an equal or a higher buffer capacity compared to the plasma sample is practicable to maintain a certain pH during the assaying procedure.
Advantageously, fibrinogen assaying is ef~scted from an undiluted and, possibly also heparin-containing, plasma sample, i.e. a sample, in which heparin is present and not neutralized.
The invention also relates to a reagent for assaying the fibrinogen content largely independently of thrombin inhibitors o* a possibly undiluted and possibly also heparin-containing plasma sample, s~
containing thrombin, prothrombin fragments having a "thrombin-like activity" or mixtures -thereof, for enzymatically converting fibrinogen in a buffer having a pH in -the range of from A -to 7.3 and subsequ~nt detection of the fibrin formad by determining -the coagulation time or the fibrinogen cleavage products.
This reagent serves for the simple assaying of fi~rinogen, without having to put up with a cumbersome dilu-tion of plasma samples or neutralization or removal of heparin from the plasma samples.
I'he invention will now be e~plained in more de-tail by way of -the following examples.
Assaying of fibrinogen a-t pH 6 and pH 8:
For reasons of comparison, meizothrombin and thrombin were used at pH 6 and pH 8, and the coagulation times were determined in dependence on the fibrinogen content in plasma samples. Meizothrombin was produced by activating human prothrombin with immobilized ecarin (Pentapharm) according to the protocol of S-tocker and Muller (Thromb. Haemostasis 65 (6), Abstract 855 (1991)). Plasma-containing samples having different fibrinogen contents were used for the tests.
50 ~1 plasma sample were admixed with 100 ~1 buffer (0.2 M Tris HCl buffer, pH 8, or 0.2 M phosphate buffer, pH 6) and 150 ~1 meizothrombin (15 NIH
analogous U/ml) or thrombin (10 NIH U/ml, Thrombin Reagent, Immuno), and -the coagulation ~imes at 37C
were determined by means of a Schnitger-Gross coagulometer (Amelung). The determining method was carried out both in the presence and in the absence of heparin (1 U/ml).
From the Tables it becomes apparP.nt that at pH 6, the course of the calibration curves for fibrinogen is independent of the heparin content in the sample..
However, by a content of heparin in the sample the coagulation times are extended at pH 8, both, for meizothrombin and for thrombin as coagulation enzymes.
Meizothrombin pH 8 pH 6 Fibrinogen without withwithout with content (mg/ml) hep. hep. hep. hep.
310 8.0 10.630.~ 30.1 238 11.1 15.150.7 49.1 94 26.0 35.5166.4 157.5 62.5 35.6 >250 >250 >250 Thrombin pH 8 pH 6 Fibrinogen without withwithout with content (mg/ml) hep. hep. hep. hep.
373 11.6 >20018.6 17.3 310 16.1 >20024.1 24.1 238 25.6 >20028.4 30.1 100 50.0 >20085.9 89.8
Claims (9)
1. In a method of assaying fibrinogen largely independently of thrombin inhibitors by enzymatic conversion with one of thrombin, prothrombin fragments having a thrombin-like activity and mixtures thereof, and subsequent detection of the fibrin formed or of the fibrinogen cleavage products, the improvement comprising carrying out said enzymatic conversion of fibrinogen at a pH ranging from 4 to 7.3 and detecting said fibrin formed by determining the respective one of the coagulation time and the fibrinogen cleavage products.
2. A method as set forth in claim 1, wherein said enzymatic conversion of fibrinogen is carried out in a buffer having a molarity of buffer salts ranging from 0.02 to 0.5 M and upon increasing the ionic strength by adding salts.
3. A method as set forth in claim 2, wherein said salt is NaCl.
4. A method as set forth in claim 2, wherein said molarity of buffer salts ranges from 0.1 to 0.25 M.
5. A method as set forth in claim 1, wherein said fibrinogen assaying is effected in an undiluted plasma sample.
4. A method as set forth in claim 5, wherein said plasma sample contains heparin.
7, A reagent for assaying the fibrinogen content largely independently of thrombin inhibitors in a plasma sample containing one of thrombin, prothrombin fragments having a thrombin-like activity and mixtures thereof for enzymatic conversion of fibrinogen and subsequent detection of fibrin formed via the respective one of coagulation time and fibrinogen cleavage products in a buffer having a pH ranging from 4 to 7.3.
8. A reagent as set forth in claim 7, wherein said plasma sample is undiluted.
9. A reagent as set forth in claim 8, wherein said plasma sample further contains heparin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ATA999/92 | 1992-05-15 | ||
| AT0099992A AT399055B (en) | 1992-05-15 | 1992-05-15 | METHOD FOR DETERMINING FIBRINOGENS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2096215A1 true CA2096215A1 (en) | 1993-11-16 |
Family
ID=3504619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002096215A Abandoned CA2096215A1 (en) | 1992-05-15 | 1993-05-13 | Method of assaying fibrinogen and a reagent therefor |
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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851836A (en) * | 1994-09-02 | 1998-12-22 | Nippon Shoji Kaisha Ltd. | Method for determining fibrinogen and reagent for determination thereof |
| US9213035B2 (en) | 2008-05-22 | 2015-12-15 | Ethicon, Inc. | Protein assay |
| CN110257475A (en) * | 2019-06-28 | 2019-09-20 | 深圳市国赛生物技术有限公司 | Fibrinogen detection reagent and preparation method thereof and detection reagent product |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1221479B1 (en) * | 1999-09-27 | 2006-11-22 | Sysmex Corporation | Method for stabilizing thrombin |
| US6448024B1 (en) * | 2000-10-03 | 2002-09-10 | Roche Diagnostics Corporation | Method, reagent, cartridge, and device for determining fibrinogen |
| RU2462721C2 (en) * | 2008-05-22 | 2012-09-27 | Этикон, Инк. | Method for thrombin and fibrinogen activity and functionality test |
| FR3028204B1 (en) | 2014-11-10 | 2017-04-21 | Sidel Participations | "THERMOPLASTIC CONTAINER MANUFACTURING FACILITY INCORPORATING A GRINDING DEVICE AND SUCH A GRINDING DEVICE" |
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 CZ CZ93878A patent/CZ87893A3/en unknown
- 1993-05-12 EP EP93890097A patent/EP0570354B1/en not_active Expired - Lifetime
- 1993-05-12 DE DE59308942T patent/DE59308942D1/en not_active Expired - Fee Related
- 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
- 1993-05-13 CA CA002096215A patent/CA2096215A1/en not_active Abandoned
- 1993-05-14 SK SK486-93A patent/SK48693A3/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 JP JP5112801A patent/JPH0646898A/en active Pending
- 1993-05-14 HU HU9301401A patent/HU216866B/en not_active IP Right Cessation
- 1993-05-14 FI FI932196A patent/FI932196A/en unknown
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851836A (en) * | 1994-09-02 | 1998-12-22 | Nippon Shoji Kaisha Ltd. | Method for determining fibrinogen and reagent for determination thereof |
| US9213035B2 (en) | 2008-05-22 | 2015-12-15 | Ethicon, Inc. | Protein assay |
| US9896716B2 (en) | 2008-05-22 | 2018-02-20 | Ethicon, Inc. | Protein assay |
| CN110257475A (en) * | 2019-06-28 | 2019-09-20 | 深圳市国赛生物技术有限公司 | Fibrinogen detection reagent and preparation method thereof and detection reagent product |
Also Published As
| Publication number | Publication date |
|---|---|
| AT399055B (en) | 1995-03-27 |
| SK48693A3 (en) | 1993-12-08 |
| NO309902B1 (en) | 2001-04-17 |
| CZ87893A3 (en) | 1994-02-16 |
| MX9302827A (en) | 1994-05-31 |
| DK0570354T3 (en) | 1999-05-31 |
| FI932196A (en) | 1993-11-16 |
| EP0570354B1 (en) | 1998-09-02 |
| HUT64146A (en) | 1993-11-29 |
| NO931773D0 (en) | 1993-05-14 |
| HU9301401D0 (en) | 1993-09-28 |
| ATE170569T1 (en) | 1998-09-15 |
| HU216866B (en) | 1999-09-28 |
| ATA99992A (en) | 1994-07-15 |
| NO931773L (en) | 1993-11-16 |
| EP0570354A1 (en) | 1993-11-18 |
| DE59308942D1 (en) | 1998-10-08 |
| FI932196A0 (en) | 1993-05-14 |
| JPH0646898A (en) | 1994-02-22 |
| ES2123043T3 (en) | 1999-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5051357A (en) | Method and assay using inactivation of factors Va and VIIIa by activated Protein C to diagnose thrombic disease or assay for Protein C and kit therefor | |
| US4692406A (en) | Process and a reagent for the simultaneous determination of fibrinogen and fibrinogen fission products in plasma | |
| JP3055933B2 (en) | Improved stable coagulation control | |
| JP2003517610A (en) | Hematological assays and reagents | |
| AU705596B2 (en) | Method for specifically detecting a coagulation factor V which has an increased stability toward activated protein C in the activated state | |
| US5766869A (en) | Factor V ratio blood test for susceptibility to thromboembolism | |
| Eisen | Kinin formation and fibrinolysis in human plasma | |
| CA2096215A1 (en) | Method of assaying fibrinogen and a reagent therefor | |
| WO1993010262A1 (en) | Protein s chromogenic assay | |
| US5529905A (en) | Method of assaying plasma proteins with prothrombin fragments | |
| US5476771A (en) | Test for quantitative thrombin time | |
| EP0440775B1 (en) | Factor ix chromogenic assay | |
| AU654962B2 (en) | Factor VIII:Ca chromogenic assay | |
| WO1993025220A1 (en) | Test for quantitative thrombin time | |
| Lawson et al. | A sensitive fluorescent assay for determining α2-plasmin inhibitor using a synthetic substrate | |
| Gjønnæss | Cold promoted activation of factor VII | |
| Kandall et al. | Determinants of Prothrombinase Activity and Modification of Prothrombin Conversion by Thrombin‐Treated Factor V | |
| Ribeiro et al. | Inhibition of ADP-Ribose Pyrophosphatase-I by Nitric Oxide-Generating Systems: A Mechanism Linking Nitric Oxide to Processes Dependent on Free ADP-Ribose | |
| Watanabe et al. | Antithrombin activity of intact human platelets | |
| EP0318571B1 (en) | Determination of components active in proteolysis | |
| Engel et al. | Molecular changes during prothrombin activation | |
| Zucker-Franklin et al. | Partial purification of intermediate coagulation product I and a study of its properties | |
| Villanueva et al. | Conformational integrity of human α-thrombin | |
| CA2422523A1 (en) | Method for measuring the activity of the blood clotting factor xiiia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |