CA1316088C - Method for the determination of plasminogen - Google Patents
Method for the determination of plasminogenInfo
- Publication number
- CA1316088C CA1316088C CA000549409A CA549409A CA1316088C CA 1316088 C CA1316088 C CA 1316088C CA 000549409 A CA000549409 A CA 000549409A CA 549409 A CA549409 A CA 549409A CA 1316088 C CA1316088 C CA 1316088C
- Authority
- CA
- Canada
- Prior art keywords
- plasminogen
- streptokinase
- plasmin
- determination
- amount
- 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.)
- Expired - Fee Related
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
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/968—Plasmin, i.e. fibrinolysin
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Neurosurgery (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Abstract of the disclosure A method for the determination of plasminogen in body fluids in the presence of streptokinase using a substrate for plasmin, the action of plasmin on which results in a cleavage product which can be measured optically, which method comprises essentially simultaneous addition of streptokinase and chromogenic or fluorogenic substrate for plasmin to the body fluid, and determination of the amount of the cleavage product formed in a defined time, or of the rate of formation of the cleavage product, and from this the concentration of plasminogen, is described. When the amount of streptokinase which is used is such that the plasminogen present is in molar excess, this method can be used to determine the rate of activation of plasminogen.
Description
~3~ ~9~
A method for the determination of plasminogen The invention relates to a method for the determination of plasminogen using a chromogenic or fluorogenic su~strate andin the presence of streptokinase.
eecause of the importance of this protein for fibrinolysis, the determination of plasminogen is nowadays a normal con-stituent of the diagnosis and monitoring of peopLe at risk of thrombcsis. A deficiency of plasminogen often results in a thrombotic event such as myocardial infarc-tion, pulmonary embolism or deep vein thrombosis in the legs. The therapy of thrombolysis with streptokinase or urokinase, which is often carried out nowadays, is like-wise dependent on an adequate concentration of plasminogencapable of being activated.
Apart from immunological determinations using nephelometry or radial immunodiffusion, it is possible to carry out 2û functional determinations of plasminogen with fluorogenic or chromogenic peptide substrates after activation w;th streptokinase. These assays~utilize the fact that plas-minogen, after activation to plasmin, remains bound to streptokinase and, in plasma, this complex (activator) is not, in contrast to free plasminr ;mmediately inhibi-ted. After the introduction of chromogenic tripeptide substrates, this technique was described for the first time in Chromogenic Peptide Substrates, Chemistry and ~linical Usage, page 128~ Scully and Kakkar ed. Churchill L;vingstone, Edinburgh and New York, 1979. However, in this and other similar known methods, it is necessary to preincubate the sample with streptokinase for a rela-tively long time, about 10 min.~ in order to convert the plasma plasminogen completely into the activaeor complex, and the substrate is added separately.
1 3 ~
It has been found, surprisingly, that plasminogen can also he activated by streptokinase in the presence of a chromogenic substrate for pLasmin. After a relatively short lag period, plasmin is formed as a complex with streptokinase, and the substrate is cleaved. The onset of substrate hydrolysis is rapid even in plasma samples with low plasminogen concentrations. The advantage of a method of this type is that there is no necessity either for a preincubation or for a second pipetting step to add the substrate.
Hence the invention relates to a method for the determina-tion of plasm;nogen in body fluids in the presence of streptokinase using a substrate for plasmin, the action of plasmin on which results in a cleavage product which can be measured optically, which method comprises essenti-ally simultaneous addition of streptokinase and a chromo-genic or fluorogenic substrate for plasmin to thebody fluid, and deter-m;nation ~f the amount of the cleavage product formed in a defined ti~e, or of the rate of formation of the cleav-age product, and from this ~he concentration of plasminogen.
This method can be carried out in the following manner:
the plasminogen concentration in a sample can be deter-mined with a reagent which, in addition to a chromogenicsubstrate, contains an excess of streptokinase, preferably 1,000 U/ml. The plasminogen in the sample is thereby con-verted into plasminogen-streptokinase complexes which convert the chromogenic substrate. The evaluation in 3û this method is effected by determining the rate of forma-tion (delta E/min) of the chromophore cleaved off the chromogenic substrate. However, it it also possible to measure the time required for the development of a defined difference in extinction, preferably 0~1 E.
In both types of evaluation the plasminogen concentration in the sample is proportional to the parameters measured.
Comparison of this method (embodiment 1) with conventional ~ 3~088 methods revealed good agreement in 40 samples from patients (Example 1).
However, another possible procedure is as follows: the reagent used for this has a substantially lower strepto-kinase concentration. This entails the plasminogen content of a sample being established from the concentra-tion of plasmin produced by the action of streptokinase-plasminogen complexes on plasminogen. Measurement of the rate of formation of plasmin makes it possible not only to determine plasminogen quantitatively but also to gain information on the ability of plasminogen to be activated (embodiment 2). Evaluation is effected by determining the t;me required after addition of the reagent for a defined difference in ext;nction, preferably 0.1 E, to be reached.
It was shown on 40 plasma samples from patients that the results of embodiments 1 and 2 agreed well in 34 cases.
In 6 cases, normal plasmino~en concentrations were found with embodiment 1, whereas determinations ~ith embodiment 2 resulted ;n figures below 50X of normal (Example 2).
The methocl according to the invention is expediently car-ried out in buffered solution, for example using a phosph-ate, tris, HEPES or acetate buffer with a pH of 6 to 9 preferably a phosphate buffer, especially 0.1 mol/l K
phosphate, pH 7.5.
All chromogenic plasmin substrates are suitable as chromo-genic substrates:
U-D-CHA-NVA-Lys-pNa explanations:
H-D-Val-Leu Lys-pNA CHA cyclohexylalanine 35 H-D-NVA-CHA-Lys-pNA NVA norvaline H-D-NLEU-CHA-Arg-pNA pNA para-nitroanilide H-D-~ut-CHA-Lys-pNA NLEU norleucine H-D-NLEU-CHA-Lys-pNA 8ut epsilon-aminobutyric acid H-D-Phe-Tyr~Arg-pNA
~3~0~8 The examples which follow illustrate the invention.
Example 1 Streptokinase (aehringwerke AG, Marburg, Federal Republic of Germany) was dissolved in 0.1 mol/l K phosphate buffer, pH 7.5, in a concentration of about 1,000 internat;onal units/ml. 4 ml of this solution were mixed with 0~1 ml of a 10 mmol/l solution of the chromogenic substrate H-D-1û cyclohexylalanyl-norvalyl-lysyl-para-nitroanilide (Pentapharm AG, ~asel), and the solution was heated to 37C. 500 ~l of this solution were pipetted into 50 ~l of plasma in a cuvette which had been preheated to 37C.
The absorption at 405 nm was measured. The relation be-t~een the absorption and the time was virtually l;nearafter 30 sec. Delta E/min was determined from the l;near part of the function.
Construction of reference plots:
Serial dilutions were prepared by dilution of plasma with isotonic saline, and each dilution was treated as des-cribed above. The reference plots were obtained by use e;ther of the linear increase in extinction per minute after a lag period of 40 sec, or of the time taken to reach a defined difference in extinction~
The results are shown in the table which follows.
Concentration of delta E/min Reaction time for 30 plasminogen t%) delta E = 0.1, ;n sec 100 (initial) 0.680 18.0 0.360 31.2 0.188 55.8 12.5 0.098 112.8 0 O.OOQ
A straight line was obtained when delta E/min was plotted against the concentration. Semireciprocal plotting of ~ 3~8~
the time taken to reach a difference in extinction of 0.1 E likewise resulted in a straight line.
Example 2 The plasminogen de~ermination was carriecl out as in Example 1 but a reagent with a streptokinase concen~ration of 40 lU/mL was used. The shape of the reference plot changed as the streptokinase concentration decreased. In particular, it is possible at a low streptokinase concen-tration, fQr example 40 IU/ml, to gain information on the ability of the plasminogen ~o be activated by streptoki-nase by measuring the reaction time for a fixed delta Eof 0u1, for example. Information of this type is of importance for patients with myocardial infarction.
~hen comparative plasminogen determinations on 40 samples from patients were carried out by the mathods of Example 1 and Example 2, the plasminogen roncentration in a total of 34 samples ~as found to be in the normal range, with good agreement between the figures from the two methods.
Ho~ever, pathological figures (less than 50% of normal) were found by the method of Example 2 in 6 samples.
A method for the determination of plasminogen The invention relates to a method for the determination of plasminogen using a chromogenic or fluorogenic su~strate andin the presence of streptokinase.
eecause of the importance of this protein for fibrinolysis, the determination of plasminogen is nowadays a normal con-stituent of the diagnosis and monitoring of peopLe at risk of thrombcsis. A deficiency of plasminogen often results in a thrombotic event such as myocardial infarc-tion, pulmonary embolism or deep vein thrombosis in the legs. The therapy of thrombolysis with streptokinase or urokinase, which is often carried out nowadays, is like-wise dependent on an adequate concentration of plasminogencapable of being activated.
Apart from immunological determinations using nephelometry or radial immunodiffusion, it is possible to carry out 2û functional determinations of plasminogen with fluorogenic or chromogenic peptide substrates after activation w;th streptokinase. These assays~utilize the fact that plas-minogen, after activation to plasmin, remains bound to streptokinase and, in plasma, this complex (activator) is not, in contrast to free plasminr ;mmediately inhibi-ted. After the introduction of chromogenic tripeptide substrates, this technique was described for the first time in Chromogenic Peptide Substrates, Chemistry and ~linical Usage, page 128~ Scully and Kakkar ed. Churchill L;vingstone, Edinburgh and New York, 1979. However, in this and other similar known methods, it is necessary to preincubate the sample with streptokinase for a rela-tively long time, about 10 min.~ in order to convert the plasma plasminogen completely into the activaeor complex, and the substrate is added separately.
1 3 ~
It has been found, surprisingly, that plasminogen can also he activated by streptokinase in the presence of a chromogenic substrate for pLasmin. After a relatively short lag period, plasmin is formed as a complex with streptokinase, and the substrate is cleaved. The onset of substrate hydrolysis is rapid even in plasma samples with low plasminogen concentrations. The advantage of a method of this type is that there is no necessity either for a preincubation or for a second pipetting step to add the substrate.
Hence the invention relates to a method for the determina-tion of plasm;nogen in body fluids in the presence of streptokinase using a substrate for plasmin, the action of plasmin on which results in a cleavage product which can be measured optically, which method comprises essenti-ally simultaneous addition of streptokinase and a chromo-genic or fluorogenic substrate for plasmin to thebody fluid, and deter-m;nation ~f the amount of the cleavage product formed in a defined ti~e, or of the rate of formation of the cleav-age product, and from this ~he concentration of plasminogen.
This method can be carried out in the following manner:
the plasminogen concentration in a sample can be deter-mined with a reagent which, in addition to a chromogenicsubstrate, contains an excess of streptokinase, preferably 1,000 U/ml. The plasminogen in the sample is thereby con-verted into plasminogen-streptokinase complexes which convert the chromogenic substrate. The evaluation in 3û this method is effected by determining the rate of forma-tion (delta E/min) of the chromophore cleaved off the chromogenic substrate. However, it it also possible to measure the time required for the development of a defined difference in extinction, preferably 0~1 E.
In both types of evaluation the plasminogen concentration in the sample is proportional to the parameters measured.
Comparison of this method (embodiment 1) with conventional ~ 3~088 methods revealed good agreement in 40 samples from patients (Example 1).
However, another possible procedure is as follows: the reagent used for this has a substantially lower strepto-kinase concentration. This entails the plasminogen content of a sample being established from the concentra-tion of plasmin produced by the action of streptokinase-plasminogen complexes on plasminogen. Measurement of the rate of formation of plasmin makes it possible not only to determine plasminogen quantitatively but also to gain information on the ability of plasminogen to be activated (embodiment 2). Evaluation is effected by determining the t;me required after addition of the reagent for a defined difference in ext;nction, preferably 0.1 E, to be reached.
It was shown on 40 plasma samples from patients that the results of embodiments 1 and 2 agreed well in 34 cases.
In 6 cases, normal plasmino~en concentrations were found with embodiment 1, whereas determinations ~ith embodiment 2 resulted ;n figures below 50X of normal (Example 2).
The methocl according to the invention is expediently car-ried out in buffered solution, for example using a phosph-ate, tris, HEPES or acetate buffer with a pH of 6 to 9 preferably a phosphate buffer, especially 0.1 mol/l K
phosphate, pH 7.5.
All chromogenic plasmin substrates are suitable as chromo-genic substrates:
U-D-CHA-NVA-Lys-pNa explanations:
H-D-Val-Leu Lys-pNA CHA cyclohexylalanine 35 H-D-NVA-CHA-Lys-pNA NVA norvaline H-D-NLEU-CHA-Arg-pNA pNA para-nitroanilide H-D-~ut-CHA-Lys-pNA NLEU norleucine H-D-NLEU-CHA-Lys-pNA 8ut epsilon-aminobutyric acid H-D-Phe-Tyr~Arg-pNA
~3~0~8 The examples which follow illustrate the invention.
Example 1 Streptokinase (aehringwerke AG, Marburg, Federal Republic of Germany) was dissolved in 0.1 mol/l K phosphate buffer, pH 7.5, in a concentration of about 1,000 internat;onal units/ml. 4 ml of this solution were mixed with 0~1 ml of a 10 mmol/l solution of the chromogenic substrate H-D-1û cyclohexylalanyl-norvalyl-lysyl-para-nitroanilide (Pentapharm AG, ~asel), and the solution was heated to 37C. 500 ~l of this solution were pipetted into 50 ~l of plasma in a cuvette which had been preheated to 37C.
The absorption at 405 nm was measured. The relation be-t~een the absorption and the time was virtually l;nearafter 30 sec. Delta E/min was determined from the l;near part of the function.
Construction of reference plots:
Serial dilutions were prepared by dilution of plasma with isotonic saline, and each dilution was treated as des-cribed above. The reference plots were obtained by use e;ther of the linear increase in extinction per minute after a lag period of 40 sec, or of the time taken to reach a defined difference in extinction~
The results are shown in the table which follows.
Concentration of delta E/min Reaction time for 30 plasminogen t%) delta E = 0.1, ;n sec 100 (initial) 0.680 18.0 0.360 31.2 0.188 55.8 12.5 0.098 112.8 0 O.OOQ
A straight line was obtained when delta E/min was plotted against the concentration. Semireciprocal plotting of ~ 3~8~
the time taken to reach a difference in extinction of 0.1 E likewise resulted in a straight line.
Example 2 The plasminogen de~ermination was carriecl out as in Example 1 but a reagent with a streptokinase concen~ration of 40 lU/mL was used. The shape of the reference plot changed as the streptokinase concentration decreased. In particular, it is possible at a low streptokinase concen-tration, fQr example 40 IU/ml, to gain information on the ability of the plasminogen ~o be activated by streptoki-nase by measuring the reaction time for a fixed delta Eof 0u1, for example. Information of this type is of importance for patients with myocardial infarction.
~hen comparative plasminogen determinations on 40 samples from patients were carried out by the mathods of Example 1 and Example 2, the plasminogen roncentration in a total of 34 samples ~as found to be in the normal range, with good agreement between the figures from the two methods.
Ho~ever, pathological figures (less than 50% of normal) were found by the method of Example 2 in 6 samples.
Claims (4)
1. A method for the determination of plasminogen in body fluids in the presence of streptokinase using a sub-strate for plasmin, the action of plasmin on which results in a cleavage product which can be measured optically, which method comprises essentially simul-taneous addition of streptokinase and a chromogenic or fluorogenic substrate for plasmin to the body fluid, and determination of the amount of the cleav-age product formed in a defined time, or of the rate of formation of the cleavage product, and from this the concentration of plasminogen.
2. The method as claimed in claim 1, wherein the body fluid is plasma.
3. The method as claimed in claim 1, wherein the total concentration of plasminogen is determined by use of a molar excess of streptokinase over the amount of plasminogen present.
4. The method as claimed in claim 1, wherein the rate of activation of plasminogen is determined by use of an amount of streptokinase such that the plasminogen pre-sent is in molar excess.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863635191 DE3635191A1 (en) | 1986-10-16 | 1986-10-16 | METHOD FOR DETERMINING PLASMINOGENS |
| DEP3635191.1 | 1986-10-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1316088C true CA1316088C (en) | 1993-04-13 |
Family
ID=6311820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000549409A Expired - Fee Related CA1316088C (en) | 1986-10-16 | 1987-10-15 | Method for the determination of plasminogen |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0264753B1 (en) |
| JP (1) | JP2540563B2 (en) |
| AT (1) | ATE85364T1 (en) |
| AU (1) | AU602535B2 (en) |
| CA (1) | CA1316088C (en) |
| DE (2) | DE3635191A1 (en) |
| ES (1) | ES2053496T3 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3838529A1 (en) * | 1988-11-14 | 1990-05-17 | Behringwerke Ag | GLOBALTEST FOR DETECTING THE MAIN COMPONENTS OF THE FIBRINOLYSIS SYSTEM |
| DE19914811A1 (en) | 1999-03-31 | 2000-10-05 | Henkel Kgaa | Detergent compositions containing a bleaching agent include a combination of a cyanomethyl ammonium salt bleach activator and an enzyme |
| FR2979635B1 (en) * | 2011-09-07 | 2015-03-20 | Hyphen Biomed | PROCESS FOR DETERMINING PLASMINOGEN IN A LIQUID MEDIUM, COMPOSITIONS AND KIT THEREFOR. |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2521206A1 (en) * | 1975-05-13 | 1976-12-02 | Eckart Dr Med Jacobi | Plasminogen content measuring device - uses determination method based on fibrin formation in plasma sample |
| SE7801373L (en) * | 1978-02-07 | 1979-08-08 | Kabi Ab | EASY SPLABLE SUBSTRATE FOR QUANTIFIATION OF PROTEASES |
-
1986
- 1986-10-16 DE DE19863635191 patent/DE3635191A1/en not_active Withdrawn
-
1987
- 1987-10-10 AT AT87114820T patent/ATE85364T1/en not_active IP Right Cessation
- 1987-10-10 ES ES87114820T patent/ES2053496T3/en not_active Expired - Lifetime
- 1987-10-10 EP EP87114820A patent/EP0264753B1/en not_active Expired - Lifetime
- 1987-10-10 DE DE8787114820T patent/DE3784000D1/en not_active Expired - Fee Related
- 1987-10-15 JP JP62258482A patent/JP2540563B2/en not_active Expired - Lifetime
- 1987-10-15 AU AU79801/87A patent/AU602535B2/en not_active Ceased
- 1987-10-15 CA CA000549409A patent/CA1316088C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE3784000D1 (en) | 1993-03-18 |
| EP0264753A2 (en) | 1988-04-27 |
| JP2540563B2 (en) | 1996-10-02 |
| AU7980187A (en) | 1988-04-21 |
| JPS63105698A (en) | 1988-05-10 |
| DE3635191A1 (en) | 1988-04-21 |
| ATE85364T1 (en) | 1993-02-15 |
| EP0264753B1 (en) | 1993-02-03 |
| EP0264753A3 (en) | 1988-12-28 |
| AU602535B2 (en) | 1990-10-18 |
| ES2053496T3 (en) | 1994-08-01 |
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