AU602535B2 - A method for the determination of plasminogen - Google Patents
A method for the determination of plasminogen Download PDFInfo
- Publication number
- AU602535B2 AU602535B2 AU79801/87A AU7980187A AU602535B2 AU 602535 B2 AU602535 B2 AU 602535B2 AU 79801/87 A AU79801/87 A AU 79801/87A AU 7980187 A AU7980187 A AU 7980187A AU 602535 B2 AU602535 B2 AU 602535B2
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- AU
- Australia
- Prior art keywords
- plasminogen
- streptokinase
- plasmin
- concentration
- determination
- 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.)
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- 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
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- 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
A method for determining plasminogen in body fluids in the presence of streptokinase using a substrate for plasmin, from which an optically measurable fragment is produced by the action of plasmin, is characterised in that the body fluid is mixed essentially simultaneously with streptokinase and a chromogenic or fluorogenic substrate for plasmin, and the amount of the fragment produced in a particular time, or the rate of formation of the fragment, and from this the concentration of plasminogen, is determined. When the plasminogen present is in molar excess compared with streptokinase this method can be used to determine the rate of activation of plasminogen.
Description
i i l, Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
Class Application Number: Lodged: Int. Class ii
A
A
d
I
j 6 Complete Specification Lodged: Accepted: Published: Piority tRl SRilated Art:
(I
ft Name of Applicant: Address of Applicant: Actual Inventor: Address for Service: BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany HANS-JURGEN KOLDE EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled: A METHOD FOR THE DETERMINATION OF PLASMINOGEN The following statement is a full description of this invention, including the best method of performing it known to U r -L B .i Y James Murray Association. ames urray Registered Patent Attorney To: TIHE COMMISSIONER OF PATENTS, BEHRINGWERKE AKTIENGESELLSCHAFT 86/B 034 Ma 582 4Dr. Ha/Li.
A method for the determination of plasminogen The invention relates to a method for the determination of plasminogen using a chromogenic or fluorogenic substrate and in the presence of streptokinase.
Because of the importance of this protein for fibrinolysis, the determination of pLasminogen is nowadays a normal constituent of the diagnosis and monitoring of people at risk of thrombosis. A deficiency of pLasminogen often t results in a thrombotic event such as myocardial infarction, pulmonary embolism or deep vein thrombosis in the I legs. The therapy of thrombolysis with streptokinase or urokinase, which is often carried out nowadays, is likewise dependent on an adequate concentration of plasminogen capable of being activated.
Apart from immunological determinations using nephelometry or radial immunodiffusion, it is possible to carry out functional determinations of plasminogen with fluorogenic or chromogenic peptide substrates after activation with streptokinase. These assays utilize the fact that plasminogen, after activation to plasmin, remains bound to streptokinase and, in plasma, this complex (activator) is not, in contrast to free plasmin, immediately inhibited. After the introduction of chromogenic tripeptide substrates, this technique was described for the first time in Chromogenic Peptide Substrates, Chemistry and Clinical Usage, page 128, Scully and Kakkar ed. Churchill Livingstone, Edinburgh and New York, 1979. However, in this and other similar known methods, it is necessary to preincubate the sample with streptokinase for a relatively long time, about 10 min., in order to convert the plasma plasminogen completely into the activator complex, and the substrate is added separately.
i i' Prokurist uri ppa. Stein ppa. Bug i2 2 It has been found, surprisingly, that pLasminogen can also be 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- Stion of plasminogen in body fluids in the presence of t streptokinase using a substrate for plasmin, the action it.I 15 of plasmin on which results in a cleavage product which can be measured optically, which method comprises essentially simultaneous addition of streptokinase and a chromo- V genic or fluorogenic substrate for plasmin to thebody fluid, and determination of the amount of the cleavage product formed in a defined time, or of the rate of formation of the cleav-
S
e age product, and from this the concentration of plasminogen.
This method can be carried out in the following manner: the plasminogen concentration in a sample can be deter- S 25 mined with a reagent which, in addition to a chromogenic substrate, contains an excess of streptokinase, preferably 1,000 U/ml. The plasminogen in the sample is thereby converted into plasminogen-streptokinase complexes which convert the chromogenic substrate. The evaluation in this method is effected by determining the rate of formation (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 r k .1 i *r L- -ilili-i i.iill ilii-CY-iii iL~. i i: j 3 methods revealed good agreement in 40 samples from patients (Example 1).
~I I I~t r However, another possible procedure is as follows: the reagent used for this has a substantially lower streptokinase concentration. This entails the plasminogen content of a sample being established from the concentration of plasmin produced by the action of streptokinaseplasminogen 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 Evaluation is effected by determining the time required after addition of the reagent for a 15 defined difference in extinction, 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 plasminogen concentrations were found with embodiment 1, whereas determinations with embodiment 2 resulted in figures below 50% of normal (Example 2).
The method according to the invention is expediently carried out in buffered solution, for example using a phosphate, tris, HEPES or acetate buffer with a pH of 6 to 9, preferably a phosphate buffer, especially 0.1 moL/l K phosphate, pH All chromogenic plasmin substrates are suitable as chromogenic substrates:
II
I It I (t I C H-D-CHA-NVA-Lys-pNa H-D-Val-Leu-Lys-pNA H-D-NVA-CHA-Lys-pNA H-D-NLEU-CHA-Arg-pNA H-D-But-CHA-Lys-pNA H-D-NLEU-CHA-Lys-pNA H-D-Phe-Tyr-Arg-pNA explanations: CHA cyclohexylalanine NVA norvaline pNA para-nitroanilide NLEU norleucine But epsilon-aminobutyric acid -4- The examples which follow illustrate the invention.
Example 1 Streptokinase (Behringwerke 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 international units/mL. 4 ml of this solution were mixed with 0.1 mL of a 10 mmol/l solution of the chromogenic substrate H-Dcyclohexylalanyl-norvalyl-lysyl-para-nitroanilide (Pentapharm AG, Basel), and the solution was heated to 37 0 C. 500 pl of this solution were pipetted into 50 pL of plasma in a cuvette which had been preheated to 37 0
C.
The absorption at'405 nm was measured. The relation be- 15 tween the absorption and the time was virtually linear t t after 30 sec. Delta E/min was determined from the linear S ;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 described above. The reference plots were obtained by use either 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.
SConcentration of delta E/min Reaction time for plasminogen delta E 0.1, in sec 100 (initial) 0.680 18.0 0.360 31.2 0.188 55.8 12.5 0.098 112.8 0 0.000 A straight line was obtained when delta E/min was plotted against the concentration. Semireciprocal plotting of 5 the time taken to reach a difference in extinction of 0.1 E likewise resulted in a straight line.
Example 2 The plasminogen determination was carried out as in Example 1 but a reagent with a streptokinase concentration of 40 IU/ml was used. The shape of the reference plot changed as the streptokinase concentration decreased. In particular, it is possible at a low streptokinase concentration, for example 40 IU/ml, to gain information on the ability of the plasminogen to be activated by streptokinase by measuring the reaction time for a fixed delta E Sof 0.1, for example. Information of this type is of importance for patients with myocardial infarction.
When comparative plasminogen determinations on 40 samples from patients were carried out by the methods of Example 1 and Example 2, the plasminogen concentration in a total of 34 samples was found to be in the normal range, with Ir good agreement between the figures from the two methods.
'I However, pathological figures (less than 50% of normal) were found by the method of Example 2 in 6 samples.
Claims (1)
- 6- THE CLAIMS.DEFINING THE INVENTION ARE AS FOLLOWS: R~a1xxxx ax 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 es ntia..lly 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. ct 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. DATED this 14th day of October 1987. BEHRINGWERKE AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS O 50 QUEEN STREET a MELBOURNE. VIC. 3000. -3
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3635191 | 1986-10-16 | ||
| DE19863635191 DE3635191A1 (en) | 1986-10-16 | 1986-10-16 | METHOD FOR DETERMINING PLASMINOGENS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU7980187A AU7980187A (en) | 1988-04-21 |
| AU602535B2 true AU602535B2 (en) | 1990-10-18 |
Family
ID=6311820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU79801/87A Ceased AU602535B2 (en) | 1986-10-16 | 1987-10-15 | A 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 |
| CA1316088C (en) | 1993-04-13 |
| EP0264753A3 (en) | 1988-12-28 |
| ES2053496T3 (en) | 1994-08-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |