Method of determining activity of antitrombin-bm

专利摘要:
The invention relates to medicine, in particular to a method for determining the activity of antithrombin-BM. Citrate plasma is diluted with 0.9% NaCl solution. An antithrombin (AT) reaction mixture is prepared. Solutions of the AT reaction mixture and 0.9% NaCl in one case and diluted plasma and the AT reaction mixture in the other case are introduced into the plastic cuvettes. These mixtures are incubated for 5 minutes and mixed with 1.9 mmol of chromozyme TH. Within 30 seconds, the initial extinction is determined. After 30, 60 and 90 seconds, the measurement is repeated. From the difference between the extinction coefficients, the average value is determined. Thus, a total antithrombin activity is obtained. By repeating the measurement with a lower concentration of heparin, AT-III o is obtained. From the difference between CO total antithrombin activity and with AT-III, the amount of antithrombin-BM is obtained, table 5. 1 il. you
公开号:SU1271379A3
申请号:SU823450446
申请日:1982-06-08
公开日:1986-11-15
发明作者:Лилль Хельмут；Шренк Юрген；Вундервальд Петер
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:


cm
This invention relates to a method for determining the activity of a new thrombin inhibitor which is called anti-thrombin BM ().
The definition of AT-BM is based on the use of differences in specificity with respect to AT-III in the case of different cofactors. Antitrombin cofactor VM, thrombin M, the determined substrate of thrombin are added to the test solution and, based on the amount of the cleavage products of the substrate thrombin, the activity of antithrombin BM,
Example, 9h. svetotob early blood mixed with 1 h. 0.11 mol / L sodium citrate and centrifuged at 3000 rpm. the obtained citrate plasma is diluted with 1.0 ml of Q, a solution of Ns-Cl,
5 MP of a solution containing 0.1 M Tris / HCl, pH 8.1; 0.15 M NaCl, 0, OGM EDTA, 1% polyethylene glycol, 6.5 IU aprotinin / ml and I, 75 USP heparin / ml, are mixed with 0.25 ml of thrombin solution with O., 5 units / ml and left within 30 minutes (AT is the reaction mixture).
The determination is carried out according to the following scheme at 25 ° C in a cuvette with a layer thickness of 1 cm and at a wavelength of Hg405HM
The extinction increment compared to air is measured. Pipet 0.10 MP of 0.9% NaCl and 2 AT-Re stock mixes with a pipette into one plastic cuvette, and 0.10 ml of diluted plasma and 2 ml of the AT reaction mixture5 into the other cuvette, and mix them for 5 minutes. min. Then, the contents of each cuvette were mixed with 1.9 mmol of the TH chromozyme, tos-gly-proarg-pM. (0.20 ml), the initial extinction was determined for 30 s and the stopwatch was simultaneously turned on. After 30, 60 and 90 seconds, the determination is repeated. The mean value is determined from the difference between the coefficients of tinction (AE / AEC).
Thus, total anti-trombinbp activity is obtained.
By repeating the measurement with her heparin concentration lowered to 0.2 USP / ml, concentrations of AT-III are obtained. From the difference between total anti-thrombin activity and AT-IIT, an amount of AT-BM is obtained.
Example 2; Use traces of reagent solutions
1. Buffer: Tris / HC1 100 mmol / l, pH 8.1; dextran sulfate (mv, 500,000) 1 ml / 100 ml; aprotinin 6.5 IU / ml (inhibitor unit; trypsin, Chromosin TN, 25 ° C; NaCl 150 mmol / l.
2o Thrombin 0.5 U / ml.
3. Chromozyme TH (tos-gly-pro-argpNA) 1.9 mmol / l,
4, Mixture of reagents: Tris / HC
90 mmol / l, 1; dextran sulfate 0.9 mg / 100 I-OT; aprotinin 5.9 IU / ml; NaCl 136 1 1 mol / l; thrombin 0.045 units / ml.
Preparation of a mixture of reagents.
Mix in a plastic container and leave to stand for about 30 minutes at 25 ° C with 10 ml of buffer (solution 1) and 1 MP of thrombin (solution 2).
Preparation, tests.
To determine 1 hour, citrate plasma is diluted with 200 hours of 0.9% NaCl (e.g., 30 ml of plasma and 10 ml of 0.9% NaCl solution).
Conditions for determining: Hg wavelength 405 nm; plastic cuvette, layer thickness 1 cm; measuring temperature 25 ° C.
Measurement versus air (extinction increment)
At least one idle value with thrombin (t) is required for each measurement series. Pipette 0.05 ml of 0.9% NaCl and J ml of solution 4 with one pipette and 0.05 ml of diluted plasma into the other. and 1 ml of solution 4, mixed, inclusive for 5 minutes at 25 ° C. Then the contents of each cuvette are mixed with solution 3 (TL 0.1 ml sample with 0.1 mi) and AE / 30 s is calculated; AEts / 30 s - Dirt / ZO with p. 1id..5, / ml AEft / / 30 s X 95,
For determination, AT-III solutions (concentration 12.5 IU / ml at 25 ° C) and AT-BM (concentration 12.1 IU / ml at 25 ° C) are prepared.
This concentration corresponds to the normal AT-III content in human gchasme.
In order to show the specificity of the determination in the presence of AT-AT-III, the determination is carried out with both the AT-BM solution and the mixture of both solutions.
AT-VM measurement „
1 part of the AT-BM solution is diluted with 100 hours of physiological NaCl solution (0.05 ML samples + 5.0 ppm of NaCl solution). From this solution is prepared the distance dilution with physiological saline solution (9 hours). AT-BM solution + I (NaCl solution; 8 + 2; 7 + Zl.d. up to the final dilution 1: 1000). Constant volumes (0.05 ml) of the prepared dilutions are then used in a test, according to which proportional inhibition values are measured within selected limits (see drawing). Measurement of AT-VM in the presence of 5 AT-III. 0.05 ml of AT-BM solution was diluted with 0.05 MP of AT-III solution and 4.95 mp of physiological saline solution (1: 100 respectively, dilution of both components). A series of dilutions are prepared from the resulting solution in the same manner as described above. Constant volumes (0.05 mp) of the prepared dilutions are then used in the test (see drawing). As can be seen from the data shown in the drawing, the presence of AT-III does not affect the measurement result, only 30 AT-BMs are measured. J Similar results are obtained when using dextran sulfate, complex effects of mucopolysaccharide with polysulfuric acid, pentosol polysulphate sulfates, or short-chain heparin derivatives. Example 3. The effect of thrombin concentration on the determination of AT-III. An aqueous solution of AT-III O was used as a sample. The reagent of example 1 was used: Tris buffer, pH 8.1; heparin, 1.75 US P / ml; substrate 0,19 mmol / l. The results of the determination are given in Table 45. 1 .. T a b l and c a 1
17.0 17.2 17.3
11.9 12.04 12.11 137 p with p p p p in Xr S pa pa pa re s
Example 6. The effect of dextran sulfate concentration on the determination of nitrombin-BM.
As a test, a water AT-BM solution was used. The reagent of Example 2 was used: Tris buffer, pH 8.1; trom bin, 0.05 u / ml substrate
0, J9 mmol / l.
The results of the determination are given
in tab. 4. 4 Example A. Effect of thrombin concentration on the determination of AT-VM with heparin. A water target of antithrombin-BM was taken as a test. Used asset in example 1: Tris buffer, 8,1; heparin 1.75 US P / ml; substrate 0.19 mmol / l. The results of the determination are shown in Table. 2. Table 2 Substrate Determination of AT-BM with heparin, IU / ml, in the presence of trobin B, units / ml of brain, TN 16.5 21.30 22.35 238 11.58 14.90 15.60 Example 5. Infant the concentration of thrombin on the definition of at-VM extransulfate. As a sample, an aqueous creator of antithrombin-BM was used. The test material of example 1 was used: Tris buffer, 8.1; dextra disulfate, 10 μg / ml; stratum, 0.10 mmol / l. The results of the determination are abl. 3. Table 3 Determination of AT-BM Substrate with dextran sulfate, .IU / ml, in the presence of thrombin, u / ml
12713796
Substrate
Table 4
Determination, IU / ml, in the presence of dextran sulfate, Mfcr / ml
Chromozyme TH 36.14 44.70 43.76 S 2238 25.29 31.29 31.43
Example 7 Dependence of thrombin activity on concentration. Concentration of the chromozyme TH, mmol / l 0,019 0,038 Thrombin activity, dU / min The concentration of thrombin in the test was. Thrombin in the test was 0.015 U / ml. As shown by experimental data, the test can be carried out at a substrate concentration of 0.019-0.2585 mmol / l. Since the substrate S 2238 has almost identical K, the value of 9 x X 10 mol / l, for this substrate is really the same boundary region. The calculation of the concentrations of thrombin, heparin or dextran sulfate in the test. The determination of the concentration of thrombin (U / kp) was made, based on the LLE / ZO signal measured in the test, as follows:. V X 2 & x V where V is the volume applied in the test (2.30 ml); .
; subacid-hromozyme TH (toe-glypro-agp pHA m-value 2x10 mol / l.
Table 5 0.076 0.114 0.152 0.190 0.285 0.144 0.156 0.158 0.157 0.155 — the extinction coefficient for p Nitroaniline (9.70 cm µmol. V is the sample volume of thrombin (AT-reactive mixture 2.00 ml); 2 is a factor necessary for to obtain LU / MIN. If, for example, a thrombin A E signal was obtained at 0.500 / 30 s, then the thrombi concentration in the AT reaction mixture was 0.12 U / ml. At a further dilution, the mixture used in the test (2 , 30 ml), the concentration of thrombin in the dough becomes 0.10 Y / ml. The heparin preparation is characterized by the number of USP-dinicles per 1 ml. For example, the concentration of heparin is 1.00 USP / ml in the buffer solution gives a concentration of 0.95 USP / ml in the AT reaction mixture (i.e., 5.0 ml of the buffer solution is mixed with 0.25 ml of thrombin solution). Then the test results in a heparin concentration of 0.83 USP / ml, as in the test the AT-reactive mixture is diluted from 2 to 2.30 ml)

权利要求:
Claims (1)
[1]
The claims The method of determining the activity of antithrombin-VM, which consists in the fact that to the test citrate plasma or serum add 0.01-0.1 units / ml of thrombin; 0.076-0.285 mmol / L of its substrate (toz — gly — pro — arg-pNA or H — D-phe-pip -arg-pNA) in a buffer with a pH of 8.1 and 1 25 mg / ml of dextransulfate or short chain derivatives of heparin and by the amount of thrombin substrate cleavage products formed, the value of antithrombin activity is judged Order 6258/60
Circulation 778 Subscription
VNIIIPI of the USSR State Committee for Inventions and Discoveries 113035, Moscow, Zh-35, Raushskaya nab., 4/5
Production and printing company, Uzhhorod, st. Project, 4

类似技术:

---

公开号 | 公开日 | 专利标题

---

Hemker et al.1995|Thrombin generation in plasma: its assessment via the endogenous thrombin potential

---

Ødegård et al.1975|Heparin cofactor activity measured with an amidolytic method

---

SU1271379A3|1986-11-15|Method of determining activity of antitrombin-bm

---

Ødegård et al.1976|Antifactor Xa activity measured with amidolytic methods

---

US4692406A|1987-09-08|Process and a reagent for the simultaneous determination of fibrinogen and fibrinogen fission products in plasma

---

Bergström et al.1978|Determination of vitamin K sensitive coagulation factors in plasma. Studies on three methods using synthetic chromogenic substrates

---

US4496653A|1985-01-29|Process for the determination of antithrombin-BM

---

Teien et al.1975|Heparin assay in plasma: A comparison of five clotting methods

---

EP1562047B1|2008-05-14|Reagent kit for detecting lupus anticoagulant

---

Kahle et al.1978|Antithrombin III I. Evaluation of an automated antithrombin III method

---

US20090087870A1|2009-04-02|Hematological assay and kit

---

Handeland et al.1983|Simplified assay for antithrombin III activity using chromogenic peptide substrate: Manual and automated method

---

CN108982865B|2021-05-11|Thrombus elastography heparin quantitative detection kit and preparation method thereof

---

CA2155503C|2008-10-14|A method for detecting defects in protein c/protein s mediated blood clotting

---

Hoffmann et al.1988|Automated nephelometry of fibrinogen: analytical performance and observations during thrombolytic therapy.

---

US5985582A|1999-11-16|Thrombin-based assay for antithrombin III

---

Mitrophanov et al.2020|Controlled multifactorial coagulopathy: effects of dilution, hypothermia, and acidosis on thrombin generation in vitro

---

Bounameaux et al.1980|Monitoring of Heparin Treatment. Comparison between Thrombin Time, Activated Partial Thromboplastin Time, and Plasma Heparin Concentration, and Analysis of the Behavior of Antithrombin III.

---

Morse et al.1971|Automated fibrinogen determination

---

Bartl et al.1979|Determination of the biological activity of heparin by use of a chromogenic substrate

---

Ødegård et al.1976|Antithrombin III, heparin cofactor and antifactor Xa in a clinical material

---

Goodnight JR et al.1980|Measurement of antithrombin III in normal and pathologic states using chromogenic substrate S-2238: comparison with immunoelectrophoretic and factor Xa inhibition assays

---

Saleem et al.1975|Improved micromethod for plasma fibrinogen unaffected by heparin therapy

---

AU622339B2|1992-04-02|Method and reagent for the determination of antithrombin iii

---

Yamada et al.1979|A new APTT assay employing a chromogenic substrate and a centrifugal autoanalyzer

同族专利:

---

公开号 | 公开日

---

DE3038163A1|1982-05-06|

---

JPS5793915A|1982-06-11|

---

EP0049861A3|1983-07-27|

---

EP0049861B1|1985-05-29|

---

US4379142A|1983-04-05|

---

ES8205813A1|1982-08-16|

---

AT13489T|1985-06-15|

---

ES505829A0|1982-08-16|

---

DD201975A5|1983-08-24|

---

EP0049861A2|1982-04-21|

---

DE3170739D1|1985-07-04|

---

JPS5932443B2|1984-08-09|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

SE392038B|1971-09-08|1977-03-14|Kabi Ab|PROCEDURE FOR INSULATION OF ANTITROMBIN FROM BLOOD OR BLOOD PRODUCTS|

---

US4087415A|1976-06-09|1978-05-02|William L. Wilson|Antithrombin III|

---

JPS597693B2|1978-01-07|1984-02-20|Green Cross Corp|US4689323A|1983-09-26|1987-08-25|Miles Laboratories, Inc.|Covalently bound heparin--antithrombin-III complex|

---

FR2553518B1|1983-10-13|1986-04-18|Choay Sa|NEW CONJUGATES DEVELOPED BY FIXING A LIGAND ON AN INSOLUBLE SUPPORT, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS|

---

JPH0545600B2|1984-03-23|1993-07-09|Kowa Co|

---

HU199883B|1984-09-21|1990-03-28|Boehringer Ingelheim Int|Process for producing anticoagulant proteins and pharmaceutical compositions comprising same|

---

FR2646775A1|1989-05-11|1990-11-16|Centre Nat Rech Scient|USE OF CASEINOGLYCOPEPTIDE K, IN PARTICULAR COW MILK, FOR THE MANUFACTURE OF A COMPOSITION, IN PARTICULAR A MEDICINAL PRODUCT, FOR THE PREVENTION AND TREATMENT OF THROMBOSIS|

---

US5849703A|1990-08-27|1998-12-15|G. D. Searle & Co.|Pre-formed anticoagulant heparin/TFPI complexes|

---

US6291427B1|1990-08-27|2001-09-18|G.D. Searle & Co.|Anticoagulant combination of LACI and sulfated polysaccharides|

---

DE4203980A1|1992-02-11|1993-08-12|Max Planck Gesellschaft|METHOD FOR DETERMINING HIRUDINE AND SYNTHETIC THROMBINE INHIBITORS|

---

WO1993025220A1|1992-06-05|1993-12-23|Reid Thomas J Iii|Test for quantitative thrombin time|

---

US5476771A|1993-02-22|1995-12-19|The United States Of America As Represented By The Secretary Of The Army|Test for quantitative thrombin time|

---

AU6670198A|1997-02-28|1998-09-18|The Texas A & M University System|Heparan sulfate/heparin interacting protein compositions and methods of use|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

DE19803038163|DE3038163A1|1980-10-09|1980-10-09|THROMBINE INHIBITOR, ITS PRODUCTION AND USE|

---