SU1161878A1 - Method of determing activity of thrombin - Google Patents

Abstract

A method for determining the activity of thrombin by interacting with fibrinogen and then determining its activity according to the level of fibrinoppegid A and B in the test and control samples, characterized in that, in order to increase the specificity of the determination, fibrinogen treated with 5-dimethylamine sulfone is added to the test sample additionally added, and it can also determine its specificity. concentrations of 2-5 mg / ml, then determine the fluorescence intensity in the test solution after separation, fibrin, and the activity of thrombin () is determined by the formula where Od and 3, the fluorescence intensity of the test and control samples ;, the degree of amplification of the flume of the orimeter; To a coefficient equal to the ratio of the magnitude of the eating fluorescence intensity to the activity of thrombin in the standard sample.

Description

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00 The invention relates to the field of medicine and can be used in the diagnosis of disorders of the blood clotting system. The purpose of the invention is to increase the specificity of the analysis of the activity of thrombin. The essence of the invention is as follows. A solution of dansylated fibrinogen is used as a substrate for determining the activity of thrombin. Dansylation of fibrinogen is carried out according to the method, the essence of which is that the amino groups of fibrinogen react with the fluorescent dye Dansyl chloride in an alkaline medium to form a covalent bond. Under the influence of thrombin, fluorescently labeled low molecular weight fibrinopeptides A and B are cleaved from the dansylated fibrinogen molecules. After the proteins are precipitated from the reaction mixture with 30% trichloroacetic acid, the amount of fibrinopeptides in the supernatant is determined fluorometrically. When determining the amount of thrombin in biological fluids, the results of the determination do not reflect the presence in Hjix of other nivomolecular peptides due to the absence of a fluorescent label in the latter. Examples of specific application of the method. Example 1.0 Determination of the activity of thrombin in the model system. Two ml of a solution of dansylated fibrinogen were poured into A tubes. 0.1 ml of a thrombin solution was added to each tube in the following ratios; 1 s 11 1.g2; 1: A | 1: 8. After 15 min, the reaction was interrupted by the addition of 2.0 ml of 30% TCA cells. Samples were centrifuged for 10 minutes at 1300 °. The supernatant was fluorimetric on a Fica-55 microfluorometer. Excited fluorescence wavelength 365 nm; The wavelength of fluorescence registration is 520 nm. In the control, 2.0 ml of 0.9% NaCl solution was added to 2.0 ml of fibrinogenar. The data are given in Table. 1. Results are expressed in units of relative activity. The activity of thrombin is calculated according to the formula -G. (-F p / where OQ and O) j is the fluorescence intensity of the test and control samples; h is the degree of amplification of the device; K is the proportionality coefficient, calculated experimentally for a specific type of fluorimeter by dividing the fluorescence intensity of the test sample by the number of units of thrombin activity in the sample. Example 2. In 4 samples, 0.5 ml of a dansylated fibrinogen solution containing 105-2-1 mg of protein was added to 0.5 ml of the investigated citrate plasma. For recalification, a ml of a 1.28% calcium chloride solution was added. After 15 world reactions were interrupted by the addition of 2.0 ml of a 30% aqueous solution of TCA acid. In the control, instead of a solution of calcium chloride, 0.1 ml of 0.9% NaCl solution was added. The samples were centrifuged for 10 min at 1300, the supernatant was fluorimetric on a Fica-55 microfluorometer, excitation wavelength 365 nm, fluorescence registration wavelength 520 nm. The results were expressed in relative fluorescence units (Table 2). Comparison of these data with the results of Example 1 shows that the activity of thrombin in plasma samples was low5, corresponding to large dilutions of thrombin (1: 8 - 1:16), however, causing a sufficient working fluorescence intensity of the fibrinopeptides when the sensitivity of the instrument is set to medium. I Example 3 In order to compare the sensitivity of the determination of protein and its derivatives by the methods of Lowry and the proposed method (fluorimetrically), the following studies were carried out. Dansylated β-fibrinogen dilutions containing in the sample from 2800 to 0.14 µg of protein were prepared. Protein was determined in samples according to the Lowry method and fluorimetrically on a Fica-SS microfluorometer at a fluorescence excitation wavelength of 365 nm and a recording wavelength of 520 nm. The data obtained are presented in Table. 3. Example D. The thrombin-fibrinogen reaction was set up in the following system. To 2.0 ml of a 0.5% solution of daxylated fibrinogen, 2.0 ml of 40% urea solution and 0.2 ml were added. thrombin solution with an activity of 10 s. The sample was incubated at 90 min. After incubation, 2.0 ml of 8% perchloric acid solution was added to the sample to precipitate fibrinomonomers and thrombin. The sample was centrifuged for 15 min., At 8000 ppm / min. The supernatant containing fibriyepeptides A and Bf was aspirated and corrected to 7.5 with the addition of 0.12 ml of a 10% KOH solution. The salt precipitate was removed by repeated centrifugation. . Fibrinopeptides A and B were diluted 3, 12 and 120 times with physiological solution, samples were analyzed by Lowry method as in the prototype and fluorimetrically; as in the proposed method, the output of fibrinopeptides A and B was about 1:60 of the weight taken fibrinogen, which coincides with the known theoretical analysis data. Thus, the obtained data showed that for the successful application of the Lowry method for the clinical, determination of fibrinoppeptides A and B as specific indicators of the activity of thrombin in thrombin-fibrinogenic acid tion of their content in the sample should be vshe 10 g (tab. 2a, 3), which coincides with izves GOVERNMENTAL dann E literature. Fluorimetric analysis allowed us to increase the sensitivity of the method by 10. Such a significant increase in the sensitivity of the method and the data show that the content of dansilicon fibrinogenic tissue in the samples, depending on the activity of thrombin, can vary widely (6-6 x 10 µg). However, the optimum content fluctuations dansi7S4 lyrovennogo fibrinogen in the sample, focused on one side on low values of thrombin activity, and on the other - on the rational consumption of the reagent (as shown by examples 1 and 2) is 2-5 mg. At the same time, it is possible to analyze the activity of thrombin by the level of fibrinopepgid A and B, which it is splitting off from fibrinogen, not only in model systems (as in the prototype), but also in biological samples (plasma serum), Example 5, In order to detect the specificity of label A and B fibrinogen peptides with various fluorescent probes in the technique of the proposed method were applied preparations of fibrinogen labeled with magnesium analyonaphthalene sulfonate (ANS), fluorescemin (FC) and dimethrin aminaphapixulphonyl chloride (bottom), which is one of tnositelno widely employed fluorescent probes. 5 parallel studies in the model were applied thrombin solutions (Kaunas) with proportionally decreasing activity (40, 20 and to units,). The fluorescent fluorescent fluorescence of fibrinopeptides A and B in similar experimental systems (Hitachi, Japan) were presented in tabl, 4, Pritser 6. 1.5 ml of 1% aqueous solution of dansylated fibrinogen was added to the studied digital plasma ml. 0 , 1 ml of 1 28% calcium chloride solution. After 15 mn, the reaction was precredited by adding 2.0 ml of a 30% solution of TCA acid. In the control, instead of a solution of calcium chloride, added xy 0.1 ml of 0, NaCl solution. The samples were centrifuged for 10 min at 1300,: The supernatant was fluorinated by fluorica using a Fica-55 fluoride, the length was not 365 nm, the wavelength was regisgrated; 520 them. The results are expressed in units of relative fluorescence: control 3 units, sample - 18 units.

Dependence of fibrinopeptide fluorescence on the amount of dansylated fibrinogen in the sample

Table 1

Intensity

62 fluorescence

The amount of dansilylated fibrino10 gene in the sample, mg

Comparative data of the sensitivity of the method of Lowry and fluorescence analysis in the determination of protein

More More 0.48

Enstantsi on Loury scale scale FEC FEC Fluorescence, used 200 120

82.2

15

table 2

Table 3

O O 26 3.2 3.2 1.4

The results of determining the activity of thrombin in the technique of the proposed method using fibrinogen labeled with various fluorescent probes (used units). ANS 1.4 .. FC 4.74.1 day 16.29.0

Table 4 1, 4.1, - 4.7

Claims (1)

METHOD FOR DETERMINING THE ACTIVITY OF THROMBIN by interacting with fibrinogen and then determining its activity by the level of fibrinopeptides A and B in experimental and control samples, characterized in that, in order to increase the specificity of the determination, fibrinogen treated with 5-dimethylamino aminosulfane is additionally introduced into the test sample the final concentration of 2-5 mg / ml * then determine the fluorescence intensity in the test solution after separation, fibrin, and thrombin activity (C _Tr ) is determined by the formula < where 0 _about and - the fluorescence intensity of the experimental and control samples ;. h is the gain of the fluorimeter; X is a coefficient equal to the ratio of the magnitude of the fluorescence intensity to the activity of thrombin in a standard sample. is for determinant-10 applies