SK48693A3 - Method of determination of fibrinogen - Google Patents

Abstract

A method for determination of fibrinogen by enzymatic conversion using thrombin, prothrombin fragments with a thrombin-like activity or mixtures thereof and subsequent detection of the resulting fibrin or of the fibrinogen degradation products is characterised in that the enzymatic conversion of the fibrinogen is carried out at a pH in the range from 4 to 7.3.

Description

Technical field

The invention relates to a method for the determination of fibrinogen by enzymatic reaction with thrombin, prothrombin fragments with thrombin like activity or mixtures thereof and then detecting the resulting fibrin e.g. fibrinogen cleavage products. Further, the invention includes an agent for determining the fibrinogen content of undiluted and optionally heparin containing plasma samples.

BACKGROUND OF THE INVENTION

The determination of fibrinogen in plasma is of great importance in the study of clotting disorders. For the determination of fibrinogen, immunological as well as functional methods such as e.g. clotting tests. In clotting assays, the fibrinogen content is determined by measuring the time at which a clot is formed.

A conventional assay method is described by Clauss C Acta Haemat. 1.7,237-246, 1957) Oxalate plasma is diluted in pH buffer

7.4 and concentrated thrombin solution was added. Finally, the time up to the point of precipitation is determined. However, the clotting time of a plasma sample that has reacted with thrombin is proportional to the fibrinogen content only when a sufficiently large excess of thrombin to the plasma has been chosen to overcome the plasma interfering factors. Antithrombin III (CAT III) is considered to be a disruptive factor which, in particular, due to lieparin, effectively suppresses the action of thrombin. Heparin is usually an anticoagulant and is used as an additive in blood collection or as a therapeutic. The heparin content of the plasma sample may lead to prolonged clotting times and thus to an incorrect result. :

The action of antithrombin III in the plasma will be due to the high concentration of thrombin and due to the diluted plasma and thus due to the reduction of the AT III concentration e.g. heparin strongly reduced. However, if the plasma is not diluted, the effect of antithrombin is several times stronger and the clotting time is prolonged.

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Dilution of plasma and the associated further processing step entails a considerable burden in routine experiments. In addition to higher labor costs and other necessary processing of potentially infectious plasma, each dilution is a major source of error. In addition, the dilution must often be carried out to match the content of the phi-riogen, which makes it difficult to work with automatic analyzers.

Usually, plasma dilution is performed using veronal buffer. as this buffer of fibrin precipitates, which in particular of fibrinogen facilitates the determination of narcotics with an addictive nature, the substances are considerably more difficult.

facilitates the formation of measurable plasma with low Veronal content, but at the same time it is also the use of this

Further attempts to overcome antithrombin III interfering factor e.g. Heparin, comprising neutralizing or resp. removal of heparin. Clinical Chemistry 29, 614-617 (1983) discloses a method according to which heparin is neutralized in a plasma sample by the addition of polybrene. The plasma is diluted in a pH 7.4 buffer and transferred to a photometer after the addition of thrombin. Thereafter, the reaction rate is measured imetrically. While adding the snake venom enzyme with llombin-like activity (batroxobin) instead of thrombin, fibrinogen in plasma can be determined independently of the interfering factors mentioned. These snake venom enzymes are not at least inhibited by AT III e.g. heparin.

The use of proteins which are not considered natural clotting enzymes, e.g. snake venom enzymes, in the method of determining human proteins is problematic. The reactions between the enzyme of the non-mammalian animal and the human protein are '4 k'. ? ť

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often non-specific. In accordance with the principle of biochemical analytics. Compare and explore just the same with the same is the pursuit of it. using only mammalian enzymes and among other human enzymes as the reaction partner for the determination of human proteins.

Thrombin can be produced by activation of prothrombin. As intermediates, they arise during activation of meisotrombin (MT) and meisotrombin (desFl). However, double-stranded thrombin intermediates exhibit protease activity in the presence of prothrombin, which is similar to thrombin-like activity). Thus, for example, they are capable of cleaving substrates which could be converted by thrombin. Thrombin like activity is also a protease activity that allows cleavage of thrombin substrates. whose ionic forces. inhibitors. effectors, allosteric side effects, etc. need not necessarily be consistent with these parameters for thrombin.

Activity of bovine enzyme meisotrombin. meisothrombin t

(desF1) thrombin was compared in J.of Biol. Chemistry 265. 10693 10701 (1990) at pH 7.4. Meizotrombin shows only 2% fibrinogen clotting activity of Thrombin. However, this reduced activity may also again be 10 3 15 of the MT content (desF1) in the MT preparation. 14% thrombin activity was found for MT (desF1).

The enzymatic conversion of fibrinogen to fibrin is usually performed at a pH in the range of 7.4 to 8.0. This pH range is usually used in the thrombin erizymatic reactions because it corresponds to the physiological pH range. The optimum pH value for thrombin activity is close to pH 8.

It is an object of the present invention to provide a method for the determination of fibrinogen by clotting assays, which would overcome the disadvantages of the known methods and could in particular also be carried out in undiluted plasma samples independently of AT III content e.g. heparin.

SUMMARY OF THE INVENTION

According to the invention, this object is achieved by a method for the determination of fibrinogen independent of thrombin inhibitors by enzymatic conversion of thrombin, prothrombin fragments, with thrombinlike activity or mixtures thereof and then detecting the resulting fibrin riapres. fibrinogen cleavage products, characterized in that the enzymatic conversion of fibrinogen is carried out at a pH range of 4 to 7.3 and detection of the formed fibrin is performed by clotting times or fibrinogen cleavage. The resulting fibrin clot is then detected by photometric or turbidimetric determination of clotting time . The prothrombin fragments with thrombin-like achivity include, in particular, mixtures of MT, MT (des Fl) or these substances alone. The concentration, for example, of the mixtures of enzymes used, is chosen such that a linear relationship is ensured over the widest possible range of fibrinogen content in the sample.

by establishing products. Detection with

Thus, proteins produced by the gene technique which, by virtue of their structure, exhibit thrombin-like activity and exhibit poor antithrombin III activity compared to thrombin, are suitable for the method of the invention. .

The performance of the method of determination in said pH range makes this so far independent of the abovementioned TT III and heparin interfering factors that the determination of fibrinogen can be carried out in a simple dilution and optionally heparin-containing plasma sample. thus improving the test result by eliminating a potential source of error.

The invention is based on the discovery that it is possible to suppress inhibition by ATII / heparin at a pH of 4 to 7.3, whereby thrombin activity to the enzymatic conversion of fibrinogen is achieved through suboptimal conditions. A somewhat reduced activity of thrombin even has the advantage that the clotting times are not too short and are easily noticeable.

Using thrombin, thrombin-like activity prothrombin fragments, or mixtures thereof, it has been found that thrombin activity within said pH range, preferably at a pH of 5-7 or most preferably at a pH of about 6, is optimal in determining fibrinogen independently in thrombin inhibitors.

The enzymatic conversion of fibrinogen is preferably performed in a buffer with a molarity of buffer salts in the range of 0.02 to 05 M, most preferably with a buffer of 0.1 to 0.25 M. The ionic strength can optionally be increased by the addition of salts such as sodium chloride. Thrombit has been found to be insensitive to AT, especially at low ionic strengths

III-Inhibition i. However, an environment with a buffering capacity equal to or higher than the plasma sample is also useful for carrying out the method of the invention in order to maintain the determined pH during the assay method.

Preferably, the determination of fibrinogen is carried out from undiluted and optionally also. heparin containing plasma samples in which it is also present in neutralized form.

The invention also relates to an agent for the determination of the fibrinogen content over a wide range independent of thrombin inhibitors of a sample optionally diluted and optionally also heparin containing thrombin-containing plasma. prothrombin fragments with thrombin like activites or mixtures thereof for enzymatic conversion of fibrinogen in a buffer at a pH in the range of 4-7.3 and then for detecting the fibrin formed by determining clotting time, e.g. fibrinogen cleavage products. The reagent serves for the simple determination of fibrinogen, without prolonged dilution of plasma samples or neutralization of e.g. removing heparin from plasma samples.

The invention will be explained in more detail by the following examples.

DETAILED DESCRIPTION OF THE INVENTION.

Determination of fibrinogen at pH 6 and pH 8 =

For comparison, meisotrombin and thrombin are used at pH 6 and pH 8 and the clotting time is determined depending on the fibrinogen content in the plasma samples. Meisothrombin is prepared by activation of human prothrombin by iniobilated ecarin (P. pentapharm) according to the procedure of Srocker and Müller (Thromb.Haemostasis 65 (6), abstract 855 (1991). Samples containing plasma with different fibrinogen contents were prepared for testing.

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/ µl plasma samples were mixed with 100 µl of buffer (0.2 M

Tris IICl buffer, pH 8 or 0.2 M phosphate buffer, pH 6) and 150 µl of meisotrombin (15 µl analog / µl) µl / ml. > Thrombin Reagent (Immuno) and assayed by ScIm and Gross-Coagulometer is performed both in the absence and presence of heparin (1 µM).

or thrombin C 10 NIH at 37 ° C is determined by time (Fa. Amelting). Determination ´ -T. It is apparent from the tables that the progression of the fibrinogen relationship curves at pH 6 is independent of the heparin content of the sample. However, the heparin content of the sample increases the clotting times at pH 8, both for meisotrombin and also for thrombin as a clotting enzyme.

Claims (4)

PATENT CLAIMS A method for the determination of fibrinogen in a wide range independent of thrombin inhibitors by enzymatic conversion of thrombin. prothrombin fragmentary with thrombin like activity or mixtures thereof and then detecting the resulting fibrin or fibrinogen cleavage products. characterized by. that the enzymatic conversion of fibrinogen is carried out at a pH in the range of 4 to 7.3 and the detection of fibrin is performed by determining the clotting time e.g. fibrinogen cleavage products. Method according to claim 1, characterized in that. The method according to claim 1, wherein the enzymatic conversion of fibrinogen is carried out in a buffer with a molarity of buffer salts in the range of 0.02 to 0.5 M, preferably 0.1 to 0.25 M, as well as after increasing the ionic strength by addition of salts such as NaCl. Method according to claim 1, characterized in that the determination of fibrinogen is carried out from diluted and optionally also heparin-containing plasma samples. 4. Reagent for the determination of fibrinogen content largely independent of thrombin inhibitors, optionally undiluted and, for example, also heparin containing plasma samples. containing thrombin, protroinbin fragments with thrombin like activity, or mixtures thereof for enzymatic conversion of fibrinogen and then for detecting the resulting fibrin by clotting time e.g. fibrinogen cleavage products in pH 4 to 7.3 buffer?