EP0527197A1

EP0527197A1 - A METHOD OF DETERMINING QUANTITATIVELY FIBRINOGEN, FIBRONECTIN, alpha2-ANTIPLASMIN OR A TRANSGLUTAMINASE

Info

Publication number: EP0527197A1
Application number: EP19910909533
Authority: EP
Inventors: Birger Blombäck; Birgitta Hessel
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-05-03
Filing date: 1991-05-03
Publication date: 1993-02-17
Also published as: JPH05508472A; SE9001582D0; WO1991017444A1

Abstract

Le fibrinogène, la fibronectine, l'alpha2-antiplasmine et le Facteur XIII sont évalués quantitativement par un procédé de réaction qui consiste à: faire réagir du fibrinogène avec de la fibronectine ou de l'alpha2-antiplasmine, le Facteur XIII fonctionnant comme catalyseur; pré-évaluer les quantités de tous les constituants de réaction utilisés à l'exception de la quantité recherchée; et évaluer la quantité recherchée à l'aide d'une technique d'immunoadsorption connue, en particulier par l'adsorption du fibrinogène ou de la fibronectine sur une surface et par la réaction dutit fibrinogène ou de ladite fibronectine avec l'autre constituant de réaction et ensuite avec un anticorps agissant spécifiquement contre la substance devant être évaluée, lequel anticorps peut être marqué; après quoi la quantité dudit anticorps est mesurée selon une technique connue, éventuellement par la réaction avec un anticorps secondaire marqué.Fibrinogen, fibronectin, alpha2-antiplasmin and Factor XIII are quantitatively evaluated by a reaction method which consists in: reacting fibrinogen with fibronectin or alpha2-antiplasmin, Factor XIII functioning as a catalyst; pre-evaluate the quantities of all the reaction constituents used except the quantity sought; and evaluate the quantity sought using a known immunoadsorption technique, in particular by the adsorption of fibrinogen or fibronectin on a surface and by the reaction of the fibrinogen or said fibronectin with the other reaction constituent and then with an antibody acting specifically against the substance to be evaluated, which antibody can be labeled; after which the quantity of said antibody is measured according to a known technique, optionally by reaction with a labeled secondary antibody.

Description

A Method of Determining Quantitatively Fibrinogen, Fibronectin. o_₂-Antiplasmin or a translutaminase

The present invention to a method of determining quan- titatively fibrinogen, fibronectin, α_-antiplasmin or a transglutaminase. A known and important transglutamin¬ ase is Factor XIII.

Fibrinogen, fibronectin, α_-antiplasmin and Factor XIII are all important blood constituents and it is essen¬ tial to be able to determine the concentration of these constituents in blood and in other tissues. It has been found that Factor XIII is a transglutaminase in blood, the primary role of which is to cross-link protein chains in the fibrin network subsequent to its formation. This results partly in cross-linking bet¬ ween the γ-chains in the fibrin and partly also be¬ tween the α-chains themselves. Factor XIII is also able to catalyse specifically the incorporation of certain plasma proteins in the fibrin network. For instance, fibronectin and α -antiplasmin are linked to the fibrin by Factor XIII catalysis; a ino groups in the fibrin fulfil a donator function in both cases.

The concentration of fibrinogen in the blood is also important, insomuch as a deficiency of fibrinogen will lead to bleeding conditions, whereas elevated concen¬ trations predict cardiovascular diseases. Fibronectin is a protein which is apparently essential to cell proliferation and wound healing. The concentration of fibronectin is also lowered with infectious conditions, such as sepsis. α_-antiplasmin is the most important inhibitor of fibrinolysis in blood. Increased con¬ centrations of this inhibitor or increased incorpora- tion in the fibrin coagulum has been observed in throm- botic conditions.

SUBSTITUTE Consequently, it is essential to be able to determine the concentrations of these constituents or substances in blood and other tissues. Although methods are exist for determining the concentration of fibrinogen in blood, these methods are much too time-consuming and are non-responsive and/or non-specific. There are no functional methods by means of which the concentrations of fibronectin, α_-antiplasmin and Factor XIII can be determined quantitatively.

B. Blomback, R. Procyk, L. Adamson and B. Hessel (Thrombosis Research 37, pages 613-628, 1985) and R. Procyk, L. Adamson, M. Block and B. Blomback (Thrombosis Research 40, pages 833-852, 1985) show that fibrinogen and fibronectin react in the presence of

Factor Xllla, particularly in the latter publication. This reaction takes place in bulk phase and there are no suggestions to the effect that this reaction can be used for the quantitative analysis of the reactants.

It is also shown in Biol. Chem. Hoppe Seyler 1987, June: 368 (6): 669-74, J. Biol. Chem. 1988 J.uly: 25: 263 (21): 10464-9; Blood 1986 July: 68 (1): 95-101 and Biochim Biophys Acta 1988 November 17: 967 (2): 304-13 that thro bin-activated Factor XIII (Factor

Xllla) on polystyrene spheres in the presence of fibro¬ nectin is not able to bind fibrin to the spheres; but that fibronectin is able to cross-link to cellular locations on a matrix with Factor Xllla acting as a catalyst. It is also shown that Factor XIII cross¬ links fibrin on itself and a limited number of sub¬ strates. Factor XIII also has a catalyzing effect in solutions containing fibrinogen and fibronectin, forming two types of cross-linked polymers, namely hybridoligomers and fibrinogenoligomers.

SUBSTITUTE It has now surprisingly been found that these bulk phase reactions (reactions in solution) can be used in principle for assaying fibrinogen, fibronectin, α₂-antiplasmin and transglutaminase in practice. When the reactions are carried out as surface-bound reac¬ tions, it is surprisingly found that the reaction rate is increased considerably and that, at the same time, the concentrations of the reactants can be signifi¬ cantly.decreased. The surface-bound reaction has also been found more specific.

It has also been surprisingly found that fragments of the fibrinogen molecule will not disturb the surface- bound reaction. When quantitatively determining fibri- nogen in a known manner, the fibrinolytic fragment of the fibrinogen molecule has had a disturbing effect on the assay analysis. This disturbance is not found when practicing the present method, despite the fact that potential cross-linking locations are found in such fragments as Ds. Heparin can also have a disturbing effect in several known assaying methods. This is not the case when practicing the present invention, how¬ ever.

It has now been found that fibrinogen, fibronectin, α_- antiplasmin or a transglutaminase, particularly Factor XIII can be assayed functionally in a simple, but very precise fashion by means of a reaction which involves:

- reacting fibrinogen with fibronectin or α_-antiplasmin with a transglutaminase acting as a catalyst;

- using the reaction components in pre-determined quantities, with the exception of the quantity sought for; and

- determining the quantity sought for with the aid of

SUBSTITUTE an immunosorption technique in a known manner, par¬ ticularly by absorbing fibrinogen or fibronectin on a surface and then reacting the fibrinogen or fibro¬ nectin-with the other reaction component andc then with a labelled antibody which is specific there- against the substance to be determined whereafter the amount of labelled antibody is measured in a known manner.

It has been found that several reactions take place in the organism which are dependent on enzymes which possess a transglutaminase activity. Such enzymes are present both in intracellular fluids and in tissue fluids, such as blood plasma and lymph. There are differences in specificity between these enzymes. A common feature of all these reactions, however, is the chemical reaction mechanism which involves the estab¬ lishment of a covalent bond between a glutamin amino- acid rest (acceptor) in a polypeptide chain and a lysine a ino acid rest (donator) in another polypeptide chain, in accordance with the following:

SUBSTITUTE Thus, covalent bonds are obtained between the protein chains. Many different components possessing an amino function can serve as donators in this reaction, for instance dansyl cadaverine, putrescine, etc. Glutamine is required for the acceptor function. The specificity of the various enzymes is contingent partly on the amino acid sequence around the glutamic acid rest in a polypeptide chain, and partly on the structure of the amino function rest. The course followed by the reac- tion is accelerated when the reactant groups in the proteins are juxtaposed.

The invention will now be described in more detail with reference to the accompanying drawings, in which

Figure 1 is a schematic illustration of the course followed by the reaction when determining, or assaying, fibrinogen;

Figure 2 is a diagramme which illustrates the relation- ship between colour intensity and fibrinogen concentra¬ tion;

Figure 3 is a diagramme which shows the relationship between colour intensity and fibronectin concentration, and also the effect of iodo acetamide on the reaction with Factor XIII;

Figure 4 is a diagramme which shows the relationship between colour intensity and activity for Factor Xllla; Figure 5 is a diagramme which shows the relationship between the colour intensity and the activity of Factor Xllla.

Figure 6 illustrates the relationship between absorb- ency and fibrinogen concentration in plasma; Figure 7 illustrates the relationship between absorb- ency and fibronectin concentration in plasma and serum; and

Figure 8 illustrates the activity of Factor XIII after different reaction times.

Quantitative Determination of the — Fibrinogen Concentration

When carrying out the analysis, fibronectin is adsorbed on a surface in a known manner (see Figure la) . This surface may consist of a plastic material, such as polystyrene. Materials that are suitable for this purpose are available commercially, for example "Titer- tecplattor", latex spheres, etc. A sample solution containing fibrinogen is then added (Figure lb) ) . A calcium chloride solution and Factor XIII, suitably in an activated form, for instance a thrombin activated form, is then added. A thrombin-activity inhibitor, for instance hirudin, is then added to inhibit the thrombin excess used in the activating process or possibly generated in the sample. Factor Xllla (activated Factor XIII) now catalyses the incorporation of the fibrinogen into the surface-bound fibronectin

(Figure lc)) . The fibronectin-bound fibrinogen is then caused to react with an antibody (for instance from goats) which is specific against fibrinogen, so-called antifibrinogen (Figure Id)) and the antibody is combined with the fibrinogen on the surface (Figure le) ) . The resultant fibronectin-fibrinogen-antifibri¬ nogen is now caused to react with a so-called secondary antibody (e.g. rabbit-anti-goat-IgG) , labelled with peroxidase (Figure If) , whereafter the enzyme is visi- bilized by adding a substrate for the peroxidase

(Figure lg) (HRP = "horse-radish-peroxidase") . The fibrinogen-bound peroxidase (HRP) splits or cleaves the substrate, therewith producing a yellowish colour. This part-stage of the process involving the use of an enzyme-labelled antibody and its visibilisation, is known as the enzyme immunosorption technique and is

SUBSTITUTE well known.

Quantitative Determination of the - Fibronectin Concentration

Fibrinogen that is essentially free from fibronectin and Factor XIII is adsorbed on a surface of the kind described with reference to the quantitative determin¬ ation of fibrinogen. A fibronectin-containing solution is applied to the surface, together with calcium chlor¬ ide, thrombin-activated Factor XIII and hirudin, this latter to neutralize excess thrombin. In this case, Factor Xllla catalyses the incorporation of fibronectin into the surface-bound fibrinogen, analogously with that described with reference to the quantitative determination of fibrinogen. The fibrinogen-bound fibronectin is then caused to react with an antibody (e.g. from goats) which is specific against fibro¬ nectin, whereafter a reaction takes place with a secon- dary antibody labelled with peroxidase, analogously with that described with reference to the quantitative determination of fibrinogen. Should the sample solu¬ tion containing fibronectin also contain fibrinogen, this fibrinogen shall be removed by coagulating said fibrinogen with a snake-venom enzyme, for instance batroxobine. This coagulation process should not be effected with thrombin, since Factor XIII may be pre¬ sent in the sample solution. In such case, the enzyme is activated and causes the fibronectin present in the sample solution to be incorporated in the precipitated coagulum. This drawback is not experienced when coagu¬ lation is effected with batroxobin.

Quantitative Determination of α--Antiplasmin

Fibrinogen is caused to be adsorbed on a surface in the

SUBSTITUTE same manner as that described with reference to the quantitative determination of fibronectin. A solution containing α_-antiplasmin is applied to the surface, followed-by calcium chloride, thrombin-activated Factor XIII and hirudin, this latter to neutralize excess thrombin. Subsequent to a reaction time, the fibrino- gen-bound o_₂-antiplasmin is visibilised with a specific antibody against α_-antiplasmin and a secondary anti¬ body labelled with peroxidase, analogously with that described with reference to the quantitative determin¬ ation of fibrinogen and fibronectin respectively. Should the sample solution contain fibrinogen and fibronectin, as is the case with blood plasma, it is necessary to remove the fibrinogen by coagulation with a snake—venom enzyme, and to remove fibronectin by adsorption on gelatine coupled to an appropriate poly- mer matrix, e.g. Sepharos^®, in a known manner.

Determination of the Activity of Factor XIII

Fibrinogen that is essentially free from fibronectin and Factor XIII is adsorbed on a surface, analogous with the method described with reference to the quant¬ itative determination of fibronectin and Q._-anti- plasmin. An excess quantity of fibronectin is then added, together with calcium chloride and hirudin. A sample solution containing Factor XIII is then added. If active Factor XIII is present in the sample, the fibronectin will be incorporated on the surface of the fibrinogen and can be visibilised with a specific anti¬ body against fibronectin and a secondary antibody labelled with peroxidase, e.g. HRP, as described with reference to the quantitative determination of fibro¬ nectin. The total activity of Factor XIII can be determined by first treating the sample solution with batroxobin, to remove the fibrinogen by coagulation.

SUBSTITUTE Thrombin is then added, to convert Factor XIII to an active form.

The described embodiments concerned with the quantita- tive determination of fibrinogen, fibronectin, α._- antiplasmin and Factor XIII have initially been the only embodiments and thus the preferred embodiments. However, it has recently been found possible to use a specific, labelled antibody, particularly an antibody labelled with "horse-radish-peroxidase", instead of using a specific antibody and a secondary antibody labelled with peroxidase. This latter embodiment is now the preferred embodiment for quantitatively deter¬ mining the fibrinogen, fibronectin, α_-antiplasmin concentration and the activity of Factor XIII.

Thus, when determining the fibrinogen concentration, there is now used an antibody which is specific against fibrinogen, a so-called antifibrinogen, which is label- led, preferably with "horse-radish-peroxidase" (HRP) , a so-called primary antibody, instead of a specific antibody against fibrinogen which combines with the fibrinogen, whereafter the resultant product is reacted with a so-called secondary antibody.

Correspondingly, there is used when quantitatively determining the fibronectin concentration a labelled fibronectin antibody, while when determining the con¬ centration of α_-antiplasmin, there is used a labelled α_-antiplasmin antibody. Thus, when determining the activity of Factor XIII, there is preferably used a primary labelled antifibronectin antibody. The primary antibody is preferably a HRP-labelled antibody.

The invention will now be described in more detail with reference to a number of Examples.

SUBSTITUTE Example 1

Quantitative Determination of Fibrinogen

5 200 μl of a fibronectin solution (10 μg/ml) were intro¬ duced into the wells of a microtitre plate ("Titer- tec") . The plates were allowed to stand overnight at room temperature. The wells were then emptied and washed repeatedly with a solution of TNE-BSA-buffer 0 (0.05 M-tris-0.10 M NaCl-l mM EDTA, pH 7.4, containing 0.1% bovine serum albumin) . A solution of bovine albumin (30 mg/ml) was then poured into the wells with the intention of blocking those locations on the well surfaces not saturated with fibronectin. Subsequent to 5 washing with a solution of TNE-BSA-buffer, there were added 150 μl of a sample solution containing fibrinogen (between 0.15 and 20 μg/ml) and thereafter 20 μl CaCl_ solution (0.2 M) and 5 μl hirudin (1000 ATU/ml) (ATU = antithrombin units) . Finally, 25 μl thrombin activated

20 Factor XIII (3.2 units/ml) were added, such that the final concentration was 0.4 E/ml. The plates were shaken slowly for 1.5 hours at a temperature of 37°C. The plates were then washed with a buffer solution and 200 μl of goat-antifibrinogen-IgG in suitable concen-

25 tration were added. Subsequent to incubation at room temperature for 18 hours, the plates were washed with buffer solution and 200 μl rabbit-antigen-IgG labelled with peroxidase were added. Subsequent to incubation for 2 hours at room temperature, 100 μl of peroxidase

^'30 substrate were added. The mixture was incubated for an additional 1.5 minutes, whereafter the reaction was interrupted by adding 100 μl of 1 M H_S0. solution. The colour intensity was read-off at 490 nm. The adsorption relationship with the fibrinogen concentra-

35 tion in the solution is shown in Figure 2.

SUBSTITUTE The experiment was repeated, but with the addition of iodo acetamide to the solution to a concentration of 0.5 mM. This addition was made prior to adding Factor XIII. I-odo acetamide inhibits Factor XIII. No dis¬ cernible colour development occurred in this experi¬ ment. The experiment shows that the incorporation is dependent on the presence of Factor XIII.

Example 2

Determining Fibronectin Concentration

200 μl of fibrinogen (10 μg/ml) , free from fibronectin and Factor XIII, were introduced into the wells of a microtitre plate. The plates were allowed to stand at room temperature overnight. The surface was washed and saturated with bovine serum albumin in the manner described with reference to Example 1. Subsequent to washing with buffer solution (as in Example 1) , 150 μl of a sample solution containing fibronectin (0.02-2.5 μl/ml) were added, followed by 20 μl of a calcium chloride solution (0.2 M) and 5 μl of hirudin (1000 ATU/ml) . Finally, 25 μl of thrombin-activated Factor XIII (3.2 units/ml) were added, such that the final concentration was 0.4 units/ml. The plates were shaken slowly for 2 hours at a temperature of 37°C. The plates were then washed with a buffer solution (as in Example 1) and 200 μl of goat-antifibronectin-IgG were added. Subsequent to incubation for 18 hours at room temperature, the wells were washed with buffer solution and 200 μl of rabbit-antigen-IgG labelled with peroxid¬ ase were added. Subsequent to incubation for 2 hours at 37°C, 100 μl of peroxidase substrate were added. The same method as that described with reference to Example 1 was then followed. The relationship of colour intensity to fibronectin concentration in the sample will be seen from Figure 3.

An experiment was also carried out in this case in which iodo acetamide was added, analogously with the aforedescribed Example 1; no colour development was observed. Thus, this reaction is also dependent on the presence of Factor XIII (Figure 3) .

Example 3

Determination of the Total Activity of Factor XIII

When Using Secondary Antibodies Labelled with Peroxidase

Microtitre plates were treated with fibrinogen in the same manner as that described in Example 2. The plates were also blocked and washed in the same manner as that described in Example 2. 150 μl of fibronectin solution

(2.5 μg/ml), 20 μl of calcium chloride (0.2 M) and 5 μl of hirudin (1000 ATU/ml) were then added. Finally, 25 μl of thrombin-activated Factor XIII (concentrations between 0.001 and 0.1 E/ml) were added and the plates incubated for 2 hours at 37°C, while slowly shaking the plates. The plates were then washed with buffer solu¬ tion (in accordance with Example 1) . Goat-antifibro- nectin IgG was then added. The process of determining the total activity was then continued in the manner described in Example 2. Figure 4 illustrates the relationship between colour intensity and activity of Factor XIII in the sample solution. Background colour was generated solely in the absence of Factor XIII.

Example 4

Determining the Total Activity of Factor XIII When Using Biotinylated Antifibronectin

SUBSTITUTE Microtitre plates were treated with fibrinogen, in accordance with Example 2. The plates were also blocked and washed in the same manner as that described in Example 2. 150 μl of fibronectin solution (2.5 μg/ml), 20 μl of calcium chloride and 5 μl of hirudin (1000 ATU/ml) were then added. Finally, thrombin-activated Factor XIII (concentrations between 0.001.0.1 E/ml) was added and the plates incubated for 2.5 hours while slowly shaking the plates. The plates were then washed with a buffer solution (as in Example 1) , whereafter 200 μl of biotinylated goat-antifibro- nectin-IgG (1 μg/ml) were added and the plates incuba¬ ted room temperature for 18 hours. The plates were then washed with buffer solution (see Example 1) , whereafter 200 μl avidine-HRP (0.5 μg/ml) were added, followed by a peroxidase substrate in accordance with the method described in Example 2. Figure 5 shows the relationship between the colour intensity and the activity of Factor Xllla in the sample solution.

Example 5

Determining the Concentration of Fibrinogen in Blood

With the intention of determining the fibrinogen con¬ centration in blood, the blood was introduced into a vessel containing an anticoagulant substance, for example 3.8% trisodiu citrate or 0.1 M EDTA. The anticoagulant is normally present in a proportion of 1 part coagulant to 9 parts of blood. The blood was centrifuged (room temperature, 30 minutes, 1500 rpm) and blood plasma removed by suction. Prior to the analysis, the plasma was diluted with a TNE-BSA-buffer, (1:1000, 1:2000 and so on) (see Example 1).

Analysis: microtitre plates were treated with fibro-

SUBSTITUTE nectin, as described in Example 1. The plates were also blocked and washed in the manner described in Example 1. 150 μl plasma diluted or thinned to different degrees, calcium chloride and hirudin were then added. 25 μl of thrombin-activated Factor XIII (final concentration 1 E/ml) (were then added and the plates incubated for one hour at 37°C, while slowly shaking the plates. The plates were washed with buffer solution, as described in Example 1. 200 μl of primary HRP-labelled fibrinogen-antibody were then added.

Subsequent to incubation for one hour at 37°C, 200 μl of peroxidase substrate were added and the mixture was incubated for a further 2 minutes, whereafter the reaction was interrupted by adding 50 μl of 4 M H₂S0,. The color intensity was read at 492 nm. The relationship between absorbence and fibrinogen con¬ centration in plasma can be seen from Figure 6.

Example 6

A comparison was made between the determination of fibrinogen concentration according to the present invention and two known functional fibrinogen assaying methods.

Plasma taken from 23 different healthy persons was treated in the manner described in Example 5 and the fibrinogen content was determined in the manner described in Example 5 and also with the aid of said two known methods, the so-called syneresis method according to Bergstrδm and the so-called "clot rate"- method according to Vermylen.

The results are set forth in the following table: The fibrinogen concentration in plasma was determined with the aid of three different, functional

SUBSTITUTE fibrinogen-assaying methods

Method Mean value Spreading (SD)

Cross-linking method* 2.89 0.444 Syneresis method (Bergstrδm) 3.08 0.381 Clot-ratemethod (Vermylen) 2.76 0.537

* In accordance with the present invention.

Example 7 Analysis of the fibrinogen concentration in the same plasma on mutually different occasions.

Plasma from one single individual was treated in the manner described in Example 5 and analyzed (also in accordance with Example 5) on nine different occasions.

The results are set forth in the following Table: Fibrinogen Concentration in the Same Plasma at Different Time Occasions

Date Fibrinogen

2.938

2.835

2.785

2.96

2.874

2.859

2.764

2.727 2.924

Mean: 2.852 SD, %: 2.852

BSTITUTE Example 8*

Fibrinogen concentration in plasma and serum subsequent to adding fibrinogen, fibrinolysis products and hepa- rin.

The plasma was treated in the manner described in Example 5. Serum was treated by treating a part of said plasma with the snake venom enzyme batroxobin (final concentration 0,5 E/ml for 60 minutes at 37°C) at room temperature, whereafter the fibrin coagulum was removed. Plasma standard is a mixed plasma from several individuals, plasma B.J. is plasma from a single individual. Fragment Ds and heparin were added to plasma and these plasmas were compared with intact plasma. Fragment Ds and fibrinogen were added to serum.

These plasmas and serums were analyzed in the manner described in Example 5.

The results are set forth in the following Table:

* 100 μg/ml, **200 μg/ml *** 5IE/ml

SUBSTITUTE Example 9

Determining Fibronectin Concentration in Blood

Plasma was treated in the manner -described in Example 5. The anticoagulant used comprised 0.1 M EDTA. Fibrinogen was extracted from this plasma by coagula¬ tion with the snake venom enzyme batroxobin (final concentration 0,5 E/ml, 37°C, for 60 minutes). The fibrin coagulum was removed and the supernatant

(serum) , and also the non-coagulated part of the plasma were used for analysis subsequent to diluting or thinning with TNE-BSA-buffer (see Example 5) . The analysis of fibronectin was effected essentially in the same manner as that described in Example 2. Microtitre plates were treated with fibrinogen and the plates blocked and washed in the manner described in Example 2. 150 μl of diluted plasma 20 μl of calcium chloride and 5 μl of hirudin were then added. 25 μl of thrombin- activated Factor XIII (final concentrations between 0.001 and 0.1 E/ml) were then added and the plates incubated for one hour at 37°C, while shaking the plates slowly. The plates were washed with buffer solution (as in Example l) . 200 μl of HRP-labelled, primary fibronectin antibodies, were then added, where¬ after the plates were incubated for one hour at 37°C; 200 μl of peroxidase substrate were then added. In¬ cubation was continued for a further two minutes, whereafter the reaction was interrupted by adding 50 μl of 4 M H_S0₄. The relationship between absorbence and fibronectin concentration i serum and plasma is set forth in Figure 7.

SUBSTITUTE Example 10.

The Activity of Factor XIII After Different Reaction Times

Microtitre plates were treated with fibrinogen, in the manner described in Example 2. Blocking and washing of the plates was also effected in the manner described in Example 2. 150 μl of fibronectin solution (10.0 μg/ml), 20 μl of calcium chloride (0.2 M) and 5 μl of hirudin (2 ATU/ml) were then added. Finally, 25 μl of thrombin- activated Factor XIII (concentrations between 0.001 and 0.1 E/ml) were added and the plates incubated over different time periods, namely 1, 1.5 and 4 hours at 37°C, while slowly shaking the plates.

The plates were then washed with buffer solution, as in Example 1. Monoclonal antifibronectin IgG was then added. The analysis was then continued in the same manner as that described in Example 2. Figure 8 shows the relationship between color intensity and the acti¬ vity of Factor XIII in the sample solutions.

Example 11

The Total Activity of Factor XIII and the Spontaneous Activity in Normal Plasmas

Plasma from a number of individuals (12 persons) was treated in accordance with Example 5. The anticoagulant used consisted of 0.1 M EDTA. The plasmas were caused to coagulate by adding the snake venom enzyme batroxo- bin (0.5 E/ml in final concentration) over one hour at 37°. The coagulum was removed. These samples were analyzed with respect to the spontaneous activity of factor XIII in the manner described in Example 3. With

SUBSTITUTE the intention of determining the total activity of Factor XIII, the batroxobin-coagulated plasmas were diluted or thinned in the ratio of 1:10 with a TNE- buffer (.Example 1) and thereafter activated with thrombin (10 E/ml plasma) for from 10 to 60 minutes. The reaction was interrupted with hirudin, whereafter an analysis was carried out essentially in the manner described in Example 3.

The results obtained are set forth in the following Table:

Mean: 1.1 0.0051 S.D.: 0.25 0.0038

Example 12

The total activity of Factor XIII and fibrinogen con¬ centration prior to and subsequent to transfusion with plasma (from the father) on a girl having a Factor XIII deficiency.

Plasma from a girl suffering from a Factor XIII defi¬ ciency and from her father were treated in the manner described in Example 5. The anticoagulant used con¬ sisted of 0.1 M EDTA. The girl received transfusion with plasma from the father and samples were subsequently taken. The plasmas were caused to coagu¬ late and were activated in the same manner as that described in Example 11. The fibrinogen concentration was determined in the manner described in Example 5 and Factor XIII was determined in the manner described in Examples 3 and 11.

The results are set forth in the following Table:

Sample

Plasma standard

Pat. before transfusion

Pat.30 min after transfusion

Pat.24 hours after transfusion

Pat.l week after transfusion

Father's plasma

SUBSTITUTE

Claims

1. A method for quantitatively determining fibrinogen, fibronectin, o__-antiplasmin or a -transglutaminase, particularly Factor XIII c h a r a c t e r i z e d by carrying out a reaction process involving

- reacting fibrinogen with fibronectin or -anti¬ plasmin with transglutaminase acting as a catalyst; - using all of the reaction components in predeter¬ mined quantities, with the exception of the sought quantity; determining the sought quantity with the aid of a immunosorption techique in a known manner, par- ticularly by adsorbing the fibrinogen or fibronec¬ tin on a surface and then reacting said fibrinogen or fibronectin with said other reaction component and then with an antibody specific against the substance to be determined, which antibody may be labelled, whereafter the amount of said antibody is measured in a known manner, optionally by reaction with a labelled secondary antibody.

2. A method according to Claim 1 for determining the fibrinogen concentration, c h a r a c¬ t e r i z e d by

- adsorbing fibronectin on a surface;

- adding a sample solution containing fibrinogen to the surface on which the fibronectin is adsorbed; - adding calcium chloride and Factor Xllla to said surface for the purpose of incorporating the fibri¬ nogen;

- reacting the fibronectin-bound fibrinogen with a specific antibody against fibrinogen; - reacting the resultant body with a so-called secon¬ dary antibody with peroxidase-labelled anti-IgG;

SUBSTITUTE - visibilizing the amount of peroxidase bound to and quantitatively-proportional to the fibronectin- fibrinogen body, by adding a substrate for peroxi¬ dase., to produce a yellow colour; - measuring the intensity of the yellow colour; and calculating the amount of fibrinogen present from said measured intensity.

3. A method according to Claim 1 for determining fibronectin, c h a r a c t e r i z e d by

- adsorbing fibrinogen on a surface; adding a sample solution containing fibronectin to the surface on which the fibrinogen is adsorbed; adding calcium chloride and Factor Xllla to said surface for the purpose of incorporating the fibro¬ nectin;

- reacting the fibrinogen-bound fibronectin with an antibody which is specific against fibronectin; reacting the resultant body with a so-called secon- dary antibody with peroxidase labelled anti-IgG;

- visibilizing the amount of peroxidase bound to and quantity-wise proportional to the fibrinogen- fibronectin body, by adding a substrate for peroxi¬ dase, to form a colour; - measuring the intensity of the colour; and

- calculating the amount of fibronectin present on the basis of the calculated colour intensity.

4. A method according to Claim 1 for determining α_- antiplasmin, c h a r a c t e r i z e d by

- absorbing fibrinogen on a surface;

- adding a sample solution containing α₂-antiplasmin to the surface on which fibrinogen is absorbed; adding calcium chloride and Factor Xllla to said surface for the purpose of incorporating the α₂- antiplasmin;

SUBSTITUTE - reacting the fibrinogen-bound α₂-antiplasmin with a specific antibody against α₂-antiplasmin;

- reacting the resultant body with a so-called secon¬ dary antibody with peroxidase labelled anti-IgG; - visibilizing the amount of peroxidase bound to and quantity-wise proportional to the fibrinogen- Q- -antiplasmin body, by adding a substrate for peroxidase, to form a colour; measuring the intensity of the colour; and - calculating the amount of fibrinogen present from said measured colour intensity.

5. A method according to Claim 1 for determining the activity of Factor XIII, c h a r a c t e r i z e d by - adsorbing fibrinogen on a surface; adding an excess quantity of fibronectin, and calcium chloride;

- adding to the surface a sample solution containing Factor XIII, wherein fibronectin is incorporated in the fibrinogen on said surface; and visibilizing fibronectin with a specific antibody against fibronectin and a secondary antibody label¬ led with peroxidase.

6. A method according to Claim 1 for determining the activity of Factor XIII, c h a r a c t e r i z e d by

- adsorbing fibronectin on a surface; adding an excess quantity of fibrinogen, and calcium chloride; - adding to the surface a sample solution containing Factor XIII, wherein fibrinogen is incorporated in the fibronectin on said surface; and visibilizing fibrinogen with a specific antibody against fibrinogen and a secondary antibody label- led with peroxidase.

SUBSTITUTE

7. A method according to Claim 5 or 6 for determining the total activity of Factor XIII, c h a r a c ¬ t e r i z e d by first removing fibrinogen from the sample solution by coagulation with a snake venom enzyme, such as batroxobin; and by converting Factor

XIII to an activated form by adding an enzyme, such as thrombin.

8. A method according to any one of Claims 1-7, c h a r a c t e r i z e d by using a specific, label¬ led antibody, particularly an antibody labelled with "horse-radish-peroxidase", instead of using a specific antibody and a secondary antibody labelled with peroxi¬ dase.

SUBSTITUTE