JPH05508472A - Quantitative measurement method for fibrinogen, fibronectin, α2-antiplasmin or transglutaminase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Fibrinogen, fibronectin, α2-antiplasmin or transglucose Quantitative measurement method for Taminase The present invention uses fibrinogen, fibronectin, α! - anti-blasmin or tiger This invention relates to a method for quantitatively measuring the concentration of glutaminase. Known important tigers Sglutaminase is an XlIr factor.

Fibrinogen, fibronectin, α,-antiblasmin and X[I[factor are all important blood components, and measurement of the concentrations of these components in blood and tissue is important. It is very important to be able to Factor XlIr is a blood transaminase Its main role is to bind protein chains within the fibrin network after it is formed. It has been found that cross-linking is the key. As a result, some of the fibrin Cross-linking occurs between the γ-chains and also partly between the α-chains. XI [factor I is It also catalyzes the specific uptake of certain plasma proteins into the fibrin network. do.

For example, fibronectin and α2-antiplasmin are catalyzed by X[rI. binds to fibrin; in both cases the amino groups in fibrin act as donors fulfills the functions of

Blood levels of fibrinogen are also important. This is because fibrinogen is insufficient. This can lead to bleeding, while high concentrations can lead to cardiovascular disease. be.

Fibronectin is apparently an essential protein for cell proliferation and wound healing. fib Ronectin concentrations also decrease with infectious conditions such as sepsis. α, − anti-plus Min is the most important inhibitor of blood fibrinolysis phenomena. This inhibition occurs in thrombotic conditions. An increased concentration or increased uptake of the harmful agent into the fibrin clot has been observed.

Therefore, it is possible to measure the concentrations of these components or substances in blood and tissue. Very important. There are several methods for measuring blood fibrinogen concentration. These are too time consuming and/or not specific. fibronecti A functional protein that can quantitatively measure the concentrations of α2-antiplasmin, α2-antiplasmin and factor XIII. There is currently no way.

B., Blombaeck, R., Procyk, L., and Adamson. B.

He5sel (Thrombosis Re5earch 37.613- 628°1985) and R, Procyk, L, Adamson, M, Blo. ck and B, Blombaeck (Thrombosis Re5ea rch 40, 833-852.1985), especially the latter paper It was revealed that fibrinogen and fibronectin react in the presence of factor Ia. I'm doing it. This reaction occurs in a mixed phase, so the reactants can be measured quantitatively. I can't think of a good way to use this reaction for analysis.

Also, Biol, Chem, Hoppe 5eyler 1987. Ju ne:368 (6):669-74. J, Biol, Chem, 19 88 July:25+263(21)+10464-9;Blood 198 6 July: 68 (1): 95-101 and Biochia+B iophysActa 1988 November 1 7: 967 (2 ): 304-13, in the presence of fibronectin, on a polystyrene spherical surface, Thrombin-activated xru factor (XII [factor a) binds fibrin to the sphere However, fibronetatin is produced by the catalytic action of factor X1lla. It has been shown that it can cross-link to cellular sites on Trix. Also, the xm factor is Allowing the fibrin to make its own cross-links or to make several substrate cross-links I also know that. Factor X1ll also stimulates fibrinogen and fibronectin. catalytic activity in a solution containing hybrid oligomers and fibrinogen. Two types of cross-linked polymers called oligomers are made.

Surprisingly, these reactions (reactions in solution), which are basically completely mixed, can be used. In fact, fibrinogen, fibronectin, α2-antiplasmin and It is now possible to enable the quantification of transglutaminase. anti Surprisingly, the reaction rate increases considerably when the reaction is carried out as a surface-bound reaction. , and at the same time the concentration of reactants was found to decrease significantly. In addition, surface-binding reactions was also found to be highly specific.

Furthermore, fragments of fibrinogen molecules do not perturb surface-binding reactions. I found out that this is also surprising. Conventionally known fibrino In the quantitative analysis of fibrinogen, fragments of fibrinogen molecules have a disruptive effect on the quantitative analysis. effect. Using the method according to the invention, crosslinking is possible in fragments such as D3. No such disturbance is seen, even though there are some areas where it is possible to do so.

Heparin may also have a disruptive effect on some known assay methods. However, the book This does not happen when the method according to the invention is implemented.

Fibrinogen, fibronectin, α2-antiplasmin or transglucose Taminases, especially factor X It is now known that it can be functionally quantified in a simple but highly accurate manner. D - Fibrinogen is converted to fibronectin using transglutaminase as a catalyst. or α2-antiplasmin; -Use a predetermined amount of the reaction component, apart from the amount you are trying to determine. ru: and Quantification of the amount of interest can be determined using known immunoabsorption methods, especially using fibrinose on the surface. This is done using a method that absorbs gen or fibronectin. In other words, these frames Reacting fibrinogen or fibronectin with other reaction components and then quantifying The target substance is reacted with a specific labeled antibody, and then the amount of labeled antibody is known. Measure using the following method.

In living organisms, several reactions depend on enzymes with transglutaminase activity. It is known that reactions occur. These enzymes are involved in intracellular body fluids, plasma and lymph fluids. It is present in all tissues and body fluids. These enzymes have different specificities or ing. However, there are common characteristics among these reactions that make their chemical reactions As shown in the figure below, the reaction mechanism is that the glutamine amino acid residue ( Covalent bond to lysine amino acid residue (donor) H in other polypeptide chains (acceptor) and other polypeptide chains The components that form the In this way, covalent bonds between protein chains are obtained. Danzil cadaverine, butres Many components with amino groups, such as syn, act as donors in this reaction. Ku.

Glutamine is required as an acceptor group. The specificity of various enzymes is due in part to polypeptide Amino acid sequence near the glutamic acid residue in the tide chain, partially the structure of the amino group residue is determined as a condition. The reaction progresses faster when the reacting groups in the protein are juxtaposed. be done.

The present invention will now be explained in more detail with reference to the accompanying figures.

Figure 1 is a diagram showing the progress of the reaction that occurs when fibrinogen is measured or quantified. Figure 2 shows the correlation between color intensity and fibrinogen concentration. is a diagram; Figure 3 shows the phase relationship between color intensity and fibronectin concentration as well as XI[I Diagram showing the influence of acetamide on the reaction with; Figure 4 is a diagram showing the correlation between color intensity and X1rlA factor activity. ; FIG. 5 is a diagram showing the correlation between color intensity and Xrl[a factor activity.

Figure 6 illustrates the correlation between absorbance and plasma fibrinogen concentration: Figure 7 illustrates the correlation between absorbance and plasma and serum fibronectin concentrations: FIG. 8 illustrates the activity of Xl[factor I at various times after the reaction.

Quantitative measurement of fibrinogen concentration When performing the analysis, fibronectin is adsorbed onto the surface in a well-known manner (Figure 1 (see a). This surface consists of a plastic material such as polystyrene. this eye A well-suited material is, for example, “Titer-tecplattor” latte. It is commercially available as a spherical surface. Then, add the sample solution containing fibrinogen. Add (Figure 1b). Calcium chloride solution and HI [factor, e.g. thrombin] Add in active form, such as active form. Thrombin-activity inhibitors, such as hirudin thrombin used in excess during the activation process or may be generated in the sample. Not added to inhibit thrombin. In this way, X[I[am child (activation factor xrrr) is involved in the uptake of fibrinogen into surface-bound fibronectin. (Figure 1c). Fibronectin-bound fibrinogen is then Antibodies specific for fibrinogen (e.g. from goats), so-called anti-fibrinogen The antibody becomes reactive with fibrinogen (Figure 1d), and the antibody binds to fibrinogen on the surface. (Fig. 1e). The resulting fibronectin-fibrinogen-anti Fibrinogen was labeled with veloxidase (Figure If), a so-called secondary antibody ( For example, rabbit-anti-goat-1gG), the enzyme then reacts with (Fig. 1g) (H, RP = “Horserradish peroxidase”). Fibrinogen-binding peroxidase (HRP) cleaves or cleaves the substrate, producing a yellowish color.

Partial steps of the process, including the use of enzyme-labeled antibodies and their visualization, are immunosorbent and It is well known.

Quantitative measurement of fibronectin concentration Fibrinogen, which does not contain any fibronectin and xm factor, is adsorption onto similar types of surfaces as mentioned in connection with the quantitative determination of gen. centre The fibronectin-containing solution was mixed with calcium chloride, thrombin-activated X[r[factor, It is applied to the surface along with hirudin, which neutralizes excess thrombin. this place In this case, factor X1lla is determined as described for quantitative fibrinogen measurement. sea urchin, catalyzes the incorporation of fibronectin into surface-bound fibrinogen . Fibrinogen-bound fibronectin then acts on fibronectin. It reacts with specific antibodies (e.g. from goats) and then fib A peroxidase similar to that mentioned in connection with the quantitative measurement of linogen. A reaction with the labeled secondary antibody occurs. Fibronectin-containing sample solution or fibrils When it also contains nogen, snake venom enzymes such as batroxobin are used to remove this fibrinoid. Coagulate and remove the gen. There is a possibility that factor XI [I] is present in the sample solution. Therefore, it is necessary to prevent thrombin from acting on this coagulation process. child In some cases, the enzyme is activated to precipitate the fibronectin in the sample solution. Let it be taken inside. If effectively clotted with batroxobin, this procedure There is no need to create.

Quantitative measurement of α2-antiplasmin Fibrinose was measured in the same manner as described for the quantitative measurement of fibronectin. Gen becomes adsorbed on the surface. α, - Apply a solution containing anti-blasmin to the surface. followed by calcium chloride, thrombin-activated XUt factor, and excess Add hirudin to neutralize thrombin. After the reaction time is over, Brinogen-binding α,-antiplasmin is combined with fibrinogen and fibronectin. In the same way as described for the quantitative measurement of α2-antiplasmin and Visualize with a peroxidase-labeled secondary antibody. As in the case of plasma When the sample solution contains fibrinogen and fibronectin, fibrinogen It is necessary to remove the fibronectin by coagulating it with snake venom, and the fibronectin is coupled to a suitable polymer matrix such as 5epharos. The well-known method of adsorbing fibronectin and xm factor onto gelatin Quantification of fibronectin and α,-anti-blasmin, free of fibrinogen Adsorb to the surface in the same manner as described for the target concubine. Then excessive fib Add ronectin along with calcium chloride and hirudin. Then, X[rl Add sample solution containing factors. If active factor XI is present in the sample, fibrous Nectin will be incorporated onto the surface of fibrinogen and fibronectin As mentioned above, specific antibodies against fibronectin and quantitative measurement of fibronectin The sea urchin can be visualized with a secondary antibody labeled with a peroxidase such as HRP. φ. Full activity of factor XlEr removes fibrinogen by coagulation be quantified by first treating the sample solution with batroxobin to Can be done. Thrombin is then added to convert the xtrt factor into its active form.

Fibrinogen, fibronectin, α,-antiblasmin and XlEr factor The aspects described in connection with the quantitative measurement of The situation was as follows. However, recently specific antibodies and labeled antibodies, especially Instead of using an antibody and a peroxidase-labeled secondary antibody, It was found that it is possible to use antibodies labeled with "oxidase". Currently, fibrinogen, fibronectin, C2-antiplasmin concentration Recommended embodiment for quantitative measurement of factor and xm factor activity. In this way, now When measuring fibrinogen concentration, the Instead of an antibody, the product reacts with a so-called secondary antibody. against fibrinogen labeled with “horseradish peroxidase” (HPR) Specific antibodies, so-called anti-fibrinogen, ie anti-fibrinogen antibodies are used.

Correspondingly, for quantitative determination of fibronectin concentration, labeled fibronectin α2-antiplasmin antibody was used, whereas when measuring the concentration of α2-antiplasmin, a labeled α2-antibody was used. Plasmin antibodies are used. Thus, in the case of measuring the activity of factor XlEr, A primary labeled anti-fibronectin antibody is used. -The next antibody should be HP if possible. R-labeled antibodies are preferred.

In the following we will try to describe the invention in more detail by giving some examples.

Example 1 Quantitative measurement of fibrinogen Fibronectin solution (1011g/ml) 200μl microtiter The sample was injected into a well plate (“Titer-tec”). plate at room temperature I left it overnight. Then, empty the valve hole and add THE-BSA-buffer (0.05M-). Lith-〇, 10M NaCl-1a + M EDTA, pH 7.4, 0.1% The cells were washed repeatedly with calf serum albumin (containing calf serum albumin). Then fill it with fibronectin. Calf albumen solution (3 0 mg/ml) into the valve hole. After washing with THE-BSA buffer, the fibrils were Add 150 μl of sample solution containing Nogen (between 0.15 and 20 Mg/ml). , then 2oμl of CaC1! solution (0,2M) and 5μm (7)l Nirudine (Ioo.

Add ATU/ml) (ATU=antithrombin units).

Finally, 25 μM thrombin activating X1ll factor (3.2 units/ml) was added. The final concentration was 0.4E/ml.

Plates were gently shaken for 1.5 hours at 37°C. Then buffer the plate Wash with solution and add goat anti-fibrinogen-1gG 200μm at an appropriate concentration. I got it. After incubating for 18 hours at room temperature, the plates were washed with buffer and 200 μl of rabbit-antigen-Igo labeled with oxidase was added. at room temperature After incubation for 2 hours, 100 μl of peroxidase substrate was added.

This mixture was incubated for an additional 1.5 minutes, then IM HzSOt The reaction was stopped by adding 100 μl of solution. Color intensity was read at 490 nm. Sucking Figure 2 shows the correlation between the yield and the fibrinogen concentration in the solution.

The experiment was repeated by adding iodoacetamide to the solution to 0.5 mM. This attachment The addition was performed before the addition of factor XlEr.

Iodoacetamide inhibits XffI factor. The coloration seen in this experiment is distinguishable. It was impossible. This experiment shows that uptake depends on the presence of factor X[iI. won.

Example 2 Measuring fibronectin concentration Fibrinogen (10 Mg/ n11) 200 μm was injected into the valve hole of the microtiter plate. plate It was left at room temperature overnight. Wash the surface as described for Example 1 and apply the calf serum solution. The valve hole was filled with rubumin. After washing with buffer (as in Example 1), the fibrous Add 150 μl of a sample solution containing nectin (0.02-2.5 μm/m), and then 20 μl of calcium chloride solution (0.2M) and 5 μl of hirudin (lO0 0 AT U/ml) was added. Finally, 25 μl thrombin-activated XlE Add r factor (3.2 units/ml) to give a final concentration of 0.4 units/ml. did.

The plates were gently shaken for 2 hours at 37°C. Then wash the plate with buffer. As in Example 1), goat anti-fibronectin-IgG was added. room After 18 hours of incubation at room temperature, the valve hole was rinsed with buffer and the peroxidizer was removed. 200 μl of rabbit-antigen-Igo labeled with enzyme was added. Incubate at 37℃ for 2 hours. After incubation, 100ul of peroxidase substrate was added. after that, The same method as described in connection with Example 1 was applied.

Figure 3 shows the correlation between color intensity and fibronectin concentration in the sample.

Similar to Example 1 above, an experiment was conducted in which iodide acetamide was added, but no color was developed. I couldn't see it. Therefore, this reaction also depends on the presence of factor XII (Fig. 3).

Example 3 Measurement of total activity of factor X1rl using peroxidase-labeled secondary antibody fixed Microtiter plates were prepared with fibrinogen in the same manner as described in Example 2. Processed with. The plates were also blocked and washed as in Example 2. Then 150 μl of fibronectin solution (2,5 μg/mD, 20 μm calcium chloride ( 0.2 M) and 5 μm Hirudin (1000 A T U/+ni) were added.

Finally, add 25 μl of trompon-activated X1rl factor (concentrations 0.001 and 0.1 E/ml) and incubate the plate at 37°C for 2 hours with gentle shaking. Incubated. The plates were then washed with buffer (according to Example 1). One Goat anti-fibronectin IgG was then added. Then use the method described in Example 2 to Continued the process of measuring activity. Figure 4 shows the color intensity and the Xrf [factor activity] in the sample solution. This diagram illustrates the correlation between gender. The background color is the absence of factor XIII. When using biotinylated fibrinogen produced in measurement Microtiter plates were treated with fibrinogen according to Example 2. In example 2 Plates were blocked and washed as described. Then add 150 μl of fibronectin solution (2,5 μg/ml), 20 μl calcium chloride and 5 μl Hirudin (100 OATU/ml) was added. Finally, thrombin Add activated Xlr [factor (concentration of 0,OOl to 0.1 E/ml) The cells were incubated for 2.5 hours with gentle shaking.

Then, wash the plate with buffer (as in Example 1), then add 200 μI of biotin. Add tinylated goat anti-fibronectin-1gG (1g/ml) and plate was incubated for 18 hours at room temperature. Next, wash the plate with buffer solution.Example 1 ), then add 200 μl of avidin-HPR (0.5 ttg/ml). peroxidase substrate was added according to the method described in Example 2. In Figure 5 The correlation between color intensity and X1tl factor activity in the sample solution is shown.

Example 5 Measurement of blood fibrinogen concentration When measuring blood fibrinogen concentration, an anticoagulant, such as 3.8% citric acid, Blood was injected into a container containing trisodium acid or 0.1 MEDTA. Normal anti The ratio of coagulant is 1 part blood to 9 parts. Centrifuge the blood at room temperature for 30 minutes at 150 °C. Plasma was removed by aspiration (0 rpm'). Plasma was TNE-BSA-buffered before analysis. solution (1:1000°, 1:2000, etc.) (see Example 1).

Analyze the microtiter plate with fibronectic tissue as described in side 1. Processed with Plates were blocked and washed as in Example 1. Then various concentrations 150 μl of diluted plasma and 11 calcium chloride and hirudin were added. the Add another 25 μl of thrombin-activating x11 factor (final concentration IE/ml). The plate was then incubated at 37°C for 1 hour with gentle shaking.

Wash the plate with buffer as described in Example 1, then 200 μl of primary HRP- Labeled fibrinogen-antibody was added. After incubating for 1 hour at 37°C , add 200 μl of peroxidase substrate and incubate the mixture for an additional 2 minutes. It was incubated. Then 50 μ [4. The reaction was stopped by adding M H85O. . The color intensity was read at 492 nm. Absorbance and plasma fibrinogen concentration Correlation with 5! The I section can be known from FIG.

Example 6 Fibrinogen concentration measurement according to the invention and known functional fibrinogen quantification We compared two methods for

Plasma collected from 23 healthy subjects was treated as described in Example 5 and treated with fibrinogen. The content was determined by the method described in Example 5 and also by two known methods, the so-called eer The so-called plug precipitation method according to Gstrom and the "clot rate" method according to Vermylen. Measurements were also carried out using the "degree" method.

The results are shown in the following table: Plasma fibrinogen concentration was measured using three different functional fibrinogen quantification methods. I measured it.

Crosslinking method’ 2.89 0.444 Plug precipitation method (Bergstrom) 3.08 0.381 Clot velocity method (V ermylen) 2.76 0.537 zero Method according to the invention Example 7 Analysis of fibrinogen concentration in the same plasma at different time points Plasma from one subject was processed as described in Example 5 and analyzed at nine different time points. (or according to f!li5). The results are shown in the following table: Fibrinogen concentration at different time points in the same plasma 2811-902,938 2911-902,835 51 December - 902,785 1712-902,96 141-912,859 151-912,764 81-912,727 17i month - 912,924 Average: 2.852 Standard deviation SD, %: 2.852 fibrinogen-enriched plasma and serum after addition of the method described in Example 5. Processed with. By partially treating the above plasma with the snake venom enzyme batroxobin, Treat the serum at 37°C for 60 min (final concentration 0.5E/ml), then filtrate. The brine clot was removed. Standard plasma is a mixture of plasma from several subjects; Plasma B, J, are plasma from a single subject. Fragment D, and heparin in plasma These plasmas were compared with the original plasma.

Fragment D3 and fibrinogen were added to the serum.

These plasma and serum were analyzed according to Example 5.

The results are shown in the following table: Standard plasma 2.69 Serum < 0.03 Serum + 2mg fibrinogen/ml 1.95 Serum + Fragment DS” 0 ．． 03 Standard plasma + Fragment DS” 2.66 Plasma B, J, 2.88 Plasma B, J, + heparin 2.85 bottles 100 μg/ml zero 20 0 μg/mL 0*ne 5 [E/m 1 case 9 Measurement of blood fibronectin concentration Plasma was processed as described in Example 5. The anticoagulant used contained 0.1M EDTA. nothing. Fibrinogen is extracted from this plasma by clotting with the snake venom enzyme batroxobin. The fibrils were extracted (37°C, 60 minutes, final concentration 0.5 E/ml). The supernatant (serum) and non-clotted portion of the plasma were removed with TNE-BSA-buffer. It was diluted and then subjected to analysis (see Example 5). The analysis of fibronectin is described in Example 2. It was carried out in essentially the same way as before. Fibrino microtiter plate The plates were blocked and washed as in Example 2. unintentionally 150 μl diluted plasma, 20 μl calcium chloride and 5 μl hirudin. was added. An additional 25 μl of thrombin-activated factor XIII (final concentration 0. 001 and 0.1E/ml) and shake the plate gently. Incubate for 'h at 7°C. Wash plate with buffer (as in Example 1). Next, 200 μm of HRP-labeled, -next fibronectin antibody was added, and then Plates were incubated for 1 hour at 37°C; then peroxidase 200 μm of quality was added. After incubating for another 2 minutes, the 4 MHz The reaction was stopped by adding 50 μl of SO4. Absorbance and fibrone in serum and plasma Figure 7 shows the cutin concentration.

Example 10 Activity of factor X1ll after various reaction times. The rate was treated with fibrinogen. Block and wash the plate using the method in Example 2. It was carried out in Next, 150 μl of fibronectin solution (1,0,0 Mg/m l), 20 μm calcium chloride (0.2 M) and 5 μl hirudin (2 ATU/ml) was added. Finally, add 25 μl of thrombin-activated X11 factor. (at a concentration between 0,001 and 0.1 E/ml) and shake the plate gently. Incubate at 37°C for three different times: 1 hour, 1.5 hours, and 4 hours. I bet.

The plates were washed with buffer as in Example 1.

Monoclonal anti-fibronectin IgG was then added. Then analyze as an example This was carried out using the method described in 2. Figure 8 shows the phase between color intensity and XIII activity in the sample solution. Indicates a relationship.

Example II Total activity of xrr+ factor and natural activity in normal plasma collected from a certain number of subjects (12 people) The collected plasma was processed according to Example 5. The anticoagulant used was 0°IM containing EDTA. nothing. The snake venom enzyme batroxobin (final concentration o, 5 E/ml) was added to the plasma. Allowed to solidify for 1 hour at 37°C. The coagulum was then removed. These samples Factor X was analyzed for natural activity using the method described in Example 3. of factor XIII For the purpose of measuring total activity, batroxobin-clotted plasma was diluted 1=1 with TNE-a buffer. After diluting thrombin (IOE/ml plasma) for 10 to 60 minutes, ) was activated. Essentially the same as described in Example 3 after stopping the reaction with hirudin. The analysis was carried out using the method.

The results obtained are shown in the following table: FXIII FXIr [ B, J, (standard) 1.00 0.0040B, B, 1.38 0.0050 , S, 1.46 0.0030 To, A, 0.83 0.0060 M, G, 0.96 0.0040 i, A, 1.13 0.0000 W, G, 0.74 0.0020 B 1.14 0.0050 M 1.56 0.0095 K　O,830,0090 B, U, 1.39 0.0030 [J, B, 1.13 0.014 Or, F, 0.75 0.0016 Average: 1.1 0.0051 Standard deviation S, D: 0125 0.0038 Example 12 )’Xr1 before and after plasma (from the father) infusion for girls with rfl factor deficiency Total activity of factor 1 and fibrinogen levels A girl with factor XIII deficiency and her father Plasma collected from parents was processed as described in Example 5. The anticoagulant used was 0゜1M. Contains EDTA. The sample was collected after the girl received an infusion of plasma from her father. Ta. Plasma was coagulated and activated as described in Example 11. The method described in Example 5 Fibrinogen concentration was measured and factor XIII was determined as described in Examples 3 and 11. Ta.

The results are shown in the following table: Standard plasma 1.000 2.96 Patient plasma before transfusion < 0.003 4.21 Patient plasma 30 minutes after transfusion 0.1 05 4.21 Patient plasma 24 hours after transfusion 0.050 3.46 Weeks after transfusion Patient plasma: 0.032 3.74 Father's plasma: 1.2741.51 Engraving (no changes to the content) FIG.1 This figure illustrates the progress of the reaction that occurs after measuring fibrinogen concentration. ,2 Correlation between color intensity and fibrinogen concentration FIG. 3 Fibronectin μg/ml FIG.4 Correlation between color intensity and factor activity of Xla FXIIla, mtJ/m+ FIG.5 Correlation between color intensity and factor activity of XI FXIIIa, mU/m1 FIG.6 Fibrinogen versus ω during normal face inhibition Fibrinogen, M6/ML Inhalation 1 degree/MIN L+92NM FIG.8 ×■ factor activity after different reaction times FXIII, MU/ML Engraving (no changes to the content) Summary book XII [fibrinogen is catalyzed by factor by a reaction process involving reaction with nectin or α2-antiplasmin, Fibrinogen, fibronectin, α,-antiblasmin and factor X1ll All reaction component quantities, with the exception of billable quantities, should be quantitatively determined. The determination of the claimable amount determined by Fibrinogen or fibronectin is adsorbed onto the surface, and these reaction components, and then reacted with an antibody specific to the substance to be measured. Since the two or two antibodies that will be used will probably be labeled, the amount of this antibody will be determined using a known method. , optionally reacted with a labeled secondary antibody.

Procedural amendment (Mutsu) 1.Display of the incident Fibrinogen, fibronectin, α2-antiplasmin or transglucose Quantitative method for Taminase Brombach, Pilgel (1 other person) 4. Agent 6. Number of claims that are overshadowed by the positive 7-Object of amendment Description, claims, and abstract translation □--4 8- Contents of amendment as attached Translation of the description, claims, and abstract (no change in content) Procedural amendment form hmm 1.Display of the incident 2. Name of the invention Full name (name) Blombach, Birgel (1 other person) 4. Agent 6. Number of words that make tW more than 111'XE4 7, subject of correction translation of the drawing international search report ,Gang,-E A,,1-hPro/SE 9110031B International Investigation Report PCT/SE　91100318

Claims (8)

[Claims] 1. fibrinogen, which is characterized by carrying out the following reaction process: fibronectin, α2-antiplasmin or transglutaminase, especially X Method for quantitatively measuring factor III: -Transglutaminase acts as a catalyst to convert fibrinogen into fibrous React with nectin or α2-antiplasmin; - using predetermined amounts of all reaction components except for the required amount; - the amount sought can be determined by known methods using immunoabsorption methods, in particular fibrinogen or Fibronectin is adsorbed onto the surface and then these fibrinogen or fibrinogen React the fibronectin with the other reaction components listed above, and then react with the substrate to be measured. It was measured by reacting with a specific antibody. This antibody is probably the target. After that, the amount of the above-mentioned antibody was labeled using a known method. Optionally measured by reacting with a secondary antibody. 2. The method of claim 1 for measuring fibrinogen concentration, characterized in that: : - adsorb fibronectin to the surface; - the surface to which fibronectin is adsorbed; Add sample solution containing fibrinogen to the surface; - calcium chloride and XII on the surface for the purpose of fibrinogen uptake; Add factor Ia; - fibronectin-bound fibrinogen to fibrinogen react with an antibody specific for the gene; - the conjugate thus obtained is reacted with a peroxidase -React with a so-called secondary antibody having a labeled anti-IgG; -Fibronec peroxidant that binds to the tin-fibrinogen conjugate and is quantitatively proportional to it. The amount of peroxidase can be visualized by adding a peroxidase substrate to develop a yellow color. awaken; - measure the intensity of the yellow color; and from the intensity of the color measured as above Calculate the amount of linogen. 3. The method of claim 1 for measuring fibronectin, characterized in that: - adsorb fibrinogen on the surface; - surface adsorbed with fibrinogen Add a sample solution containing fibronectin to; - calcium chloride and XII on the surface for the purpose of fibronectin uptake; Add factor Ia; - fibrinogen-bound fibronectin to fibronectin - React with a peroxidase-labeled antibody; react with a so-called secondary antibody with IgG; - a group for peroxidase; The fibrinogen-fibronectin bond is developed by adding a Visualize the amount of peroxidase bound to the body and proportional to its amount; - measure the intensity of the color; and - Calculate the amount of fibronectin present based on the calculated color intensity. 4. The method of claim 1 for measuring α2-antiplasmin, characterized in that: - adsorb fibrinogen on the surface; - adsorb fibrinogen on the surface; Add a sample solution containing α2-antiplasmin to the surface; - Calcium chloride and XI on the above surface for the purpose of incorporating α2-antiplasmin. Add factor IIa; -fibrinogen-bound α2-antiplasmin to α2-antiplasmin react with a specific antibody against plasmin; - the resulting conjugate is reacted with a peroxidase - react with a so-called secondary antibody having an anti-IgG labeled with a peroxidase. Fibrinogen-α2-anti- Visualize the amount of peroxidase that binds to the plasmin complex and is quantitatively proportional to it. to become; - measure the intensity of the color; and - Calculate the amount of fibrinogen present from the color intensity measured as described above. 5. The method of claim 1 for measuring the activity of factor XIII, characterized in that: - Adsorption of fibrinogen to the surface; - Excess amount of fibronectin and potassium chloride. Add lucium; - Add a sample solution containing factor XIII to the surface, where fibronectin is incorporated into fibrinogen on the surface; and -Specific antibody against fibronectin and secondary antibody labeled with peroxidase Visualize fibronectin with. 6. The method of claim 1 for measuring the activity of factor XIII, characterized in that: - Adsorbs fibronectin to the surface; - Excess amount of fibrinogen and carbon chloride Add lucium; - Add a sample solution containing factor XIII to the surface, where fibrinogen is incorporated into fibronectin on the surface; and -Specific antibody against fibrinogen and secondary antibody labeled with peroxidase Visualize fibrinogen. 7. First, fibrinol is removed by coagulation with snake venom enzymes such as batroxobin. Gen is removed from the sample solution and then an enzyme such as thrombin is added to remove factor XIII. 7. A method according to claim 5 or 6, characterized in that the offspring are converted into an active form. 8. Instead of using specific antibodies and peroxidase-labeled secondary antibodies, specific, labeled antibodies, especially antibodies labeled with “horseradish peroxidase” 8. A method according to any one of claims 1 to 7, characterized in that it is used.