WO2005038045A1

WO2005038045A1 - Method and kit for measuring the activity of factor xiii

Info

Publication number: WO2005038045A1
Application number: PCT/KR2003/002174
Authority: WO
Inventors: Seong Soo Alexander An; In Bum Suh; Kun Taek Lim; Hyun Jung Oh
Original assignee: Peoplebio, Inc.
Priority date: 2003-10-17
Filing date: 2003-10-17
Publication date: 2005-04-28
Also published as: AU2003269564A1

Abstract

The present invention relates to a method and a kit for measuring the activity of Factor XIII, more particularly a method and a kit for measuring the activity of Factor XIII in a sample so that Factor XIII may be quantitated in more reliable and convenient manner.

Description

METHOD AND KIT FOR MEASURING THE ACTIVITY OF FACTOR XIII

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to a method and a kit for measuring the activity of Factor XIII, more particularly a method and a kit for measuring the activity of Factor XIII in a sample so that Factor XIII may be quantitated in more reliable and convenient manner.

DESCRIPTION OF THE RELATED ART Some plasma factors such as Factor XIII, TAFI, fibronectin, and vitronectin help to stabilize the clot. Factor XIII and TAFI are enzymes, and fibronectin and vitronectin provides anchoring points and bridges with many other plasma proteins and cellular receptors. In plasma, Factor XIII exists as a tetramer (Mr=320, 000) , which two pairs of subdomain (A2B2) are held together by noncovalent bonds (Freyssinet J , et al . , Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XHIa, Biochem J. 169 (2) :403-10 (1978) ) . Factor XIII is the only non-proteolytic enzyme of the coagulation cascade. Thrombin activates the zymogen to the active form by removing about 4000 MW fragment from the NH₂- terminals of the both A chains. The full activity of FXIIIa can be gained by addition of CaCl₂, which binds to the B chain dimmer, and fibrinogen. The catalytic activity is located in the A subunits. A subunit contains 6 free sulfhydryl groups one of which is the active site (Chung SI, et al . , Relationships of the catalytic properties of human plasma and platelet transglutaminases (activated blood coagulation factor XIII) to their subunit structures. J Biol Chem. 10 :249 (3) :940-50 (1974) ) . Activated Factor XIII (Factor Xllla) stabilizes the polymerized fibrin clot by cross-linking fibrin monomers between Lys and Gin residues of a- and g-chains of fibrin, especially in D domain in the final stage of the coagulation cascade to form homopolymers . Factor XIII increases the stability, insolubility, elasticity and resistance against lysis of the clot. Factor XIII plays more roles, not only cross-linking fibrin. Factor Xllla crosslinks many substrates in vivo and in vitro, including fibrin, factor V, 2-antiplasmin, platelets, other connecting proteins and connective tissue. For example, Factor Xllla also catalyzes the cross-linking of the δ-chain of fibrin to α₂-plasmin inhibitor and fibronectin, as well as the cross- linking of collagen and fibronectin, which may be related to wound healing (Sakata and Aoki, J. Clin . Invest . 65:290- 297(1980); Mosher, J^". Biol . Chem, 250:6614-6621(1975); and Mosher and Chad, J. Clin . Invest . 64:781-787(1979)). The covalent incorporation of ₂-plasmin inhibitor into the fibrin network may increase the resistance of the clot to lysis (Lorand et al . , ibid.). Factor XIII belongs to a family of transglutaminase . Among the family, only plasma Factor XIII exists as zymogen in vivo (Bannerjee D, et al . , Molecular structure of plasma transglutaminase precursor. Ann N Y Acad Sci . 8:202:172- 85(1972)). Factor XIII is found in many places, plasma, platelets, megakaryocytes, monocytes, placenta, uterus, liver and prostrate tissues. Plasma factor XIII is synthesized in the liver. Mutations in Factor XIII could cause severe bleeding disorders. The Val34Leu mutation has been shown to have some protection against myocardial infarction and venus thromboebolism, on the other hand, the mutation could lead to the intracerebral hemorrhages and higher specific activity. Other polymorphism displayed its association with recurrent miscarriage and impaired wound healing. The recombinant Factor XIII has been shown to improve against unexplained bleedings. The functional levels of Factor Xllla tended to be lower in patients with tumors, liver disease, severe burns or after surgery. PMN-elastase seems to inactivate Factor XIII by enzymatic cleavage, especially at the site of inflammation. For diagnostic and clinical studies, a variety of assays have been developed for measuring Factor Xllla. For example, U.S. Pat. No. 4,601,977 discloses a method for determining blood coagulation Factor XIII in plasma by measuring the activity of its activated form Xllla by using casein and a fluorescent cadaverine derivative as a substrate. U.S.Pat. No. 5,015,588 suggests a method for quantitating Factor XIII in plasma, which comprises the steps of (a) desensitizing or removing intrinsic fibrinogen from the plasma sample to for a defibrinated plasma sample; (b) incubating the defibrinated plasma sample with thrombin and Ca²⁺ to activate Factor XIII to Factor Xllla; (c) incubating the activated plasma sample with a gluta ine substrate and a primary amine derivative of biotin to form a biotinylated reaction product; and (d) quantitating the biotinylated reaction product. In addition, U.S. Pat. No. 5,204,240 teaches a method for the determination of Factor XIII in which the sample is activated with thrombin in the presence of a fibrin- aggregation inhibitor and incubated with a substrate for

Factor XIII, with a primary amine and with a buffer substance in solution, and the ammonia which is formed is determined using NADH, glutamic dehydrogenase and ketoglutarate. U.S.Pat. No. 5,508,202 discloses a method of determining Factor XIII activity which comprises (a) mixing a sample and a fibrin precipitation inhibitor; (b) adding a thrombin solution; (c) determining the fibrin coagulation time in the presence of calcium ion; and (d) comparing the coagulation time with the normal coagulation time to determine Factor XIII activity in the sample.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION The present inventors have made intensive researches t^'o develop novel method for quantitating Factor XIII in a sample in more reliable and convenient manner, and as a result found that Factor XIII could be accurately quantitated by using as substrate and assaying system casein and fibrinogen-HRP (horse radish peroxidase) conjugate. Accordingly, it is an object of this invention to provide a method for measuring the activity of Factor XIII in a sample. It is another object of this invention to provide a kit for measuring the activity of Factor XIII in a sample. Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the standard control curve obtained by the present method; Fig. 2 represents the assay results of quantitation of FXIII in a plasma sample; Fig. 3 represents the results of the inhibition of FXIII by putrescine; Fig. 4 shows the assay results using the plasma samples from patients suffering from the diseases associated with FXIII deficiency. Fig. 5 represents the assay results of quantitation of FXIII using a platelet sample; Fig. 6 represents the assay results of quantitation of FXIII using a urine sample; and Fig. 7 shows the coagulation potential standard curve obtained by the present method.

DETAILED DESCRIPTION OF THIS INVETNION In one aspect of this invention, there is provided a method for measuring the activity of Factor XIII in a sample, which comprises the steps of: (a) contacting said sample to be measured to casein which is immobilized onto a solid substrate; (b) adding a fibrinogen-HRP (horse radish peroxidase) conjugate; (c) incubating the mixture of (b) with thrombin and Ca²⁺ to activate Factor XIII in said sample, thereby generating a casein-fibrinogen-HRP complex; (d) washing the reaction mixture of (c) ; (e) incubating the reaction mixture with a HRP substrate; and (f) measuring the activity of HRP on said casein-fibrinogen-HRP complex in the presence of a HRP substrate .

The present invention is directed to a method for measuring the activity of Factor XIII, making it possible to quantitate Factor XIII in a sample. The present method is applicable to a variety of samples derived from body tissues and biofluids, preferably, plasma, platelet, urine, seminal fluid, vaginal fluid and cerebro- spinal fluid, more preferably, plasma, platelet and urine, most preferably, plasma. One of features of the present method is that casein and fibrinogen are used as a substrate of Factor XIII. Casein is immobilized onto a solid substrate, preferably, through noncovalent interaction. It is preferred that the solid substrate is a plate used for immunoassay which is generally manufactured using a polymer such as polystyrene, polyethylene and polypropylene . According to the present method, fibrinogen is conjugated to HRP. HRP has been selected as the most suitable color- developing enzyme for the present assay system. The process for conjugation between fibrinogen and HRP is critical. Preferably, the fibrinogen-HRP conjugate is prepared by reacting maleimide- conjugated HRP with thiolated fibrinogen. This process is preferred in the senses that it can reserve Factor Xllla action sites on fibrinogen and prevent the undesired polymerization of fibrinogen. Preferably, the incubation of the step (c) is performed in a sample buffer comprising a fibrin-aggregation inhibitor. Examples of the fibrin-aggregation inhibitor includes, but not limited to, sodium, potassium, magnesium, iodide and chloride ions, protein denaturing agents (e.g., urea and sodium dodecyl sulfate) , SH reagents (e.g., dithiothreitol, dithioerythrito, 2-mercaptoethanol and β-thiodiglycol) , chelating agents (e.g., EDTA and EGTA) , gly-pro-arg-pro peptide, gly-his-arg-pro peptide, tetraethylammonium chloride, benzyltriethylammonium chloride, ammonium sulfate, sodium cholate, potassium ferricyanide, dimethyl sulfoxide, dimethylformamide, ethylene glycol and hexamethylene glycol . More preferably, the fibrin- aggregation inhibitor is gly-pro-arg-pro or gly-his-arg-pro peptides, most preferably, a mixture thereof. The preferable concentration of the fibrin-aggregation inhibitor ranges from 50 to 150 μg/ml, most preferably, about 100 μg/ml for gly-pro- arg-pro and 50 μg/ml gly-his-arg-pro peptides, respectivly. According to a preferred embodiment of this invention, the sample buffer further comprises a bovine serum albumin (BSA) and/or NaCl . The role of BSA is to reduce the unspecific interactions between casein and fibrinogen in the assay. The role of NaCl is to maintain the stable enzyme reaction during the assay. The preferable concentration of BSA ranges from 0.3% to 2%, most preferably, about 1%. The preferable concentration of NaCl ranges from 20 to 100 mM, most preferably, about 50 mM. According to a preferred embodiment of this invention, the method further comprises, after the step (c) , the step of (c') terminating the reaction for the generation of the casein- fibrinogen-HRP complex by using a thrombin inhibitor and/or Factor Xllla inhibitor. Preferably, PPACK (phenyl-pro-arg- chloromethyl ketone) is used as thrombin inhibitor and putrescine or DTT (dithiothreitol) is used as Factor Xllla inhibitor. It is more preferred that the combination of a thrombin inhibitor and Factor XIIla inhibitor is employed. According to a preferred embodiment, the washing step is carried out after the termination of step (c' ) with a washing buffer. The washing buffer contains a buffer capable of adjusting pH around 7.0, for example phosphate buffer such as phosphate buffered saline (PBS) . It is preferred that the washing buffer contains NaCl and nonionic surfactant, e.g., polysorbates such as Tween 40, 60 and 80. Another feature of this method is to use HRP as indicator for the quantity of FXIII in a sample. The enzyme catalyzing color development reaction has been well knwon, including alkaline phosphatase, β-galactosidase and HRP. The present inventors have found that HRP could provide the most sensitive and reliable assay system in the present method. Exemplary HRP substrate includes, but not limited to, TMB (3,3 ' ,5,5' -tetra-methyl benzidine) , ABTS ( [2 , 2 ' -azino-bis (3- ethylbenziazoline-6-sulfonic acid)], AEC (aminoethylcarbazole) , OPD (o-phenylenediamine) , diaminobenzidine, chloronaphthol , luminol (chemiluminescent horseradish peroxidase substrate, Sigma Inc.), 10-acetyl-3 , 7-dihydroxyphenoxazine (fluorescent horseradish peroxidase substrate, Probes. Inc) and guaiacol, most preferably, TMB. According to a preferred embodiment, the method further comprises, after the step (e) , the step of (e' ) terminating the reaction between HRP and HRP substrate. This termination may be carried out using the conventional methods for stopping enzymatic reactions . It is preferred that the termination is performed with a strong acid such as HC1, H₂S0₄ and trichloroacetic acid (TCA) , most preferably, H₂S0₄. In a preferred embodiment, the present method further comprises the step of (g) quantitating Factor XIII in the sample by use of a standard curve obtained from a normal sample.

The normal sample contains the normal Factor XIII level. The standard curve obtained by the present invention represents, preferably at least 0.97, more preferably, at least 0.98 and most preferably, at least 0.99 of the correlation coefficient between the activity of HRP and the amount of the sample.

In another aspect of this invention, there is provided a kit for measuring the activity of Factor XIII in a sample, which comprises: (a) a casein immobilized onto a solid substrate; and (b) a fibrinogen-HRP (horseradish peroxidase) conjugate. Since the present kit embodies the present method, the common descriptions between them are omitted in order to avoid the complexity of this specification leading to undue multiplicity. According to a preferred embodiment, the kit further comprises one or more components selected from the group of consisting of (c) thrombin; (d) Ca²⁺; (e) a thrombin inhibitor; (f) a Factor Xllla inhibitor; (g) a HRP substrate; (h) a standard control comprising a normal sample containing normal Factor XIII level; (i) a sample buffer comprising a fibrin- aggregation inhibitor; and (j) a washing buffer.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .

EXAMPLES EXAMPLE I: Preparation of Microtiter Plates Coated with Casein 55 mg of 0.01% NaN₃ (Fisher) were added to dimethylcasein (11 mg, Flika) dissolved in 550 ml of 200 mM MOPs buffer. The resultant was stirred well and its aliquot (100 μl) was transferred to each well of Immunex-HBX plate. The plate was incubated overnight at 4^°C in the incubation chamber and the supernatant was discarded. The plate was washed with PBS and blocked by aliquoting 315 μl of Blocking buffer (Pierce) into each well, followed by incubating at room temperature for 20 min. The plate was washed with PBS and then dried at room temperature or in weak vacuum container.

EXAMPLE II: Preparation of FXIII substrate-HRP Conjugate To 20 mg of horse radish peroxidase (HRP, Roche) dissolved in 4 ml of PBS, 30 μl of 150 mM sulfo-EMCS (N- [e- Maleimidocaproyloxy] sulfosuccinimide ester, Pierce) dissolved in DMSO were added, followed by incubating for 45 min at room temperature. Then, ethylmaleimide (Sigma) was added to have 5 mM final concentration. The resultant was dialyzed overnight in H₂0 at 4^°C or desalted using desalting column (Pierce) to obtain malei ide-conjugated HRP. The maleimide-conjugated HRP was divided into 4 batches . To 20 mg of fibrinogen (Sigma) dissolved in 4 ml of PBS, the same volume of 2-IT buffer (50 mM NaCl, 100 mM triethanolamine, 0.1 mM EDTA, pH 7.4) was added and then 200 μl of 5 mM 2-IT (iminothiolane, Sigma) were added. The solution was incubated for 30 min at room temperature and for 1 hr at

4°C. The thiolated fibrinogen was divided into 4 batches for loading to the desalting column (Pierce) . While one batch of the thiolated fibrinogen was coming through the column, it was directly added into one batch of the maleimide-conjugated HRP with stirring. The rest of the batches followed the above procedure to cross-link maleimide-conjugate HRP to the thiolated fibrinogen. Four batches were pulled together. The unreacted thiol groups in the fibrinogen were blocked with 5 mM of ethylmaleimide (Sigma) and the mixture was incubated for 30 min at room temperature. The unreacted maleimide groups in the maleimide-conjugated HRP were blocked with 1 mM mercaptoethanol

(Sigma), followed by 30 min-incubation at room temperature.

The HRP conjugated fibrinogen was desalted using desalting column or dialyzed overnight in H₂0 at 4°C. The solution containing fibrinogen-HRP was concentrated using Centricon concentrator YM-100 and Gly-Pro-Arg-Pro peptide was added to have 50 μg/ml concentration.

EXAMPLE III: Preparation of FXIII Assay Kit The present FXIII assay kit was prepared to include the following components: Standard Control, casein-coated plates, sample buffer, FXIII substrate-HRP containing Gly-Pro-Arg-Pro or Gly-His-Arg-Pro peptides, TMB (3, 3' , 5, 5' -tetramethyl benzidine) solution, thrombin cofactor, thrombin, reaction stop solution, thrombin and Factor Xllla inhibitor and washing buffer. The Standard Control was prepared by adding 1.5 μl of 60 mg/ml Gly-Pro-Arg-Pro peptide into 800 μl of human pooled normal plasma and adding 800 μl of 0.5% BSA. The diluted plasma were aiquoted and lyophilized. The sample buffer was prepared by mixing 7.5 g of BSA, 15 ml of 1 M Tris, 1.25 ml of 60 mg/ml of Gly-Pro-Arg-Pro and 0.6 ml of 60 mg/ml of Gly-His-Arg-Pro, 37.5 ml of 1 M NaCl and 75 mg of NaN₃, and making the final volume to 750 ml. The sample buffer thus obtained comprises 1 % BSA, 20 mM Tris, 50 mM NaCl,

100 μg/ml of Gly-Pro-Arg-Pro, 50 μg/ml of Gly-His-Arg-Pro and

0.01 % Na N₃. The TMB solution as a substrate of HRP was purchased from MOSS or KPL (USA) . 33 mM CaCl₂ solution (Sigma) was used as the thrombin cofactor. 1000 units/vial of thrombin (Sigma) was diluted with 10 ml of 20 mM Tris (pH 7.4) and then 2 vials of 100 units/ml of thrombin were taken out and mixed with 2 ml of 0.5% BSA. The final thrombin solution thus obtained comprises 0.25% BSA, 50 units/ml of thrombin and 20 mM Tris (pH 7.4) . The reaction stop solution was prepared using putrescine

(or DTT) and PPACK (phenyl-Pro-Arg-chloromethyl ketone) . 500 μl of 2.5 mg/ml of PPACK (Bachem Inc.) and 1.2 ml of 200 mM putrescine (Sigma) were mixed and 150 μl of 0.25 M trehalose and 150 μl of 0.5 % BSA were added. The washing buffer was prepared to include 5 X PBS, 0.24% Tween 80 and 1.75 mM NaCl. Its pH was adjusted to 6.4. When the 5 X is diluted to 1 X, the pH will rise to 7.2.

EXAMPLE IV: FXIII Assay For standard control curve, 50 μl of the assay buffer were added to the 1^st well to the 6^th well of the casein-coated plate.

50 μl of the diluted Standard Control were added to the 1^st well and mixed well. Then, the Standard Control was serially diluted from the 1^st well to the 5^th by transferring 50 μl from the 1^st well to the 2^nd well until the 5^th well. The serial dilution was not transferred to the 6^th well. The above dilution gave the following concentration gradient from 200% to 12.5%. The duplicated measurements were done . For the plasma sample to be tested, 20 μl of the diluted plasma sample were placed into empty sample wells of the casein-coated plate and 30 μL of the sample buffer were added. 20 μL of the FXIII substrate-HRP solution were added into the plasma sample and Standard Control wells and 15 μL of thrombin cofactor were then added into the plasma sample and Standard Control wells. Thereafter, 15 μl of thrombin were added into the sample and Standard Control wells for activating Factor XIII. The side of the plate was tapped gently for well mixing of the components . The reaction mixture was incubated for 20 min at 37°C with gentle shaking and the plate was covered to reduce the evaporation. After 20 min incubation, the reaction was terminated by adding 10 μl of the thrombin and FXIII inhibitor into the reaction wells . The plate was kept at room temperature for 5 min and washed 4 times with 250 μl of the washing buffer. 100 μL of HRP substrate reagent solution (TMB) were added to each well and incubated for 5 min at room temperature. The reaction was stopped with 50 μl of 0.5N H₂S0₄. The absorbance of the plate was measured at a wavelength of 450 nm within 20 minutes.

Fig. 1 shows the standard control curve. The linear equation describing the results is y = 0.3215x + 0.3148 and the correlation coefficient γ = 0.9932. Fig. 2 represents the assay results using plasma sample. The activity of FXIII linearly increased in the presence of thrombin in parallel with increased plasma amount. Therefore, it could be appreciated that the present FXIII assay kit is very effective in the quantitation of FXIII in plasma sample. Furthermore, Fig. 3 represents the results of the inhibition of FXIII by putrescine, demonstrating that the activity of FXIIIa linearly declined with increased amount of putrescine. These results also show that the present assay kit can provide a true assay of FXIII activity. Fig. 4 shows the assay results using the plasma samples from patients suffering from the diseases associated with FXIII deficiency. The patients of hemophilia A exhibited little or very low FXIII activity. The patients of delayed PT (Prothrombin time) showed the half value of FXIII activity compared to that of normal person. The present FXIII assay method can be applied to a variety of biofluids such as plasma, platelet, urine, seminal fluid, vaginal fluid and cerebro-spinal fluid. The platelet sample stored at -20^°C and 4°C showed the linear increase of FXIIIa activity with increase of platelet amount as represented in Fig. 5. Such linear relation was also observed with the assay using urine sample as demonstrated in Fig. 6. In Fig. 6, CT (control) means the unactivated platelet, where thrombin and its cofactor were not added and the temperatures are a storage temperature of platelet .

EXAMPLE V: Coagulation Assay The present assay for measuring coagulation potential was performed as follows : The new assay buffer for the coagulation potential assay was prepared by mixing 7.5 g of BSA, 15 ml of 1 M Tris, 37.5 ml of 1 M NaCl and 75 mg of NaN₃, and making the final volume to 750 ml. The assay buffer thus obtained comprises 1% BSA, 20 mM Tris, 50 mM NaCl and 0.01% Na N₃. The Standard Control for the coagulation potential assay was prepared by adding 800 μl of human pooled normal plasma and adding 800 μl of 0.5% BSA. The diluted plasma was aliquoted and lyophilized. The HRP- conjugated fibrinogen was used as substrate in the coagulation assay. The rest of the components for the Factor XIII assay were used, such as thrombin, thrombin cofactor, TMB, inhibitor, reaction stop solution. For standard control curve for the coagulation potential, 50 μl of the new assay buffer were added to the 1^st well to the 6^th well of the casein-coated plate. 50 μl of the diluted Standard Control were added to the 1^st well and mixed well. Then, the Standard Control was serially diluted from the 1^st well to the 5^th by transferring 50 μl from the 1^st well to the 2^nd well until the 5^th well. The serial dilution was not transferred to the 6^th well . The above dilution gave the following concentration gradient from 200% to 12.5%. The duplicated measurements were done . For the plasma sample to be tested, the sample plasma was diluted 20 folds with the assay buffer, and 20 μl were placed into empty sample wells of the casein-coated plate and 30 μL of the sample buffer were added. 20 μL of the substrate-HRP solution were added into the plasma sample and Standard Control wells and 15 μL of thrombin cofactor were then added into the plasma sample and Standard Control wells. Thereafter, 15 μl of thrombin were added into the sample and Standard Control wells for activating Factor XIII. The side of the plate was tapped gently for well mixing of the components. The reaction mixture was incubated for 20 min at 37°C with gentle shaking and the plate was covered to reduce the evaporation. After 20 min incubation, the reaction was terminated by adding 10 μl of the thrombin and FXIII inhibitors into the reaction wells. The plate was kept at room temperature for 10 min and washed 4 times with 250 μl of the washing buffer. 100 μL of HRP substrate reagent solution (TMB) were added to each well and incubated up to 5 min at room temperature. The reaction was stopped with 50 μl of 0.5N H₂S0₄. The absorbance of the plate was measured at a wavelength of 450 nm within 20 minutes. Fig. 7 shows the coagulation potential standard curve. The linear equation describing the results is y = 0.2448x + 0.0162 and the correlation coefficient γ = 0.9665. Accordingly, it could be recognized that the present method is highly effective in the measurement of coagulation potential .

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents .

Claims

What is claimed is:

1. A method for measuring the activity of Factor XIII in a sample, which comprises the steps of: (a) contacting said sample to be measured to casein which is immobilized onto a solid substrate; (b) adding a fibrinogen-HRP (horse radish peroxidase) conj gate; (c) incubating the mixture of (b) with thrombin and Ca²⁺ to activate Factor XIII in said sample, thereby generating a casein-fibrinogen-HRP complex; (d) washing the reaction mixture of (c) ; (e) incubating the reaction mixture with a HRP substrate; and (f) measuring the activity of HRP on said casein- fibrinogen-HRP complex in the presence of a HRP substrate.

2. The method according to claim 1, wherein said sample is selected from the group consisting of plasma, platelet, urine, seminal fluid, vaginal fluid and cerebro-spinal fluid.

3. The method according to claim 1, wherein said casein is immobilized on a plate.

4. The method according to claim 1, wherein said fibrinoge - HRP conjugate is prepared by reacting maleimide-conjugated HRP with thiolated fibrinogen.

5. The method according to claim 1, wherein said incubating is performed in a sample buffer comprising a fibrin-aggregation inhibitor.

6. The method according to claim 5, wherein said fibrin- aggregation inhibitor is selected from the group consisting of sodium, potassium, magnesium, iodide and chloride ions, protein denaturing agents, SH reagents, chelating agents, gly-pro-arg- pro peptide, gly-his-arg-pro peptide, tetraethylammonium chloride, benzyltriethylammonium chloride, ammonium sulfate, sodium cholate, potassium ferricyanide, dimethyl sulfoxide, dimethylformamide, ethylene glycol and hexamethylene glycol.

7. The method according to claim 6, wherein said fibrin- aggregation inhibitor is gly-pro-arg-pro peptide, gly-his-arg- pro peptide peptide or mixture thereof .

8. The method according to claim 1, wherein said sample buffer further comprises a bovine serum albumin and/or NaCl .

9. The method according to claim 1, wherein said HRP substrate is selected from the group consisting of TMB (3 , 3 ' , 5, 5 ' -tetra- methyl benzidine) , ABTS ( [2, 2 ' -azino-bis (3-ethylbenziazoline- 6-sulfonic acid)], AEC (aminoethylcarbazole) , OPD (o- phenylenediamine) , diaminobenzidine, chloronaphthol and guaiacol .

10. The method according to claim 9, wherein said HRP substrate is TMB.

11. The method according to claim 1, wherein after the step of

(c) said method further comprises the step of (c') terminating the reaction for the generation of said casein-fibrinogen-HRP complex by using a thrombin inhibitor and/or Factor XIIla inhibitor.

12. The method according to claim 11, wherein said terminating is performed with PPACK (phenyl-pro-arg-chloromethyl ketone) as thrombin inhibitor and/or putrescine or DTT (dithiothreitol) as Factor Xllla inhibitor.

13. The method according to claim 1, wherein after the step of (e) said method further comprises the step of (e' ) terminating the reaction between HRP and HRP substrate .

14. The method according to claim 13 , wherein said terminating is performed with a strong acid.

15. The method according to claim 1, wherein said method further comprises the step of (g) quantitating Factor XIII in said sample by use of a standard curve obtained from a normal sample.

16. The method according to claim 15, where said standard curve represents at least 0.97 of the correlation coefficient between the activity of HRP and the amount of the sample.

17. The method according to claim 16, where said standard curve represents at least 0.99 of the correlation coefficient between the activity of HRP and the amount of the sample.

18. A kit for measuring the activity of Factor XIII in a sample, which comprises: (a) a casein immobilized onto a solid substrate; and (b) a fibrinogen-HRP (horse radish peroxidase) conjugate.

19. The kit according to claim 18, wherein said kit further comprises one or more components selected from the group of consisting of (c) thrombin; (d) Ca²⁺; (e) a thrombin inhibitor; (f) a Factor Xllla inhibitor; (g) a HRP substrate; (h) a standard control comprising a normal sample containing normal Factor XIII level; (i) a sample buffer comprising a fibrin- aggregation inhibitors; and (j) a washing buffer.

20. The kit according to claim 18, wherein said casein is immobilized on a plate.

21. The kit according to claim 18, wherein said fibrinogen-HRP conjugate is prepared by reacting maleimide-conjugated HRP with thiolated fibrinogen.

22. The kit according to claim 19, wherein said fibrin- aggregation inhibitor is selected from the group consisting of sodium, potassium, magnesium, iodide and chloride ions, protein denaturing agents, SH reagents, chelating agents, gly-pro-arg- pro and gly-his-arg-pro peptides, tetraethylammonium chloride, benzyltriethylammonium chloride, ammonium sulfate, sodium cholate, potassium ferricyanide, dimethyl sulfoxide, dimethylformamide, ethylene glycol and hexamethylene glycol.

23. The kit according to claim 22, wherein said fibrin- aggregation inhibitor is gly-pro-arg-pro peptide, gly-his-arg- pro peptide peptide or mixture thereof .

24. The kit according to claim 19, wherein said sample buffer further comprises a bovine serum albumin and/or NaCl .

25. The kit according to claim 19, wherein said HRP substrate is selected from the group consisting of TMB (3,3 ' ,5,5 ' -tetra- methyl benzidine) , ABTS ( [2 , 2 ' -azino-bis (3-ethylbenziazoline- 6-sulfonic acid)], AEC (aminoethylcarbazole) , OPD (o- phenylenediamine) , diaminobenzidine, chloronaphthol and guaiacol .

26. The kit according to claim 25, wherein said HRP substrate is TMB.

27. The kit according to claim 19, wherein said thrombin inhibitor is PPACK (phenyl-pro-arg-chloromethyl ketone)

28. The kit according to claim 19, wherein said Factor Xllla inhibitor is putrescine or DTT (dithiothreitol) .