Method for evaluating active concentration of glutamine transaminase

Abstract

The invention provides a method for evaluating the activity concentration of glutamine transaminase, and belongs to the technical field of enzyme activity concentration detection. The invention provides a determination method for evaluating the active concentration of glutamine transaminase, which comprises the following steps: reacting a thrombin-loaded conjugate pad with a mixed solution of fibrinogen and a sample to be detected to obtain a fibrin clot-loaded conjugate pad; judging the activity concentration of the glutamine transaminase by comparing the permeability of the binding pad loaded with the fibrous protein clots and the permeability of the binding pad loaded with the quality control fibrous protein clots, wherein the activity concentration of the glutamine transaminase is in negative correlation with the permeability. The method disclosed by the invention is simple, convenient and rapid, low in detection cost and wide in scene application, and has important significance in preliminarily judging the activity concentration of transglutaminase in the fields of industry and agriculture, food, cosmetics and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme activity concentration detection, and particularly relates to a method for evaluating the activity concentration of transglutaminase. Background Art

[0002] Transglutaminase, also known as transglutaminase (TGase), is involved in the post-translational modification of proteins and peptides, as well as various cellular and biological processes such as coagulation, apoptosis, cell differentiation, and extracellular matrix stabilization. TGase catalyzes the amidotransfer reaction between the γ-amide group of glutamine residues and the ε-amino group of lysine in proteins, forming an ε-(γ-glutamine)-lysine heteropeptide bond, thereby altering the functional properties of proteins. It promotes intramolecular and intermolecular cross-linking of proteins, as well as cross-linking between proteins and amino acids, thereby improving protein structure and function. It has significant effects on protein properties such as foaming, emulsification, emulsion stability, thermal stability, water retention, and gelling capacity, thereby enhancing the flavor, taste, texture, and appearance of foods. Therefore, TGase has a wide range of applications in industry, agriculture, food, cosmetics, and medicine.

[0003] Currently, there are over a dozen methods for measuring transglutaminase. Commonly used methods in laboratories include protein electrophoresis, radiolabeling, fluorescence, and ammonium titration. These methods are often complex, require specialized personnel, and are time-consuming, expensive, and not portable. The national standard method (GB / T 34795-2017) relies on the principle that transglutaminase catalyzes the reaction of N-benzyloxycarbonyl-L-glutamylglycine and hydroxylamine to form L-glutamic acid-γ-monohydroxamic acid. L-glutamic acid-γ-monohydroxamic acid can be visualized with ferric chloride, exhibiting a characteristic absorption peak at 525 nm. Therefore, the enzyme activity concentration can be calculated using the external standard method based on the amount of L-glutamic acid-γ-monohydroxamic acid produced. However, the national standard method is complex and requires specialized personnel and high-precision equipment. This results in high overall costs, a long testing cycle, and limited availability, requiring testing in specialized laboratories. At present, there is still no rapid, low-cost and easy-to-use method for detecting the activity concentration of transglutaminase in China. Summary of the Invention

[0004] In view of this, the present invention provides a method for evaluating the activity concentration of transglutaminase, which can detect the activity concentration of transglutaminase quickly and at low cost.

[0005] The present invention provides a method for evaluating the activity concentration of transglutaminase, wherein a binding pad loaded with thrombin reacts with a mixture of fibrinogen and a sample to be tested to obtain a binding pad loaded with fibrin clots; reacting the binding pad loaded with thrombin with a mixture of fibrinogen and a quality control sample to obtain a binding pad loaded with a quality control fibrin clot; The activity concentration of transglutaminase is determined by comparing the permeability of the binding pad loaded with the fibrin clot and the binding pad loaded with the quality control fibrin clot: the activity concentration of the transglutaminase is negatively correlated with the permeability.

[0006] Preferably, the method for detecting the permeability comprises a capillary method; The capillary method includes placing one end of a binding pad loaded with fibrin clots in a chromatography solution, overlapping the other end of the binding pad with the chromatography pad for chromatography, and determining the transglutaminase activity concentration based on the extension length of the chromatography solution on the chromatography pad; The extension length is inversely proportional to the transglutaminase activity concentration.

[0007] The present invention provides a method for determining the activity concentration of transglutaminase based on a detection device, wherein the detection device is used to determine the activity concentration of transglutaminase; The detection device includes a carrying unit and at least one detection unit disposed in the carrying unit; The detection unit includes a groove and a test strip; The groove is used to hold a first mixed solution formed by fibrinogen and a sample to be tested or a second mixed solution formed by fibrinogen and a quality control sample; The test strip includes a conjugate pad and a chromatography pad; One end of the conjugate pad extends into the groove, and the other end overlaps the end of the chromatography pad through the barrier pad, and when the barrier pad is pulled out, the end of the conjugate pad contacts the end of the chromatography pad; The measurement steps include the following: Thrombin is loaded onto the conjugate pad, and a first mixed solution formed by fibrinogen and a sample to be tested is added to the groove so that the liquid surface contacts the end of the conjugate pad loaded with thrombin. After standing, the barrier pad is removed and chromatography is performed, and the activity concentration of the transglutaminase is determined based on the extension length of the chromatographic solution on the chromatography pad. The extension length is inversely proportional to the transglutaminase activity concentration.

[0008] Preferably, when a plurality of detection units are provided on the carrying unit, a second mixed solution formed by adding fibrinogen and a quality control sample into the groove of at least one detection unit is used for quality control testing.

[0009] Preferably, the barrier pad is placed between the conjugate pad and the chromatography pad to separate the overlapping area of ​​the conjugate pad and the chromatography pad; and the barrier pad is made of a waterproof material.

[0010] Preferably, the length of the chromatography pad is 8-15 cm and the width is 1.5-4 mm; the length of the conjugation pad is 1.5-2.5 cm; the width of the conjugation pad is 2-4 mm.

[0011] Preferably, the length of the groove is 0.5-0.8 cm, the width of the groove is 0.5-0.8 cm, and the depth of the groove is 1-1.5 cm.

[0012] Preferably, the amount of fibrinogen added is 0.5-0.6 g, and the total volume of the first mixed solution or the second mixed solution is 150-240 μL.

[0013] Preferably, the coating concentration of thrombin on the conjugate pad is above 100 U / mL.

[0014] Preferably, the standing or chromatography time is 140 to 300 seconds.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention provides a method for evaluating the activity concentration of transglutaminase, wherein a binding pad loaded with thrombin is reacted with a mixture of fibrinogen and a sample to be tested to obtain a binding pad loaded with a fibrin clot; a binding pad loaded with thrombin is reacted with a mixture of fibrinogen and a quality control sample to obtain a binding pad loaded with a quality control fibrin clot; the activity concentration of transglutaminase is judged by comparing the permeability of the binding pad loaded with the fibrin clot and the binding pad loaded with the quality control fibrin clot: the activity concentration of the transglutaminase is negatively correlated with the permeability. The present invention finds that when there is no transglutaminase, the fibrin clot formed by thrombin and fibrinogen is relatively loose, has a large porosity, and has a high permeability; when there is transglutaminase, the fibrin clot has a compact structure, a small porosity, and a low permeability, and the higher the activity concentration of transglutaminase, the tighter the fibrin clot formed, the smaller the porosity, and the lower the permeability. Therefore, the activity concentration of transglutaminase can be quickly determined based on the permeability of the fibrin clot on the binding pad. The method of the present invention can directly measure the activity concentration of transglutaminase. Compared with the traditional method, there is no need for testing in a dedicated laboratory, the detection operation steps can be reduced, and no high-precision instruments and equipment are required. The detection process for detecting the activity concentration of transglutaminase is simpler and faster, which can reduce the detection cycle, and the detection is low-cost and has a wide range of applications. It solves the technical problem of detecting the activity concentration of transglutaminase under simple conditions. It is of great significance for the preliminary judgment of the activity concentration of transglutaminase in the fields of industry, agriculture, food, cosmetics and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Schematic diagram of the front and side of the detection device, where 1 is the first groove, 2 is the second groove, 3 is the first binding pad, 4 is the second binding pad, 5 is the barrier pad, 6 is the first chromatography pad, 7 is the second chromatography pad, and 8 is the carrying unit; Figure 2 Schematic diagram of the test results of the sample to be tested and the blank PBS solution quality control sample. DETAILED DESCRIPTION

[0017] The present invention provides a method for evaluating the activity concentration of transglutaminase, comprising reacting a thrombin-loaded binding pad with a mixture of fibrinogen and a sample to be tested to obtain a binding pad loaded with a fibrin clot; reacting the thrombin-loaded binding pad with a mixture of fibrinogen and a quality control sample to obtain a binding pad loaded with a quality control fibrin clot; and determining the activity concentration of transglutaminase by comparing the permeability of the binding pad loaded with the fibrin clot and the binding pad loaded with the quality control fibrin clot; the activity concentration of the transglutaminase is negatively correlated with the permeability.

[0018] In the present invention, thrombin converts fibrinogen into fibrin monomers, which can self-polymerize to form fibrin clots, but the fibrin clots formed are relatively loose, have a large porosity, and are highly permeable. Transglutaminase can covalently link adjacent fibrin monomers to form a stable hard clot network. When the sample to be tested does not contain transglutaminase, the fibrin clot on the conjugate pad has a loose structure, a large porosity, and a high permeability; when the sample to be tested contains transglutaminase, the fibrin clot on the conjugate pad has a tight structure, a small porosity, and a low permeability. In addition, different concentrations of transglutaminase activity will result in differences in the degree of coagulation of the fibrin clot formed by fibrinogen under the action of thrombin. The higher the concentration of transglutaminase activity, the tighter the fibrin clot formed, the smaller the porosity, and the lower the permeability. Therefore, the permeability of the fibrin clot on the conjugate pad is negatively correlated with the activity concentration of transglutaminase, and the activity concentration of transglutaminase can be quickly determined based on the permeability of the fibrin clot on the conjugate pad. The results of the present embodiment show that by reacting a mixture of fibrinogen and a test sample with a conjugate pad loaded with thrombin and measuring the permeability of the fibrin clot, low, medium, and high activity concentrations of transglutaminase can be accurately distinguished. The test results are consistent with the test results of the national standard method.

[0019] In the present invention, the method for detecting permeability preferably includes a capillary method. The capillary method preferably includes placing one end of a conjugated pad loaded with a fibrin clot in a chromatography fluid, and overlapping the other end of the conjugated pad for chromatography, and determining the transglutaminase activity concentration based on the extension length of the chromatography fluid on the chromatography pad; the extension length is inversely proportional to the transglutaminase activity concentration.

[0020] In the present invention, the chromatography fluid preferably includes PBS buffer and / or HBS buffer. The reaction between the fibrinogen solution and thrombin forms two phases: a coagulated phase and an uncoagulated phase. The higher the concentration of transglutaminase activity, the tighter the fibrin clot formed, the smaller the porosity, the higher the relative content of the coagulated phase, the lower the relative content of the uncoagulated phase, the lower the permeability, and the less liquid that can permeate through. When one end of the binding pad loaded with fibrin clot is placed in the sample liquid and the other end is overlapped with the chromatography pad for chromatography, the capillary length of the chromatography fluid is shorter when the fibrin clot with a higher proportion of coagulated phase is chromatographed. Therefore, the higher the concentration of transglutaminase activity, the shorter the length of the chromatography fluid extending on the chromatography pad in the same time, and the activity concentration of the transglutaminase is further determined.

[0021] The present invention provides a method for determining the activity concentration of transglutaminase based on a detection device, characterized in that the activity concentration of transglutaminase is determined by using a detection device; The detection device includes a carrying unit and at least one detection unit disposed in the carrying unit; The detection unit includes a groove and a test strip; The groove is used to hold a first mixed solution formed by fibrinogen and a sample to be tested or a second mixed solution formed by fibrinogen and a quality control sample; The test strip includes a conjugate pad and a chromatography pad; One end of the conjugate pad extends into the groove, and the other end overlaps the end of the chromatography pad through the barrier pad, and when the barrier pad is pulled out, the end of the conjugate pad contacts the end of the chromatography pad; The measurement steps include the following: Thrombin is loaded onto the conjugate pad, and a first mixed solution formed by fibrinogen and a sample to be tested is added to the groove so that the liquid surface contacts the end of the conjugate pad loaded with thrombin. After standing, the barrier pad is removed and chromatography is performed, and the activity concentration of the transglutaminase is determined based on the extension length of the chromatographic solution on the chromatography pad. The extension length is inversely proportional to the transglutaminase activity concentration.

[0022] In the present invention, the material of the binding pad and the chromatography pad preferably includes plant fiber paper strips. In the embodiment of the present invention, the plant fiber paper strips were purchased from Xinhua Paper, with specifications of: basis weight 105 gsm, density 185 kg / m 3, thickness 0.53 mm. The conjugate pad is preferably loaded with or not loaded with thrombin. The length of the conjugate pad is preferably 1.5-2.5 cm, more preferably 1.8-2.3 cm, and most preferably 2.1 cm. The width of the conjugate pad is preferably 2-4 mm, more preferably 2.5-3.5 mm, and most preferably 3 mm. One end of the conjugate pad extends into the groove. The groove is used to hold a mixture of fibrinogen and a test sample or a quality control sample. The length of the groove is preferably 0.5-0.8 cm, more preferably 0.6-0.7 cm, and most preferably 0.6 cm. The width of the groove is preferably 0.5-0.8 cm, more preferably 0.6-0.7 cm, and most preferably 0.6 cm. The depth of the groove is preferably 1-1.5 cm, more preferably 1.1-1.3 cm, and most preferably 1.2 cm. The other end of the conjugate pad overlaps the end of the chromatography pad via a barrier pad, and when the barrier pad is removed, the end of the conjugate pad contacts the end of the chromatography pad. The barrier pad is placed between the conjugate pad and the chromatography pad to separate the overlapping area of ​​the conjugate pad and the chromatography pad. The barrier pad is preferably made of a waterproof material, such as polycarbonate (PC), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), or polystyrene. The contact between the end of the conjugate pad and the end of the chromatography pad includes the conjugate pad pressing against the chromatography pad or the chromatography pad pressing against the conjugate pad. The length of the chromatography pad is 8-15 cm, more preferably 9-13 cm, and most preferably 10 cm. The width of the chromatography pad is preferably 1.5-4 mm, more preferably 2.5-3.5 mm, and most preferably 2 mm. For ease of observation, the upper surface of the carrier unit is preferably provided with scale lines corresponding to the chromatography pad, and the test results can be divided into different zones according to the scale lines. To facilitate the installation of the test strip and prevent it from shifting, the carrying unit is preferably provided with a test strip slot that matches the test strip. The height of the test strip slot is preferably the same as the depth of the groove, so that the chromatography pad is parallel to the bottom surface of the carrying unit.

[0023] In the present invention, the determining step preferably includes the following: Thrombin is loaded onto the conjugate pad, and a first mixed solution formed by fibrinogen and a sample to be tested is added to the groove so that the liquid surface contacts the end of the conjugate pad loaded with thrombin. After standing, the barrier pad is removed and chromatography is performed, and the activity concentration of the transglutaminase is determined based on the extension length of the chromatographic solution on the chromatography pad. The extension length is inversely proportional to the transglutaminase activity concentration.

[0024] In the present invention, thrombin is loaded onto the conjugate pad. The coating concentration of thrombin on the conjugate pad is preferably greater than 100 U / mL, more preferably greater than 120 U / mL, most preferably greater than 150 U / mL, and most preferably between 160 and 180 U / mL. The loading of thrombin preferably includes dropwise addition of thrombin or freeze-dried thrombin. The solvent for thrombin preferably includes a PBS solution. The volume of the dropwise addition is preferably 10 to 20 μL, more preferably 12 to 18 μL, and even more preferably 15 μL. The freeze-dried thrombin loading method preferably comprises dissolving thrombin in a lyophilization protection solution to obtain a thrombin solution, immersing the conjugate pad in the thrombin solution, and then removing the conjugate pad and freeze-drying it. The present invention compared the effects of dropwise addition and freeze-dried loading on the conjugate pad on detection accuracy. The results showed that freeze-dried loading on the conjugate pad resulted in higher detection accuracy. Freeze-dried loading on the conjugate pad enabled complete and uniform adhesion of thrombin to the conjugate pad, improving detection accuracy.

[0025] In the present invention, after thrombin is loaded onto the conjugate pad, a mixture of fibrinogen and the sample to be tested is added to the groove so that the liquid surface contacts the end of the conjugate pad. The amount of fibrinogen added is 0.5-0.6 g, more preferably 0.52-0.58 g, and most preferably 0.54 g. The fibrinogen is preferably dissolved in HBS buffer. The sample to be tested is preferably dissolved in PBS buffer. The HBS buffer and PBS buffer can improve the stability of the test results. The volume of the first mixed solution or the second mixed solution is preferably 150-240 μL, more preferably 160-220 μL, further preferably 180-210 μL, and most preferably 200 μL. The volume of the fibrin solution in the first mixed solution or the second mixed solution is preferably 140-230 μL, more preferably 160-200 μL, and most preferably 180 μL. Adding a mixture of fibrinogen and the test sample to the groove can improve the stability of test results. This study compared the effects of adding the fibrinogen and test sample mixture to the groove with adding the fibrinogen solution first and then adding the test sample. The results showed that adding the mixture to the groove resulted in more stable test results. For easier observation, Brilliant Blue dye is preferably added to the mixture of fibrinogen and the test sample or quality control sample.

[0026] In the present invention, the barrier pad is pulled out after standing and then subjected to chromatography, and the standing time is 140~300s, more preferably 160~260s, further preferably 170~200s, and most preferably 180s. The standing temperature is preferably 36~38°C, more preferably 37°C. The time and temperature of the chromatography are preferably the same as the time and temperature of the standing. The standing can allow the binding pad loaded with thrombin to react with the mixed liquid in the groove to form a stable fibrin clot on the binding pad, thereby improving the accuracy and stability of the test results. Pulling out the barrier pad after standing can allow the end of the binding pad to contact the end of the chromatography pad, and the binding pad absorbs liquid for chromatography on the chromatography pad. Glutamine aminotransferases of different activity concentrations can be accurately distinguished by chromatography.

[0027] In the present invention, after chromatography, the concentration of transglutaminase activity is determined based on the extension length of the chromatographic fluid on the chromatography pad; the extension length is inversely proportional to the transglutaminase activity concentration. In the present embodiment, by testing various concentrations of transglutaminase, the relationship between the extension length of the chromatographic fluid on the chromatography pad and the transglutaminase activity concentration was obtained when the fibrinogen content was 0.54 g and the thrombin coating concentration was 100 U / mL, as shown in the following figure: When the extension length was greater than 7.5 cm, the transglutaminase concentration was less than 0.2 mg / mL; When the extension length was greater than 5.5 cm and less than 7.5 cm, the transglutaminase concentration was between 0.2 and 0.6 mg / mL; When the extension length is less than 3.5 cm or less than 5.5 cm, the transglutaminase concentration is greater than 0.6 mg / mL.

[0028] In the present invention, when the carrier unit is provided with multiple detection units, a second mixed solution formed by adding fibrinogen and a quality control sample to the groove of at least one detection unit is used for quality control testing. The quality control samples preferably include a low-activity concentration quality control sample with an activity concentration of 0.05 U / mL, a medium-activity concentration quality control sample with an activity concentration of 0.4 U / mL, and a high-activity concentration quality control sample with an activity concentration of 0.8 U / mL. The quality control samples also preferably include a blank PBS solution quality control sample.

[0029] In an embodiment of the present invention, the surface of the carrier unit of the detection device is provided with a scale line corresponding to the position of the chromatography pad, and 3.5 cm near the groove end is marked as a high zone or quality inspection A, 5.5 cm is marked as a middle zone or quality inspection B, and 7.5 cm is marked as a low zone or quality inspection C. There are two detection units on the carrier unit of the detection device. After the excess thrombin is lyophilized on the conjugate pad, a mixture of fibrinogen brilliant blue solution and the sample to be tested is added to the groove of one detection unit, and a mixture of fibrinogen brilliant blue solution and PBS buffer is added to the groove of the other detection unit. After reacting for 3 minutes, the conjugate pad condenses into a fibrin clot, forming a resistance to wicking. The barrier pad is pulled out so that the other end of the conjugate pad overlaps the chromatography pad, and the results of the quality inspection port and the concentration indication port are observed for 3 minutes to judge the transglutaminase activity concentration. When quality inspections A, B, and C all have liquid chromatography color development, it means that the detection device is available. Otherwise, the detection device does not meet the quality requirements and will be discarded and cannot be used. After 3 minutes of wicking, if only the high activity indicator port of the test sample shows color, and quality checks A, B, and C all show color, it indicates that the transglutaminase activity concentration is high. After 3 minutes of wicking, if both the medium and high activity indicator ports of the test sample show color, and quality checks A, B, and C all show color, it indicates that the transglutaminase activity concentration is medium. After 3 minutes of wicking, if all the low, medium, and high activity indicator ports of the test sample show color, and quality checks A, B, and C all show color, it indicates that the transglutaminase activity concentration is low. The farther the brilliant blue solution of the test sample flows on the sample chromatography pad within a certain period of time, the longer the extension length, and the lower the transglutaminase activity concentration. When the extension length is greater than the low zone, the transglutaminase concentration is approximately less than 0.2 mg / mL; when the extension length is greater than the middle zone and less than the low zone, the transglutaminase concentration is between 0.2 and 0.6 mg / mL; when the extension length is greater than the high zone and less than the middle zone, the transglutaminase concentration is above 0.6 mg / mL.

[0030] In the examples of the present invention, the method was used to detect transglutaminase at different activity concentrations. The test results showed that the method can accurately distinguish between low, medium, and high activity concentrations of transglutaminase, and the test results are consistent with those of the national standard method. Therefore, the method can quickly and accurately detect the activity concentration of transglutaminase.

[0031] In the embodiment of the present invention, the sample to be tested for transglutaminase is prepared by using PBS buffer solution. The transglutaminase (TGase) is purchased from Solebol and has an activity of 1000 U / g.

[0032] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 A detection device for rapidly detecting the activity concentration of transglutaminase The structural diagram of the detection device is shown in Figure 1 The detection device includes a carrying unit and two first detection units and a second detection unit disposed in the carrying unit; The first detection unit includes a first groove and a first test strip; the first groove is used to hold a first mixed liquid formed by fibrinogen and the sample to be tested; the first test strip includes a first conjugation pad and a first chromatography pad; one end of the first conjugation pad extends into the groove, and the other end overlaps with the end of the first chromatography pad through a barrier pad, and the barrier pad is placed between the first conjugation pad and the first chromatography pad to separate the overlapping area of ​​the first conjugation pad and the first chromatography pad; and when the barrier pad is pulled out, the end of the first conjugation pad contacts the end of the first chromatography pad.

[0034] The structure of the second detection unit is the same as that of the first detection unit. The second detection unit includes a second groove and a second test strip; the second groove is used to hold a second mixed solution formed by fibrinogen and the quality control sample.

[0035] The length of the chromatography pad is 10 cm and the width is 2 mm. The length of the binding pad is 2.1 cm and the width is 3 mm. The length of the groove is 0.6 cm, the width is 0.6 cm, and the depth is 1.2 cm. The barrier pad is made of plastic, with a length of 3 cm and a width of 0.5 cm. Scale lines are set on the surface of the carrier unit at the position corresponding to the chromatography pad, with 3.5 cm near one end of the groove marked as the high zone or quality inspection A, 5.5 cm marked as the middle zone or quality inspection B, and 7.5 cm marked as the low zone or quality inspection C. When the extension length is greater than the low zone, the transglutaminase activity concentration is low, that is, less than 0.2 mg / mL; when the extension length is greater than the middle zone and less than the low zone, the transglutaminase activity concentration is medium, that is, between 0.2 and 0.6 mg / mL; when the extension length is less than the middle zone or less than the high zone, the transglutaminase activity concentration is high, that is, above 0.6 mg / mL.

[0036] Example 2 Method for determining the activity concentration of glutamine transaminase standard The detection device in Example 1 was used to detect the activity concentration of transglutaminase. Samples with known transglutaminase activity concentrations were taken and tested according to the following steps: Load an excess of thrombin on the conjugate pad: dissolve thrombin (bovine thrombin, purchased from Solebo) in freeze-drying protection solution (purchased from Xiamen Anbang Biotechnology) to obtain a thrombin solution with a concentration of 100 U / mL. Soak two conjugate pads in the thrombin solution (for 1 min) to allow the thrombin to completely and evenly adhere to the conjugate pads. Then remove the conjugate pads and lyophilize them to obtain two conjugate pads loaded with thrombin.

[0037] Add 180 μL of 3 g / L fibrinogen brilliant blue solution (solvent HBS buffer) and 20 μL of transglutaminase test sample solution (transglutaminase purchased from Solebol, with an enzyme activity of 1000 U / g, and the solvent of transglutaminase is PBS buffer) into one groove; In another well, add 180 μL of 3 g / L fibrinogen brilliant blue solution (solvent HBS buffer) and 20 μL of blank PBS solution quality control sample.

[0038] After a 3-minute reaction, thrombin and fibrinogen on the conjugate pad formed a fibrin clot, creating a wicking resistance. The barrier pad was removed, and one end of the conjugate pad was overlapped with the chromatography pad. After 3 minutes of chromatography, the length of the chromatographic fluid extending across the two chromatography pads was observed. The experimental results showed that the quality check A, quality check B, and quality check C on the chromatography pad of the blank PBS solution quality control sample all showed color. The specific test results for each group of test samples are shown in Table 1.

[0039] Table 1 Glutamine transaminase test results at different activity concentrations

[0040] As shown in Table 1, when the transglutaminase concentration is 0.05-0.1 mg / mL, the extension length of the chromatography fluid on the chromatography pad is 8.8-10 cm.

[0041] When the transglutaminase concentration was 0.35-0.4 mg / mL, the extension length of the chromatography fluid on the chromatography pad was 5.9-6.9 cm.

[0042] When the transglutaminase concentration was 1-1.4 mg / mL, the extension length of the chromatography fluid on the chromatography pad was 2.8-3.1 cm.

[0043] Example 3 Method for establishing evaluation standard of transglutaminase activity The method of Example 2 was used to detect transglutaminase of various activity concentrations, and the relationship between the extension length of the chromatographic fluid on the chromatography pad and the transglutaminase activity concentration was as follows: When the extension length was greater than 7.5 cm, the transglutaminase concentration was less than 0.2 U / mL; When the extension length was greater than 5.5 cm and less than 7.5 cm, the transglutaminase concentration was between 0.2 and 0.6 U / mL; When the extension length is less than 5.5 cm or less than 3.5 cm, the transglutaminase concentration is greater than 0.6 U / mL.

[0044] Comparative Example 1 Method for determining the activity concentration of glutamine transaminase standard A drop-addition group and a lyophilization group were set up. The drop-addition group was tested for the activity concentration of the transglutaminase standard using the same method as in Example 2, except that the conjugate pad was overloaded with thrombin by dropwise addition of 15 μL of thrombin. The lyophilization group was tested for the activity concentration of the transglutaminase standard using the same method as in Example 2. The test results are shown in Table 2.

[0045] Table 2 Glutamine transaminase test results of the dripping group and the freeze-dried group

[0046] According to the results in Table 2, the test results of the freeze-dried group are more stable.

[0047] Comparative Example 2 Two groups were set up: one in which TGase was mixed with the fibrinogen solution first and one in which the fibrinogen solution was added first and then the TGase was added. The method for testing the activity concentration of the transglutaminase standard in the group in which the TGase was mixed with the fibrinogen solution first was the same as in Example 2. The method for testing the activity concentration of the transglutaminase standard in the group in which the fibrinogen solution was added first and then the TGase was added was the same as in Example 2, except that when adding the sample to the groove, the fibrinogen brilliant blue solution was added first, followed by the transglutaminase. The test results are shown in Table 3.

[0048] Table 3 Glutamine transaminase test results of each group

[0049] According to the results in Table 3, the test results of the group in which TGase and fibrinogen solution were mixed first were more stable.

[0050] Example 4 Verification of the accuracy of a rapid method for detecting transglutaminase activity concentration The method of Example 2 and the evaluation criteria of Example 3 were used to rapidly detect transglutaminase of different activity concentrations.

[0051] In addition, the national standard method was used to detect different activity concentrations of transglutaminase.

[0052] The test results are shown in Table 4.

[0053] Table 4 Glutamine transaminase test results at different activity concentrations

[0054] According to the results in Table 4, the method for rapid detection of transglutaminase activity concentration can quickly distinguish low, medium and high activity concentrations, and the test results are consistent with the national standard method, indicating that the method for rapid detection of transglutaminase activity concentration has high accuracy.

[0055] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for evaluating the activity concentration of transglutaminase, characterized in that: reacting the binding pad loaded with thrombin with a mixture of fibrinogen and a sample to be tested to obtain a binding pad loaded with fibrin clots; reacting the binding pad loaded with thrombin with a mixture of fibrinogen and a quality control sample to obtain a binding pad loaded with a quality control fibrin clot; The activity concentration of transglutaminase is determined by comparing the permeability of the binding pad loaded with the fibrin clot and the binding pad loaded with the quality control fibrin clot: the activity concentration of the transglutaminase is negatively correlated with the permeability.

2. The method according to claim 1, characterized in that Methods for detecting the permeability include capillary method; The capillary method includes placing one end of a binding pad loaded with fibrin clots in a chromatography solution, overlapping the other end of the binding pad with the chromatography pad for chromatography, and determining the transglutaminase activity concentration based on the extension length of the chromatography solution on the chromatography pad; The extension length is inversely proportional to the transglutaminase activity concentration.

3. A method for determining the activity concentration of transglutaminase based on a detection device, characterized in that: The activity concentration of transglutaminase is measured using a detection device; The detection device includes a carrying unit and at least one detection unit disposed in the carrying unit; The detection unit includes a groove and a test strip; The groove is used to hold a first mixed solution formed by fibrinogen and a sample to be tested or a second mixed solution formed by fibrinogen and a quality control sample; The test strip includes a conjugate pad and a chromatography pad; One end of the conjugate pad extends into the groove, and the other end overlaps the end of the chromatography pad through the barrier pad, and when the barrier pad is pulled out, the end of the conjugate pad contacts the end of the chromatography pad; The measurement steps include the following: Thrombin is loaded onto the conjugate pad, and a first mixed solution formed by fibrinogen and a sample to be tested is added to the groove so that the liquid surface contacts the end of the conjugate pad loaded with thrombin. After standing, the barrier pad is removed and chromatography is performed, and the activity concentration of the transglutaminase is determined based on the extension length of the chromatographic solution on the chromatography pad. The extension length is inversely proportional to the transglutaminase activity concentration.

4. The method according to claim 3, characterized in that When a plurality of detection units are provided on the carrying unit, a second mixed solution formed by adding fibrinogen and a quality control sample into the groove of at least one detection unit is used to perform quality control detection.

5. The method according to claim 3, characterized in that: The barrier pad is placed between the binding pad and the chromatography pad to separate the overlapping area of ​​the binding pad and the chromatography pad; the barrier pad is made of waterproof material.

6. The method according to claim 3, characterized in that: The length of the chromatography pad is 8-15 cm and the width is 1.5-4 mm; the length of the conjugation pad is 1.5-2.5 cm; the width of the conjugation pad is 2-4 mm.

7. The method according to claim 3, characterized in that The length of the groove is 0.5-0.8 cm, the width of the groove is 0.5-0.8 cm, and the depth of the groove is 1-1.5 cm.

8. The method according to claim 3, characterized in that: The amount of fibrinogen added is 0.5-0.6 g, and the total volume of the first mixed solution or the second mixed solution is 150-240 μL.

9. The method according to claim 3, characterized in that: The coating concentration of thrombin on the conjugate pad is above 100 U / mL.

10. The method according to any one of claims 3 to 9, characterized in that: The standing or chromatography time is 140 to 300 seconds.

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