Method for detecting activity of fibrinolytic inhibitor activated by blood thrombin

Abstract

The invention relates to the technical field of biology, and discloses a method for detecting the activity of a fibrinolytic inhibitor activated by blood thrombin, the detection method comprises reagents of an experimental group, a control group and a blank group, a sample to be detected is platelet-deficient plasma, the three groups of samples are added into the reagents of the experimental group, the control group and the blank group respectively, then absorbance detection is performed, and the activity of the fibrinolytic inhibitor activated by the blood thrombin is detected. The activity of the thrombin-activated fibrinolytic inhibitor can be obtained by calculating absorbance data obtained through absorbance detection. The method for detecting the activity of the thrombin-activated fibrinolytic inhibitor comprises the following steps: dynamically monitoring a curve of the absorbance of plasma along with time in real time, comparing the curve difference of whether an inhibitor of the thrombin-activated fibrinolytic inhibitor is added or not, and calculating related parameters to represent the activity of the thrombin-activated fibrinolytic inhibitor. The operation is convenient; the sample is easy to obtain and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for detecting the activity of a fibrinolysis inhibitor activated by blood thrombin. Background Art

[0002] Thrombin activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be converted into an effective inhibitor of the fibrinolytic system when activated by thrombin, plasmin or thrombin-thrombomodulin complex. TAFI exists in the normal human body, but it is also a potential risk factor for various thrombotic diseases. Literature indicates that TAFI participates in the occurrence and development of diseases by reducing fibrinolysis and anti-inflammatory mechanisms, and its activity level changes with the occurrence and development of the disease. Currently, there is no clinical coagulation test for endogenous TAFI activity, so it is necessary to establish a sensitive and specific TAFI activity detection method.

[0003] There are several methods for detecting the level of thrombin-activated fibrinolysis inhibitor in the blood, including immunoassay for detecting TAFI level, activity-based evaluation of TAFIa, and functional fibrinolysis assay for detecting TAFIa. Currently, immunoassay for detecting TAFI level adopts the principle of enzyme-linked immunosorbent assay, which is simple to operate and can be efficiently automated. However, this method mainly measures the antigen level rather than the functional activity, and cannot distinguish between the proenzyme TAFI and the enzyme TAFIa. The methods for evaluating TAFIa based on activity include direct evaluation of circulating TAFIa in vivo and direct quantification of cleavage products. Direct evaluation of circulating TAFIa in vivo mainly adopts reversed-phase high-performance liquid chromatography and fluorescence functional assay, and direct quantification of cleavage products is based on activity. The method for evaluating TAFIa can directly evaluate the level of TAFIa, but due to the short half-life of TAFIa, it needs to be placed at 0°C immediately after sampling, and specific thrombin and plasmin inhibitors need to be added when collecting samples to avoid unnecessary TAFI in vitro activation. The method has high pre-analytical requirements, and it is difficult for clinically collected samples to meet the requirements. The functional fibrinolysis experiment-based detection of TAFIa uses thromboelastometry and the classic plasma comprehensive fibrinolysis experiment. This method can better characterize the functional effect of TAFIa, but the problem is that it is affected by changes in the levels of components of the coagulation and fibrinolysis systems, there are no standardized measures, and the detection time of the classic plasma comprehensive fibrinolysis experiment is too long, the operation is complicated and cannot be automated. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies of the prior art, the present invention provides a method for detecting the activity of a thrombin-activated fibrinolysis inhibitor.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a method for detecting the activity of a thrombin-activated fibrinolysis inhibitor, characterized in that it comprises the following steps:

[0008] 1) Prepare reagents for the experimental group, control group and blank group: the reagents for the experimental group include the sample to be tested, inhibitor reagent of activated thrombin-activated fibrinolysis inhibitor (TAFIa), thrombomodulin reagent, tissue plasminogen activator reagent, actin FSL reagent and calcium ion solution; the reagents for the control group include the sample to be tested, thrombomodulin reagent, tissue plasminogen activator reagent, actin FSL reagent, calcium ion solution and distilled water; the reagents for the blank group include the test sample, actin FSL reagent, calcium ion solution and distilled water;

[0009] 2) Absorbance detection: Before the absorbance detection, the samples to be tested were first added with TAFIa inhibitor or distilled water according to the reagents of the experimental group, control group and blank group and incubated at room temperature for 5 minutes, and then other reagents were added and mixed respectively. The curve of the change of plasma absorbance over time of the experimental group, control group and blank group was automatically drawn using an optical density scanner;

[0010] 3) Process data and determine parameters: Subtract the absorbance value of the blank group from the absorbance values ​​of the experimental group and the control group, respectively, to obtain the absorbance difference at different time points, which is used to draw a curve of the absorbance difference changing with time, perform curve fitting and calculate the first-order derivative, obtain the first-order derivative curve, and determine parameters (A) TAFI overall antifibrinolytic time difference (min) = AB-DE; (B) TAFI initial antifibrinolytic time difference (min) = AC-DF; (C) TAFI comprehensive antifibrinolytic ability = Log10 (integral value of the area of ​​the control group-experimental group*100); AB is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the control group, DE is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the experimental group; AC is the difference between the extreme point and the first zero point value of the first-order derivative curve of the control group, DF is the difference between the extreme point and the first zero point value of the first-order derivative curve of the experimental group, and the control group-experimental group area is the difference between the areas under the curve between the two zero points of the reaction curves of the experimental group and the control group.

[0011] Preferably, the preparation step of the sample to be tested in step (1) is as follows: a blood sample is collected within 2 hours and centrifuged at 2500g for 15 minutes at room temperature, the upper plasma is aspirated and placed in a centrifuge tube and centrifuged again at 2500g for 15 minutes, the upper platelet-poor plasma is aspirated and frozen for storage, and warmed at 37°C for 5 minutes before testing, wherein the anticoagulant used is sodium citrate;

[0012] Preferably, in step (1), the concentration of TAFIa inhibitor is 720 nM, the concentration of tissue plasminogen activator is 0.6 μg / ml, the concentration of thrombomodulin is 5 nM, the concentration of calcium ions is 0.025 mol / L, the actin FSL reagent is a mixture of soybean lecithin, rabbit cerebroside and ellagic acid, wherein the ratio of the volume of the plasma to be tested to the actin FSL reagent is 1:1;

[0013] Preferably, in step (2), the reagents of the experimental group, control group and blank group are respectively placed in a 96-well transparent plate, the wells containing the reagents of the experimental group are experimental wells, the wells containing the reagents of the control group are control wells, and the wells containing the reagents of the blank group are blank wells, and the volume in the wells is 100 μl, so the detection system of the experimental wells includes: 35 μl of plasma, 10 μl of TAFIa inhibitor, 5 μl of thrombomodulin, 5 μl of tissue-type plasminogen activator, 35 μl of actin FSL reagent, and 10 μl of calcium ions, and 10 ul of distilled water is used to replace the TAFIa inhibitor in the control wells, and the detection system includes: 35 μl of plasma, 10 μl of distilled water, 5 μl of thrombomodulin, 5 μl of tissue-type plasminogen activator, 35 μl of actin FSL reagent, and 10 μl of calcium ions, and 20 ul of distilled water is used to replace the TAFIa inhibitor, thrombomodulin and tissue-type plasminogen activator in the blank wells, and the detection system includes: 35 μl of plasma, 20 μl of distilled water, and actin FSL reagent 35 μl, calcium ion 10 μl;

[0014] Preferably, step (2) is performed according to a method comprising the following steps: at a detection temperature of 37°C, first, 35 μl of the sample to be tested and 10 μl of the TAFIa inhibitor DS-1040 solution are added to the experimental well, 35 μl of the sample to be tested and 10 μl of distilled water are added to the control well, and 35 μl of the sample to be tested and 20 μl of distilled water are added to the blank well, and the plates are incubated at room temperature for 5 minutes. Then, 5 μl of TM solution and 5 μl of tPA solution are added to the experimental well and the control well, and then 35 μl of actin FSL solution and 10 μl of calcium ion solution are added to the experimental well, the control well and the blank well. Finally, the 96-well plate is placed in an optical density scanner, vibrated for 10 seconds, and continuous detection is started. The detection wavelength is 980 nm, the detection interval is 15 seconds, and the detection duration is 100 minutes.

[0015] Preferably, in step (3), the absorbance change data of the experimental wells, the control wells and the blank wells are first outputted through an optical density scanner, the data of the duplicate wells are averaged, and then the experimental well difference data is obtained by subtracting the average absorbance of the blank wells from the average absorbance of the experimental wells, and the control well difference data is obtained by subtracting the average absorbance of the blank wells from the average absorbance of the control wells, and then the experimental well difference data and the control well difference data are respectively subjected to curve fitting, and the fitting is performed using a feedforward neural network. The fitting effect is evaluated by R2 and the fitting curve, and R2 greater than 0.9 is a better fitting effect. A difference curve and a fitting curve are generated, and a first-order derivative and a curve are further generated based on the fitting data to obtain the extreme value point, area integral, first zero point value and second zero point value of the curve. Three parameters can be obtained in the first-order derivative curve of the control well, point A is the first derivative zero point value of the first-order derivative curve; point B is the second derivative zero point value of the first-order derivative curve; and point C is the extreme value point of the first-order derivative curve. Three parameters can be obtained from the first-order derivative curve of the experimental well, point D is the first derivative zero point value of the first-order derivative curve; point E is the second derivative zero point value of the first-order derivative curve; point F is the extreme point of the first-order derivative curve, and the parameters for calculating the activity of the thrombin-activated fibrinolysis inhibitor are: TAFI overall antifibrinolysis time difference (min) = AB-DE, TAFI initial antifibrinolysis time difference (min) = AC-DF, TAFI comprehensive antifibrinolytic ability = Log10 (integral value of the control well - experimental well area * 100), where AB is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the control well, and DE is the first zero point value and the second zero point value of the first-order derivative curve of the experimental well. AC is the difference between the extreme point and the first zero point of the first-order derivative curve of the control well, DF is the difference between the extreme point and the first zero point of the first-order derivative curve of the experimental well, the control group-experimental group area is the difference between the areas under the curve between the two zero points of the reaction curves of the experimental group and the control group, the parameter significance of the overall antifibrinolytic time difference of TAFI is the effect of all TAFI activation on the overall fibrinolytic function with or without inhibitors, the parameter significance of the initial antifibrinolytic time difference of TAFI is the effect of TAFIa activated in a cascade manner after thrombin formation on fibrinolysis with or without inhibitors, and the parameter significance of the comprehensive antifibrinolytic ability of TAFI is to reflect the antifibrinolytic effect of TAFIa in the whole process of fibrinolysis.

[0016] Compared with the prior art, the present invention provides a method for detecting the activity of thrombin-activated fibrinolysis inhibitor in blood. By real-time dynamic monitoring of the curve of the absorbance change of plasma over time, the curve difference between the addition of thrombin-activated fibrinolysis inhibitor and the addition of inhibitor of thrombin-activated fibrinolysis inhibitor is compared, and the relevant parameters representing the activity of thrombin-activated fibrinolysis inhibitor are calculated. The method has the advantages of convenient operation and easy sample acquisition. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1For comparison of different concentrations of recombinant thrombomodulin;

[0018] Figure 2 Comparison of inhibitors of activated thrombin-activated fibrinolysis inhibitors at different concentrations;

[0019] Figure 3 For comparison of different concentrations of tissue plasminogen activator;

[0020] Figure 4 Schematic diagram of the experimental group, control group and blank group of the 96-well plate;

[0021] Figure 5 It is a schematic diagram of the reaction curve of the absorbance changes of the experimental group, the control group and the blank group;

[0022] Figure 6 Schematic diagram of the difference curve and first-order derivative curve between the experimental group and the control group. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Reagent Description:

[0025] Recombinant Human Thrombomodulin(TM)

[0026] DS-1040: Inhibitor of activated thrombin-activated fibrinolysis inhibitor

[0027] Tissue plasminogen activator (tPA)

[0028] APTT actin FSL reagent

[0029] Example 1: Optimization of recombinant human thrombomodulin (TM) concentration

[0030] 1. Test sample: Normal human mixed plasma (NPP)

[0031] 2. Sample preparation process

[0032] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP; aspirate the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP); aspirate the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. The above operations were completed within 2 hours after fresh whole blood collection. 10 men and 10 women were randomly selected from the healthy population, and their PFP was fully mixed to obtain normal mixed plasma (NPP), which was aliquoted in small amounts and stored at -80°C for subsequent experimental applications.

[0033] 3. Experimental steps

[0034] Take out the NPP sample and incubate at 37℃ for 5 minutes. Prepare the solution: 0.25mol / L Ca2+ working solution and 12ug / mL tPA reagent working solution, with final concentrations of 0.025mol / L and 0.6ug / mL respectively; dilute the 3uM recombinant human TM stock solution in a gradient manner, as shown in Table 1. Add 35 microliters of NPP, 10 microliters of distilled water, 5 microliters of tPA solution, 35 microliters of actin FSL solution, 5 microliters of recombinant human TM solution of different concentrations and 10 microliters of calcium ion solution to the experimental wells of the 96-well plate, place the 96-well plate in an optical density scanner, shake and mix evenly, measure the absorbance at a wavelength of 980nm at intervals of 15 seconds, and detect for 100 minutes. The curve of the change of plasma absorbance value in the experimental well over time can be automatically drawn by the optical density scanner.

[0035] Table 1 Concentration of recombinant human TM after gradient dilution

[0036]

[0037] 4. Experimental results

[0038] When the working concentration of TM solution was higher than 100 nM, the time to reach the platform of the fibrinolysis curve was basically no longer prolonged. The 100 nM TM solution could effectively activate TAFI. The optimized working concentration of TM was 100 nM, that is, the final concentration was 5 nM.

[0039] Example 2: Optimization of concentration of inhibitor of activated thrombin-activated fibrinolysis inhibitor (DS-1040)

[0040] 1. Test sample: Normal human mixed plasma (NPP)

[0041] 2. Sample preparation process

[0042] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP; aspirate the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP); aspirate the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. The above operations were completed within 2 hours after fresh whole blood collection. 10 men and 10 women were randomly selected from the healthy population, and their PFP was fully mixed to obtain normal mixed plasma (NPP), which was aliquoted in small amounts and stored at -80°C for subsequent experimental applications.

[0043] 3. Experimental steps

[0044] Take out the NPP sample and incubate at 37℃ for 5 minutes. Prepare solutions: 0.25mol / L Ca2+ working solution, 100nM TM working solution and 12ug / mL tPA reagent working solution, with final concentrations of 0.025mol / L, 5nM and 0.6ug / mL respectively; dilute the 50uM activated thrombin activated fibrinolysis inhibitor inhibitor (DS-1040) stock solution in a gradient manner, as shown in Table 2. Add 35 microliters of NPP and 10 microliters of DS-1040 solution of different concentrations to the experimental wells of the 96-well plate, incubate for 5 minutes, then add 5 microliters of TM solution, 5 microliters of tPA solution, 35 microliters of actin FSL solution and 10 microliters of calcium ion solution, place the 96-well plate in an optical density scanner, shake and mix evenly, measure the absorbance at a wavelength of 980nm at intervals of 15 seconds, and detect for 100 minutes. The curve of the change of plasma absorbance value in the experimental well over time can be automatically drawn by the optical density scanner.

[0045] Table 2 Concentrations of inhibitor of activated thrombin-activated fibrinolysis inhibitor (DS-1040) after gradient dilution

[0046]

[0047] 4. Experimental results

[0048] When the working concentration of DS-1040 solution is lower than 7.2uM, the effect of inhibiting TAFIa is weak, which limits the detection of high concentration TAFI. When the working concentration of DS-1040 solution is higher than 7.2uM, the inhibitory effect on TAFIa is basically saturated. From the perspective of experimental cost and efficiency, the optimized working concentration of DS-1040 solution is 7.2uM, that is, the final concentration is 720nM.

[0049] Example 3: Optimization of tissue plasminogen activator (tPA) concentration

[0050] 1. Test sample: Normal human mixed plasma (NPP)

[0051] 2. Sample preparation process

[0052] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP; aspirate the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP); aspirate the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. The above operations were completed within 2 hours after fresh whole blood collection. 10 men and 10 women were randomly selected from the healthy population, and their PFP was fully mixed to obtain normal mixed plasma (NPP), which was aliquoted in small amounts and stored at -80°C for subsequent experimental applications.

[0053] 3. Experimental steps

[0054] Take out the NPP sample and incubate at 37℃ for 5 minutes. Prepare solutions: 0.25mol / L Ca2+ working solution, 100nM TM working solution and 7.2uM DS-1040 working solution, with final concentrations of 0.025mol / L, 5nM and 720nM respectively; dilute the 200ug / mL tissue plasminogen activator (tPA) stock solution in gradients, as shown in Table 3. Add 35 microliters of NPP and 10 microliters of DS-1040 solution to the experimental wells of the 96-well plate, incubate for 5 minutes, then add 5 microliters of TM solution, 5 microliters of tPA solution with different concentration gradients, 35 microliters of actin FSL solution and 10 microliters of calcium ion solution, place the 96-well plate in an optical density scanner, shake and mix evenly, measure the absorbance at a wavelength of 980nm at intervals of 15 seconds, and detect for 100 minutes. The curve of the change of plasma absorbance value in the experimental well over time can be automatically drawn by the optical density scanner.

[0055] Table 3 tPA concentration after gradient dilution

[0056]

[0057] 4. Experimental results

[0058] The purpose of adding tPA is to promote fibrinolysis and shorten the detection time. When the working concentration of tPA is higher than 12ug / mL, its curve will basically not stop fibrinolysis early, so the optimized working concentration of tPA is 12ug / mL, that is, the final concentration is 0.6ug / mL.

[0059] Example 4: Establishment of TAFI activity detection method

[0060] 1. Test sample: Normal human mixed plasma (NPP)

[0061] 2. Sample preparation process

[0062] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP; aspirate the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP); aspirate the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. The above operations were completed within 2 hours after fresh whole blood collection. 10 men and 10 women were randomly selected from the healthy population, and their PFP was fully mixed to obtain normal mixed plasma (NPP), which was aliquoted in small amounts and stored at -80°C for subsequent experimental applications.

[0063] 3. Experimental steps

[0064] Take out the NPP sample and incubate at 37°C for 5 minutes. Prepare solutions: 0.25mol / L Ca2+ working solution, 100nM TM working solution, 7.2uM DS-1040 working solution and 12ug / mL tPA working solution, with final concentrations of 0.025mol / L, 5nM, 720nM and 0.6ug / mL respectively. 35 μl NPP and 10 μl DS-1040 solution were added to the experimental wells, 35 μl NPP and 10 μl distilled water were added to the control wells, and 35 μl NPP and 20 μl distilled water were added to the blank wells. The wells were incubated for 5 minutes, and then 5 μl TM solution and 5 μl tPA solution were added only to the experimental wells and control wells. Finally, 35 μl actin FSL solution and 10 μl calcium ion solution were added to the experimental wells, control wells and blank wells. The 96-well plate was placed in an optical density scanner, shaken and mixed evenly, and the absorbance was measured at a wavelength of 980 nm at intervals of 15 seconds for 100 minutes. The curve of the change of plasma absorbance values ​​in the experimental wells, the experimental wells and the blank wells over time can be automatically drawn by the optical density scanner, and the experimental parameters are calculated. The test was carried out 3 times a day for 3 consecutive days, and one NPP sample was tested each time. Each NPP sample was tested independently. The overall antifibrinolytic time difference of TAFI, the initial antifibrinolytic time difference of TAFI, and the comprehensive antifibrinolytic ability of TAFI were calculated according to the following formulas: intra-batch precision (%) = (standard deviation / mean value) × 100%, inter-batch precision (%) = (standard deviation / mean value) × 100, and the parameters were obtained to analyze the precision results.

[0065] 4. Experimental results

[0066] The precision results are shown in Table 4. The intra-batch precision is less than 10%, and the inter-batch precision is less than 15%. The precision results are small, indicating that this method has good stability.

[0067]

[0068]

[0069] 5. Biological reference intervals

[0070] 1) Test samples

[0071] 120 cases of PFP in healthy people, 57 women and 63 men, were divided into groups according to age: 18-30 years (25 cases), 31-40 years (40 cases), 41-51 years (21 cases), 51-60 years (17 cases), and ≥61 years (17 cases).

[0072] 2) Sample preparation process

[0073] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP. Pipette the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP). Pipette the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. All the above operations should be completed within 2 hours after the fresh whole blood is collected.

[0074] 3) Experimental procedures

[0075] Take out the PFP sample and incubate at 37°C for 5 minutes. Prepare solutions: 0.25mol / L Ca2+ working solution, 100nM TM working solution, 7.2uM DS-1040 working solution and 12ug / mL tPA working solution, with final concentrations of 0.025mol / L, 5nM, 720nM and 0.6ug / mL, respectively. Add 35 μl NPP and 10 μl DS-1040 solution to the experimental wells, 35 μl NPP and 10 μl distilled water to the control wells, and 35 μl NPP and 20 μl distilled water to the blank wells, incubate for 5 minutes, then add 5 μl TM solution and 5 μl tPA solution only to the experimental wells and control wells, finally add 35 μl actin FSL solution and 10 μl calcium ion solution to the experimental wells, control wells and blank wells, place the 96-well plate on an optical density scanner, shake and mix evenly, measure the absorbance at 980 nm at intervals of 15 seconds, and detect for 100 minutes. The curve of the change of plasma absorbance values ​​in the experimental wells, the experimental wells and the blank wells over time can be automatically drawn by the optical density scanner to calculate the experimental parameters.

[0076] 4) Experimental results

[0077] The results of TAFI activity detection by age and gender are shown in Table 5. The biological reference intervals of TAFI overall antifibrinolytic time difference, TAFI initial antifibrinolytic time difference, and TAFI comprehensive antifibrinolytic ability were established as 1.71-29.95 min, 3.36-14.30 min, and 2.98-3.77 U / L·min, respectively.

[0078] Table 5 TAFI activity test results of healthy people

[0079]

[0080] Note: Quantitative data with normal distribution are expressed as mean ± standard deviation, and quantitative data with non-normal distribution are expressed as median (25th percentile, 75th percentile).

[0081] Example 5: Clinical application of TAFI activity detection method

[0082] 1. Selection of research subjects

[0083] 84 subjects (42 in the non-traumatic femoral head necrosis group and 42 in the healthy control group).

[0084] Inclusion criteria: patients aged ≥15 years, diagnosed with non-traumatic femoral head necrosis by clinicians. Exclusion criteria: patients with benign tumor lesions or general malignant tumor lesions of the hip joint, no X-ray results, history of thrombosis, history of anticoagulation or antiplatelet therapy.

[0085] 2. Acquisition of relevant information about the research subjects

[0086] The subjects' age, gender, smoking and drinking history, past medical history (malignant tumors, hypertension, diabetes, autoimmune system diseases), medication history (hormone use, anticoagulant or antiplatelet drug use), surgical history in the past six months, consultation time, X-ray imaging results, specimen collection time and other relevant clinical information were obtained based on their outpatient and inpatient medical records.

[0087] 3. Sample preparation process

[0088] Place the whole blood in a 3.2% citrate anticoagulant tube and centrifuge at 2500g for 15 minutes at 25°C. The upper plasma is PPP. Pipette the upper plasma and transfer it to a 1.5ml Eppendorf tube. Centrifuge the Eppendorf tube containing PPP again at 2500g for 15 minutes at 25°C. The upper plasma is platelet-poor plasma (PFP). Pipette the upper plasma into a 0.5ml Eppendorf tube for small aliquots and store at -80°C for subsequent experimental applications. All the above operations should be completed within 2 hours after the fresh whole blood is collected.

[0089] 4. Experimental steps

[0090] Take out the PFP sample and incubate at 37°C for 5 minutes. Prepare solutions: 0.25mol / L Ca2+ working solution, 100nM TM working solution, 7.2uM DS-1040 working solution and 12ug / mL tPA working solution, with final concentrations of 0.025mol / L, 5nM, 720nM and 0.6ug / mL, respectively. Add 35 μl NPP and 10 μl DS-1040 solution to the experimental wells, 35 μl NPP and 10 μl distilled water to the control wells, and 35 μl NPP and 20 μl distilled water to the blank wells, incubate for 5 minutes, then add 5 μl TM solution and 5 μl tPA solution only to the experimental wells and control wells, finally add 35 μl actin FSL solution and 10 μl calcium ion solution to the experimental wells, control wells and blank wells, place the 96-well plate on an optical density scanner, shake and mix evenly, measure the absorbance at 980 nm at intervals of 15 seconds, and detect for 100 minutes. The curve of the change of plasma absorbance values ​​in the experimental wells, the experimental wells and the blank wells over time can be automatically drawn by the optical density scanner to calculate the experimental parameters.

[0091] 5. Statistical analysis

[0092] Data processing and statistical analysis were performed using MATLAB software (R2023a version, MathWorks, USA) and SPSS software (26.0 version, IBM, USA). MATLAB software was used to fit the reaction curve and perform first-order derivative parameter analysis. SPSS software was used to test the normality of the measurement data. Normally distributed quantitative data were expressed as mean ± standard deviation, and non-normally distributed quantitative data were expressed as median (25th percentile, 75th percentile). The Student's T test was used to compare the two groups of quantitative data with normal distribution, and the Mann-Whitney U test was used to compare the two groups of quantitative data with non-normal distribution. The chi-square test or Fisher's exact probability method was used to assess whether the difference between the two groups of count data was statistically significant.

[0093] 6. Experimental results

[0094] 1) There were no significant differences in parameters between 42 patients with non-traumatic femoral head necrosis (NONFH) and 42 healthy controls (control), and the P values ​​were all greater than 0.05.

[0095] 2) The TAFI activity of patients with non-traumatic femoral head necrosis was higher than that of healthy people, and the difference was statistically significant.

[0096] Table 6 Demographic characteristics and clinical data of the study subjects

[0097]

[0098] Note: Quantitative data of normal distribution are expressed as mean ± standard deviation, and quantitative data of non-normal distribution are expressed as median (25th percentile, 75th percentile). The P values ​​between groups were all greater than 0.05.

[0099] Table 7 Results of TAFI activity parameters in patients with non-traumatic femoral head necrosis and healthy controls

[0100]

[0101] It can be seen from the above implementation cases that based on the reference interval of TAFI activity of healthy people established in Example 4, the TAFI activity of clinical patients is detected and the TAFI activity of patients is compared with that of healthy people, which can be used to evaluate the TAFI activity of patients.

[0102] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the activity of a thrombin-activated fibrinolysis inhibitor, characterized in that: The following steps are involved: 1) Prepare reagents for the experimental group, control group and blank group: the reagents for the experimental group include the sample to be tested, inhibitor reagent of activated thrombin-activated fibrinolysis inhibitor (TAFIa), thrombomodulin reagent, tissue-type plasminogen activator reagent, actin FSL reagent and calcium ion solution; the reagents for the control group include the sample to be tested, thrombomodulin reagent, tissue-type plasminogen activator reagent, actin FSL reagent, calcium ion solution and distilled water; the reagents for the blank group include the test sample, actin FSL reagent, calcium ion solution and distilled water; 2) Absorbance detection: Before the absorbance detection, the samples to be tested were first added with TAFIa inhibitor or distilled water according to the reagents of the experimental group, control group and blank group and incubated at room temperature for 5 minutes, and then other reagents were added and mixed respectively. The curve of the change of plasma absorbance over time of the experimental group, control group and blank group was automatically drawn using an optical density scanner; 3) Process data and determine parameters: Subtract the absorbance value of the blank group from the absorbance value of the experimental group and the control group, respectively, to obtain the absorbance difference at different time points, which is used to draw a curve of the absorbance difference changing with time, perform curve fitting and calculate the first-order derivative, obtain the first-order derivative curve, and determine the parameters (A) TAFI overall antifibrinolytic time difference (min) = AB-DE; (B) TAFI initial antifibrinolytic time difference (min) = AC-DF; (C) TAFI comprehensive antifibrinolytic ability = Log10 (integral value of the area of ​​the control group-experimental group * 100); AB is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the control group, DE is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the experimental group; AC is the difference between the extreme point and the first zero point value of the first-order derivative curve of the control group, DF is the difference between the extreme point and the first zero point value of the first-order derivative curve of the experimental group, and the control group-experimental group area is the difference between the areas under the curve between the two zero points of the reaction curves of the experimental group and the control group.

2. The method for detecting the activity of thrombin-activated fibrinolysis inhibitor according to claim 1, characterized in that: The preparation steps of the sample to be tested in step (1) are as follows: a blood sample is collected within 2 hours and centrifuged at 2500g for 15 minutes at room temperature, the upper layer of plasma is aspirated and placed in a centrifuge tube and centrifuged again at 2500g for 15 minutes, the upper layer of platelet-poor plasma is aspirated and frozen for storage, and warmed at 37°C for 5 minutes before testing, wherein the anticoagulant used is sodium citrate.

3. The method for detecting the activity of thrombin-activated fibrinolysis inhibitor according to claim 1, characterized in that: In the step (1), the concentration of TAFIa inhibitor is 720 nM, the concentration of tissue plasminogen activator is 0.6 μg / ml, the concentration of thrombomodulin is 5 nM, the concentration of calcium ions is 0.025 mol / L, and the actin FSL reagent is a mixture of soybean lecithin, rabbit cerebroside and ellagic acid, wherein the volume ratio of the plasma to be tested to the actin FSL reagent is 1:

1.

4. The method for detecting the activity of thrombin-activated fibrinolysis inhibitor according to claim 1, characterized in that: In the step (2), the reagents of the experimental group, the control group and the blank group are respectively placed in a 96-well transparent plate, the wells containing the reagents of the experimental group are experimental wells, the wells containing the reagents of the control group are control wells, and the wells containing the reagents of the blank group are blank wells. The volume in the wells is 100 μl, so the detection system of the experimental wells includes: 35 μl of plasma, 10 μl of TAFIa inhibitor, 5 μl of thrombomodulin, 5 μl of tissue-type plasminogen activator, 35 μl of actin FSL reagent, and 10 μl of calcium ions. In the control wells, 10 ul of distilled water is used to replace the TAFIa inhibitor. The detection system includes: 35 μl of plasma, 10 μl of distilled water, 5 μl of thrombomodulin, 5 μl of tissue-type plasminogen activator, 35 μl of actin FSL reagent, and 10 μl of calcium ions. In the blank wells, 20 ul of distilled water is used to replace the TAFIa inhibitor, thrombomodulin and tissue-type plasminogen activator. The detection system includes: 35 μl of plasma, 20 μl of distilled water, and actin 35 μl of FSL reagent and 10 μl of calcium ion.

5. The method for detecting the activity of thrombin-activated fibrinolysis inhibitor according to claim 1, characterized in that: The step (2) is performed according to a method comprising the following steps: at a detection temperature of 37°C, first, 35 μl of the sample to be tested and 10 μl of the TAFIa inhibitor DS-1040 solution are added to the experimental wells, 35 μl of the sample to be tested and 10 μl of distilled water are added to the control wells, and 35 μl of the sample to be tested and 20 μl of distilled water are added to the blank wells, and the plates are incubated at room temperature for 5 minutes. Then, 5 μl of the TM solution and 5 μl of the tPA solution are added to the experimental wells and the control wells. Then, 35 μl of the actin FSL solution and 10 μl of the calcium ion solution are added to the experimental wells, the control wells and the blank wells. Finally, the 96-well plate is placed in an optical density scanner, vibrated for 10 seconds, and continuous detection is started. The detection wavelength is 980 nm, the detection interval is 15 seconds, and the detection duration is 100 minutes.

6. The method for detecting the activity of thrombin-activated fibrinolysis inhibitor according to claim 1, characterized in that: The step (3) first outputs the absorbance change data of the experimental wells, the control wells, and the blank wells through an optical density scanner, and averages the data of the duplicate wells. Then, the experimental well difference data is obtained by subtracting the average absorbance of the blank wells from the average absorbance of the experimental wells, and the control well difference data is obtained by subtracting the average absorbance of the blank wells from the average absorbance of the control wells. Then, the experimental well difference data and the control well difference data are respectively subjected to curve fitting, and the fitting is performed using a feedforward neural network. The fitting effect is evaluated by R2 and the fitting curve. R2 greater than 0.9 is a better fitting effect. A difference curve and a fitting curve are generated. Further, a first-order derivative and a curve are generated based on the fitting data to obtain the extreme point, area integral, first zero point value and second zero point value of the curve. Three parameters can be obtained in the first-order derivative curve of the control well. Point A is the first derivative zero point value of the first-order derivative curve; Point B is the second derivative zero point value of the first-order derivative curve; and Point C is the extreme point of the first-order derivative curve. Three parameters can be obtained from the first-order derivative curve of the experimental well. Point D is the first derivative zero point value of the first-order derivative curve; point E is the second derivative zero point value of the first-order derivative curve; point F is the extreme point of the first-order derivative curve. The parameters for calculating the activity of thrombin-activated fibrinolytic inhibitor are: TAFI overall antifibrinolytic time difference (min) = AB-DE, TAFI initial antifibrinolytic time difference (min) = AC-DF, TAFI comprehensive antifibrinolytic ability = Log10 (integral value of control well-experimental well area*100), wherein AB is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the control well, DE is the difference between the first zero point value and the second zero point value of the first-order derivative curve of the experimental well, AC is the difference between the extreme point and the first zero point value of the first-order derivative curve of the control well, DF is the difference between the extreme point and the first zero point value of the first-order derivative curve of the experimental well, the control group-experimental group area is the difference in the area under the curve between the two zero points of the reaction curves of the experimental group and the control group, the parameter meaning of TAFI overall antifibrinolytic time difference is the effect of all TAFI activation on the overall fibrinolytic function in the presence or absence of inhibitors, the parameter meaning of TAFI initial antifibrinolytic time difference is the effect of TAFIa activated in a waterfall manner after thrombin formation on fibrinolysis in the presence or absence of inhibitors, and the parameter meaning of TAFI comprehensive antifibrinolytic ability is to reflect the antifibrinolytic effect of TAFIa in the whole process of fibrinolysis.

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