Thrombin-antithrombin III complex assay kit and its preparation method and application

Abstract

The present invention relates to a thrombin-antithrombin III complex antigen complex and a preparation method thereof and a thrombin-antithrombin III complex assay kit, wherein the thrombin-antithrombin III complex is cross-linked with thrombin and a cross-linking agent Sulfo-EGS, and then cross-linked with antithrombin III. The thrombin-antithrombin III complex assay kit is a chemiluminescence method, using the thrombin-antithrombin III complex as a proofreader, and the kit has the advantages of high sensitivity, not easily interfered, good stability, and strong specificity. The detection method of the thrombin-antithrombin III complex of the present invention achieves the possibility of less detection sample usage and faster detection, and provides a better choice for clinical TAT quantification.

Description

Technical Field

[0001] The invention belongs to the field of medical diagnosis, and in particular relates to a thrombin-antithrombin III complex enzymatic chemiluminescence assay kit and a preparation method and application thereof. Background Art

[0002] The sign of coagulation is the formation of thrombin. Thrombin can promote the conversion of fibrinogen in plasma into insoluble fibrin, which is closely related to the formation of thrombus. However, the half-life of thrombin produced in the human body is short, so it is very difficult to directly measure thrombin. After prothrombin is activated, it can quickly covalently link with antithrombin III to form thrombin-antithrombin III complex (TAT), which has a long half-life (10 to 30 minutes), so the concentration of thrombin can be reflected by measuring the concentration of TAT.

[0003] In general cases, prothrombin is converted into thrombin, which is quickly neutralized by antithrombin III. Direct measurement of prothrombin content cannot detect the generation of thrombin in the blood. In contrast, TAT can detect a very small degree of proenzyme conversion. Since TAT can be used as a molecular marker for activation of the coagulation system and can sensitively reflect the coagulation status in the body, it is of great significance for monitoring the coagulation status under physiological and pathological conditions of the body and the diagnosis of thrombotic diseases.

[0004] There are relatively few TAT detection methods developed so far, mainly the tube chemiluminescence method. As a chemiluminescence immunoassay technology with obvious advantages in immunoassay technology, it has significant advantages in terms of accuracy and sensitivity of the measurement results. At present, the only imported related product on the market is the Thrombin-Antithrombin III Complex Detection Kit (chemiluminescence method) produced by Sysmex Medical Electronics (Shanghai) Co., Ltd., which is used for the quantitative detection of thrombin-antithrombin III complex (TAT) in human plasma. However, there is currently no mature method in China for the detection of thrombin-antithrombin III complex using chemiluminescence technology. Summary of the invention

[0005] One of the purposes of the present invention is to provide a thrombin-antithrombin III complex, which antigen can be used to prepare a thrombin-antithrombin III complex assay kit using a chemiluminescent method, so that the kit has the characteristics of high sensitivity, good stability, and low susceptibility to interference.

[0006] A thrombin-antithrombin III complex is formed by cross-linking thrombin with a cross-linking agent Sulfo-EGS and then cross-linking with antithrombin III. The dosage ratio of thrombin to the cross-linking agent Sulfo-EGS is 5 to 50:1, and the dosage ratio of thrombin to antithrombin III is 1:0.8 to 1.5.

[0007] In some of the embodiments, the mass ratio of the blood enzyme to the cross-linking agent Sulfo-EGS is 6-40:1, 7-30:1, 8-20:1, and more preferably 9-10:1.

[0008] In some embodiments, the dosage ratio of thrombin to antithrombin III is 1:1 to 1.1.

[0009] Another object of the present invention is to provide a method for preparing the above thrombin-antithrombin III complex.

[0010] The method for preparing the above-mentioned thrombin-antithrombin III complex comprises the following steps:

[0011] A. Take thrombin and replace the stock solution with PBS buffer, add Sulfo-EGS, incubate at room temperature for 0.8-1.5h to activate, and obtain a mixed solution;

[0012] B. Take 10 mg of antithrombin III and replace the stock solution with PBS buffer, then add all of it to the above mixed solution and incubate at room temperature for 1 h to 1.5 h for cross-linking;

[0013] C. After cross-linking is completed, desalting and purification are performed, and mixing is performed to obtain a solution of thrombin-antithrombin III complex.

[0014] In some embodiments, in step A, the incubation is 1 h to 1.5 h, preferably 1 h.

[0015] In some embodiments, in step B, the incubation is 1 h to 1.2 h, preferably 1 h.

[0016] Another object of the present invention is to provide a thrombin-antithrombin III complex assay kit.

[0017] A thrombin-antithrombin III complex assay kit comprises the above-mentioned thrombin-antithrombin III complex as a calibrator.

[0018] In some embodiments, at least one of the following is also included:

[0019] A solution of magnetic microspheres conjugates coated with thrombin monoclonal antibodies,

[0020] A solution of an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody,

[0021] Substrate liquid.

[0022] In some of the embodiments, the magnetic microsphere conjugate coated with thrombin monoclonal antibody is a conjugate of magnetic particles and thrombin-antithrombin III complex antibody; the diameter of the magnetic particles is 1000 to 3000 nanometers; in the solution of the magnetic microsphere conjugate coated with thrombin monoclonal antibody, the concentration of the magnetic microsphere conjugate coated with antithrombin monoclonal antibody is 0.1 to 0.3 mg / mL.

[0023] In some embodiments, the magnetic microsphere conjugate coated with thrombin monoclonal antibody is prepared by washing, connecting, blocking and diluting activated magnetic microparticles with a diameter of 1000-3000 nanometers and thrombin monoclonal antibody.

[0024] In some embodiments, the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody is a conjugate of alkaline phosphatase and anti-thrombin III monoclonal antibody.

[0025] In some of the embodiments, in the solution of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody, the concentration of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody is 0.4-0.6 μg / mL.

[0026] In some of the embodiments, the thrombin-antithrombin III complex assay kit provided by the present invention has a calibrator: the thrombin-antithrombin III complex is diluted with a 0.05 mol / L Tris-HCl diluent (containing 0.15 mol / L NaCl, 1 w / v% BSA, 0.1 w / v% Tween-20, 0.05 w / v% ProClin300) with a pH of 7.5 to a concentration of 4 to 20 ng / mL.

[0027] Another object of the present invention is to provide a method for preparing the above-mentioned thrombin-antithrombin III complex assay kit, comprising the following steps:

[0028] (1) Preparation of tosyl-coated magnetic microparticles coated with monoclonal antibody against thrombin:

[0029] Take tosyl-modified magnetic microparticles, wash the magnetic microparticles with PBS (phosphate) buffer, remove the supernatant by magnetic separation, resuspend with BBS (borate) buffer, add ammonium sulfate solution, disperse evenly to obtain mixed solution 1, then add thrombin monoclonal antibody to obtain mixed solution 2; then place mixed solution 2 on a rotary shaker for incubation, remove the supernatant by magnetic separation, wash with Tris buffer, remove the supernatant, then add BSA solution for blocking treatment, remove the supernatant by magnetic separation, wash with Tris buffer, and finally resuspend with Tris buffer to obtain tosyl-modified magnetic microparticles coated with thrombin monoclonal antibody.

[0030] The concentration of the PBS buffer is 0.01M and the pH is 7.4; the pH value of the borate buffer is 9.0-10.0, and the concentration of the borate buffer is 0.08-0.12 mol / L; the pH of the ammonium sulfate buffer is 9.0-10.0, and the concentration of the ammonium sulfate buffer is 2-4 mol / L; in the mixed solution 1, the concentration of the activated magnetic particles is 9-11 mg / mL; in the mixed solution 2, the concentration of the thrombin monoclonal antibody is 180-220 μg / mL.

[0031] (2) Preparation of enzyme-labeled conjugates labeled with antithrombin III monoclonal antibody:

[0032] Take 10 mg of anti-thrombin III monoclonal antibody, replace the liquid in the original antibody with PBS (phosphate) buffer, then add 1 mg of DSG, incubate at room temperature for 1 hour to activate the antibody, and obtain mixed solution 3; take 10 mg of alkaline phosphatase, replace the liquid in the original alkaline phosphatase with PBS (phosphate) buffer, immediately add all of it to mixed solution 3, incubate at room temperature for 1 hour, cross-link, and obtain mixed solution 4; after completion, desalt and purify the mixture 4, add an equal volume of glycerol, mix well, and obtain an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody.

[0033] (3) Preparation of thrombin-antithrombin III complex:

[0034] Take 10 mg of thrombin and replace the stock solution with PBS buffer, add 0.2-2 mg of Sulfo-EGS, incubate at room temperature for 1 hour for activation, and obtain mixed solution 5; take 8-15 mg of antithrombin III and replace the stock solution with PBS (phosphate) buffer, immediately add all of it to mixed solution 5, incubate at room temperature for 1 hour for cross-linking. After the cross-linking is completed, desalt and purify, mix well, and obtain a solution of thrombin-antithrombin III complex. The concentration of the PBS buffer is 0.01M and the pH is 7.4.

[0035] Another object of the present invention is to provide a method for detecting a thrombin-antithrombin III complex, comprising the following steps:

[0036] Obtaining samples to be tested;

[0037] The above thrombin-antithrombin III complex was used as a calibrator to prepare a standard curve;

[0038] Add the sample to be tested and the magnetic microsphere conjugate coated with thrombin monoclonal antibody into the reaction tube for the first incubation treatment and washing;

[0039] Add a solution of an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody, perform a second incubation treatment to form a double antibody sandwich immune complex, and wash;

[0040] Add substrate solution and measure the luminescent signal;

[0041] The luminescent signal value of the sample to be tested is calculated using the standard curve to obtain the concentration of the thrombin-antithrombin III complex.

[0042] In some of the embodiments, the volume ratio of the solution of the magnetic microsphere conjugate coated with antithrombin monoclonal antibody to the sample to be tested is 4.5-5.5:1; and the time of the first incubation treatment is 4-6 minutes.

[0043] In some of the embodiments, the volume ratio of the solution of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody to the sample to be tested is 4.5-5.5:1, and the time of the second incubation treatment is 4-6 minutes.

[0044] The kit provided by the present invention connects thrombin monoclonal antibody to the surface of magnetic microspheres as a solid phase reagent, and forms an enzyme-labeled antibody sandwich immune complex after capturing the thrombin-antithrombin III complex in the sample and adding enzyme-labeled antithrombin III monoclonal antibody reagent.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] The present invention provides a method for preparing a thrombin-antithrombin III complex, by controlling a reasonable ratio and incubation time, a thrombin-antithrombin III complex with a good signal value is obtained, the problem of no commercial thrombin-antithrombin III complex available is solved, and the antigen complex is used as a calibrator, and finally the preparation of a thrombin-antithrombin III complex assay kit by an enzymatic chemiluminescence method is realized. The kit has the advantages of high sensitivity, good stability, and good repeatability of test results, provides a better choice for clinical TAT (thrombin-antithrombin III complex) quantification, and has high cost performance compared with imported reagents, and has certain application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Schematic diagram of the detection process of the thrombin-antithrombin III complex assay reagent of the present invention.

[0048] Figure 2 : Schematic diagram of the results of the influence of different cross-linking agents on the cross-linking effect in Example 2 of the present invention.

[0049] Figure 3 : Schematic diagram of the results of preparing thrombin-antithrombin III complex under different mass ratios of thrombin to Sulfo-EGS.

[0050] Figure 4: Schematic diagram of the results of preparing thrombin-antithrombin III complex under different ratios of thrombin and antithrombin III.

[0051] Figure 5 : Schematic diagram of the results of preparing thrombin-antithrombin III complex under different cross-linking agent incubation times at room temperature.

[0052] Figure 6 : Schematic diagram of the results of preparing thrombin-antithrombin III complex under different incubation time conditions with antithrombin III after thrombin cross-linking.

[0053] Figure 7 : Schematic diagram of the linearity test results of the thrombin-antithrombin III complex (TAT) assay kit in Example 3 of the present invention.

[0054] Figure 8 : Schematic diagram of the comparison test results between the thrombin-antithrombin III complex assay kit used in Example 4 of the present invention and the kit of Sysmex Corporation. DETAILED DESCRIPTION

[0055] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0056] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0057] The terms "comprises" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps or components, but may optionally include steps not listed, or may optionally include other steps or components inherent to these processes, methods, products or apparatus.

[0058] In the present invention, "multiple" refers to two or more. "And / or" describes the association relationship of the associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. Among them, 1w / v% = 10g / L.

[0059] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0060] Example 1

[0061] This embodiment provides a thrombin-antithrombin III complex assay kit, which includes (1) a solution of a magnetic microsphere conjugate coated with a thrombin monoclonal antibody; (2) a solution of an enzyme-labeled conjugate labeled with an antithrombin III monoclonal antibody; (3) a substrate solution; and (4) a calibrator.

[0062] The magnetic microsphere conjugate coated with thrombin monoclonal antibody is a conjugate of magnetic microparticles and thrombin monoclonal antibody; the diameter of the magnetic microparticles is 1000 nanometers to 3000 nanometers; in the solution of the magnetic microsphere conjugate coated with thrombin monoclonal antibody, the concentration of the magnetic microsphere conjugate coated with thrombin monoclonal antibody is 0.2 mg / mL. The concentration of the thrombin monoclonal antibody in the magnetic microsphere conjugate is 4 μg / mL.

[0063] In the solution of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody, the concentration of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody is 0.5 μg / mL.

[0064] The substrate solution is a substrate solution containing APS-5; the manufacturer of the substrate solution is Guangzhou Wondfo Biotech Co., Ltd.

[0065] The preparation of the thrombin-antithrombin III complex is obtained by coupling thrombin and antithrombin III. The calibration standard: dilute the prepared thrombin-antithrombin III complex with a diluent to a concentration of 4 ng / mL and 20 ng / mL, and store at 2-8°C. The diluent can be a diluent with a pH of 7.5 containing 0.15 mol / L NaCl, 1 w / v% BSA, 0.1 w / v% Tween-20, 0.05 w / v% ProClin300 and 0.05 mol / L Tris-HCl.

[0066] The preparation method of the thrombin-antithrombin III complex assay kit comprises the following steps:

[0067] (1) Preparation of tosyl magnetic particles coated with thrombin monoclonal antibody:

[0068] Take 20 mg of tosylated magnetic microparticles, wash the magnetic microparticles with PBS (phosphate) buffer, remove the supernatant by magnetic separation, resuspend with 1.9 mL BBS (borate) buffer, add 0.1 mL of ammonium sulfate solution, disperse evenly, and obtain mixed solution 1, then add 0.4 mg of thrombin monoclonal antibody to obtain mixed solution 2. Then place mixed solution 2 on a rotary shaker for incubation for 18 hours (hours); after magnetic separation and removal of the supernatant, wash the magnetic microparticles with Tris buffer, remove the supernatant, add 2 mL of blocking solution containing BSA for blocking treatment, place on a rotary shaker for incubation for 8 hours, after magnetic separation and removal of the supernatant, wash the magnetic microparticles with Tris buffer, and finally resuspend the magnetic microparticles with 2 mL of Tris buffer to obtain tosylated magnetic microparticles coated with thrombin monoclonal antibody.

[0069] Thrombin monoclonal antibody (antibody produced by thrombin immunity, also known as thrombin antibody): Greenmoab, catalog number: GMA-020.

[0070] Anti-thrombin III monoclonal antibody (antibody produced by anti-thrombin III immunization, also known as anti-thrombin III antibody) Greenmoab, catalog number: MA-800.

[0071] Magnetic particles: JSR, catalog number: MS160 / Tosyl.

[0072] Thrombin: Sigma, catalog number: 10602400001.

[0073] Antithrombin III: Sigma, catalog number: A2221.

[0074] The concentration of the PBS buffer is 0.01 M and the pH is 7.4.

[0075] The borate buffer has a concentration of 0.1 mol / L and a pH of 9.5.

[0076] The concentration of the ammonium sulfate buffer solution is 3 mol / L, and the pH value is 9.5.

[0077] The formula of the buffer solution for blocking the magnetic particles and the thrombin monoclonal antibody is 1w / v% BSA solution.

[0078] The Tris buffer contains 0.15 mol / L NaCl, 1 w / v% BSA, 0.1 w / v% Tween-20, 0.05 w / v% ProClin300, 0.05 mol / L Tris-HCl, and has a pH value of 7.5.

[0079] (2) Preparation of enzyme-labeled conjugates labeled with anti-thrombin III monoclonal antibodies:

[0080] Take 10 mg of anti-thrombin III monoclonal antibody, replace the liquid in the original antibody with PBS (phosphate) buffer, then add 1 mg of DSG, incubate at room temperature for 1 hour, activate the antibody, and obtain mixed solution 3; take 10 mg of alkaline phosphatase, replace the liquid in the original alkaline phosphatase with PBS (phosphate) buffer, immediately add all of it to mixed solution 3, incubate at room temperature for 1 hour, cross-link, and obtain mixed solution 4. After completion, desalt and purify the mixture 4, add an equal volume of glycerol, mix well, and obtain an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody. The concentration of the PBS buffer is 0.01M and the pH is 7.4. DSG is from ThermoFisher.

[0081] (3) Preparation of thrombin-antithrombin III complex:

[0082] Take 10 mg of thrombin and replace the stock solution with PBS buffer, add 1 mg of Sulfo-EGS (ethylene glycol bis (sulfosuccinimidyl succinate)), incubate at room temperature for 0.8 to 1.5 hours for activation, and obtain mixed solution 5; take 10 mg of antithrombin III and replace the stock solution with PBS (phosphate) buffer, immediately add all of it to mixed solution 5, incubate at room temperature for 1 to 2 hours for cross-linking. After the cross-linking is completed, desalt and purify, mix well, and obtain a solution of thrombin-antithrombin III complex. The concentration of the PBS buffer is 0.01M and the pH is 7.4. Sulfo-EGS is from ThermoFisher.

[0083] (4) Preparation of calibrators: The thrombin-antithrombin III complex prepared in this example was diluted with 0.05 mol / L Tris-HCl calibrator diluent having a pH of 7.5 and containing 0.15 mol / L NaCl, 1 w / v% BSA, 0.1 w / v% Tween-20, and 0.05 w / v% ProClin300 to a concentration of 4 ng / mL and 20 ng / mL, and stored at 2-8°C.

[0084] Example 2 Comparison of Antigen Coupling Conditions

[0085] The changes of this example compared with Example 1 include the comparison of the preparation conditions of the thrombin-antithrombin III complex:

[0086] (1) Selection of cross-linking agent:

[0087] Three experimental groups were set up. In each group, 10 mg of thrombin was used to replace the stock solution with PBS buffer, and 1 mg of Sulfo-EGS, Sulfo-MBS and DSS were added respectively, and incubated at room temperature for 1 hour to activate and obtain a mixed solution; 10 mg of antithrombin III was used to replace the stock solution with PBS (phosphate) buffer, and all were immediately added to the above three mixed solutions, incubated at room temperature for 1 hour for cross-linking. After the cross-linking was completed, desalting and purification were carried out, and mixing was carried out to obtain 10 mL of thrombin-antithrombin III complex solution.

[0088] (2) Comparison of mass ratio conditions between thrombin and Sulfo-EGS:

[0089] Three experimental groups were set up. In each group, 10 mg of thrombin was taken to replace the stock solution with PBS buffer, and 0.125 mg, 0.2 mg, 0.5 mg, 0.83 mg, 1 mg, 1.25 mg, 2 mg, and 5 mg of Sulfo-EGS were added, so that the mass ratio of thrombin to Sulfo-EGS in each group was 80:1, 50:1, 20:1, 12:1, 10:1, 8:1, 5:1, and 2:1. The mixture was incubated at room temperature for 1 hour for activation to obtain a mixed solution. 10 mg of antithrombin III was taken to replace the stock solution with PBS (phosphate) buffer, and all of them were immediately added to the above three mixed solutions, incubated at room temperature for 1 hour for cross-linking. After the cross-linking was completed, desalting and purification were performed, and mixing was performed to obtain 10 mL of thrombin-antithrombin III complex solution.

[0090] (3) Comparison of the ratio of thrombin and antithrombin III:

[0091] Three experimental groups were set up. In each group, 10 mg of thrombin was taken to replace the stock solution with PBS buffer, 1 mg of Sulfo-EGS was added, and the mixture was incubated at room temperature for 1 hour for activation to obtain a mixed solution; 5 mg, 8 mg, 10 mg, 12 mg, 15 mg, and 18 mg of antithrombin III were taken to replace the stock solution with PBS (phosphate) buffer, and all of them were immediately added to the above three groups of mixed solutions, so that the ratio of thrombin and antithrombin III was 1:0.5, 1:0.8, 1:1, 1:2, 1:1.5, and 1:1.8, and the mixture was incubated at room temperature for 1 hour for cross-linking. After the cross-linking was completed, the mixture was desalted and purified, and mixed to obtain 10 mL of thrombin-antithrombin III complex solution.

[0092] (4) Comparison of cross-linking agent room temperature incubation time:

[0093] Two experimental groups were set up, one of which was to set different crosslinker incubation times: 10 mg of thrombin was replaced with PBS buffer, 1 mg of Sulfo-EGS was added to the two groups, and they were incubated at room temperature for 0.5 h, 0.8 h, 1 h, 1.5 h, and 2 h, respectively, to activate and obtain a mixed solution; 10 mg of antithrombin III was replaced with PBS (phosphate) buffer, and all of it was immediately added to the mixed solution, incubated at room temperature for 1 h for crosslinking. After the crosslinking was completed, desalting and purification were performed, and mixing was performed to obtain 10 mL of thrombin-antithrombin III complex solution. The concentration of the PBS buffer was 0.01 M, and the pH was 7.4. Sulfo-EGS was from ThermoFisher.

[0094] The other group is the incubation time of thrombin with antithrombin III after cross-linking: that is, 10 mg of thrombin was replaced with PBS buffer, 1 mg of Sulfo-EGS was added to the two groups, and they were incubated at room temperature for 1 hour respectively to activate and obtain a mixed solution; 10 mg of antithrombin III was replaced with PBS (phosphate) buffer, and all of it was immediately added to the mixed solution, incubated at room temperature for 0.8, 1h, 1.2h, 1.5h, and 2h for cross-linking. After the cross-linking is completed, desalting and purification are carried out, and mixing is carried out to obtain 10 mL of thrombin-antithrombin III complex solution. The concentration of the PBS buffer is 0.01M and the pH is 7.4. Sulfo-EGS is from ThermoFisher.

[0095] During the detection, the thrombin-antithrombin III complex prepared under different cross-linking agent conditions, different thrombin to Sulfo-EGS mass ratio conditions, different thrombin and antithrombin III ratio conditions, and different cross-linking agent room temperature incubation time conditions were diluted 200 times and tested using the thrombin-antithrombin III complex assay kit of Example 1, and clinical samples with a TAT concentration of 4 ng / mL (near the cut-off value) were used as controls. The test results are as follows: Figure 2-5 The luminescence value>95000 (the luminescence value obtained from clinical samples with a concentration of about 4ng / mL) meets the requirements. The luminescence value reflects the TAT concentration. The higher the luminescence value, the better the cross-linking effect.

[0096] according to Figure 2 The test results show that different cross-linking agents have a great influence on the cross-linking effect, among which Sulfo-EGS cross-linking agent has the best cross-linking effect. Figure 3As shown in the figure, thrombin-antithrombin III complex was prepared under different mass ratios of thrombin and Sulfo-EGS crosslinker. When the mass ratio of thrombin to Sulfo-EGS was 80:1, the luminescence was lower than the control, which did not meet the requirements. As the ratio of Sulfo-EGS crosslinker increased, the luminescence value increased, and the crosslinking effect was improved. When the mass ratio of thrombin to Sulfo-EGS was 10:1, the crosslinking effect reached the highest value. When the crosslinker ratio was increased, the crosslinking effect decreased. Therefore, the mass ratio of thrombin to Sulfo-EGS was 10:1, which was the optimal ratio of thrombin and crosslinker Sulfo-EGS. Figure 4 The thrombin-antithrombin III complex was prepared at different ratios of thrombin and antithrombin III. When the ratio of thrombin and antithrombin III was below 1:0.8, the luminescence value was lower than the control. As the ratio of thrombin and antithrombin III increased, the cross-linking effect was improved, and when the ratio of thrombin and antithrombin III was 1:1, it was relatively optimal. When the ratio of antithrombin III was increased, the signal decreased until it was lower than the control group. Figure 5 As shown in the figure, when the crosslinker is incubated at room temperature for 0.5h, the luminescence is lower than that of the control group. As the incubation time increases, the luminescence value increases, and the crosslinking effect increases. When the crosslinker is incubated at room temperature for 1h, 1.5h, and 2h, the luminescence value is greater than that of the control group and tends to be balanced. Therefore, the crosslinker is optimal when the incubation time is 1h, which is short and has a good crosslinking effect. Figure 6 As shown, when the incubation time with antithrombin III after thrombin cross-linking was 0.8h, the luminescence was lower than that of the control group. As the incubation time was prolonged, the luminescence value increased, and the cross-linking effect was improved. When the incubation time with antithrombin III was 1h, 1.2h, and 1.5h, the luminescence value was greater than that of the control group and tended to be balanced. However, when the incubation time with antithrombin III was increased to 2h, it decreased instead. Therefore, the optimal incubation time of the cross-linking agent is 1h, which is short and has a good cross-linking effect. The above-mentioned instrument is a fully automatic chemiluminometer, and the reaction process is as follows Figure 1 The diluent formula used for diluting the antigen is 0.15 mol / L NaCl, 1 w / v% BSA, 0.1 w / v% Tween-20, 0.05 w / v% ProClin300 in 0.05 mol / L Tris-HCl, with a pH value of 7.5.

[0097] Example 3: Performance evaluation of the kit of the present invention

[0098] 1. Standard curve

[0099] The thrombin-antithrombin III complex described in Example 1 was diluted with a standard diluent to prepare calibration solutions S0-S9 of different concentrations, with concentrations of 0, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 4.0 ng / mL, 9.0 ng / mL, 18 ng / mL, 36 ng / mL, 60 ng / mL, and 120 ng / mL, respectively, and stored at -20°C for use. Then, the calibrator was detected using the kit provided in Example 1 of the present invention, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The standard curve was obtained by fitting with the concentration as the horizontal axis and the luminescence intensity as the vertical axis.

[0100] 2. Linear

[0101] The basic serum sample (concentration ≤ blank limit) or the calibration diluent (try to use the real high-value serum sample) with added antigen is used as the high-concentration sample, and the basic serum sample or the calibration diluent is used as the low-concentration sample. The high-value sample close to the upper limit of the linear range is diluted to several concentrations according to a certain ratio (for example: linear sample preparation table), and the low-value concentration sample must be close to the lower limit of the linear range. The sample of each concentration is tested 2-3 times and the average value is calculated. The concentration value of the analyte (as the horizontal axis) and the average value of the test result (as the vertical axis) are fitted with a straight line using the least squares method. And calculate the correlation coefficient r within the linear range, and the acceptance standard is correlation coefficient r ≥ 0.99.

[0102] Figure 7 Linear analysis of thrombin-antithrombin III complexes prepared under different conditions. Group 1: Antigen coupling conditions: cross-linking agent is Sulfo-EGS, the mass ratio of thrombin to Sulfo-EGS is 10:1, the ratio of thrombin to antithrombin III is 1:1, the cross-linking agent incubation time at room temperature is 1h, and the incubation time of thrombin with antithrombin III after cross-linking is 1h. Group 2: Antigen coupling conditions: cross-linking agent is Sulfo-EGS, the mass ratio of thrombin to Sulfo-EGS is 50:1, the ratio of thrombin to antithrombin III is 1:1, the cross-linking agent incubation time at room temperature is 1h, and the incubation time of thrombin with antithrombin III after cross-linking is 1h. Group 3: Antigen coupling conditions: cross-linking agent is Sulfo-EGS, the mass ratio of thrombin to Sulfo-EGS is 10:1, the ratio of thrombin to antithrombin III is 1:0.8, the cross-linking agent incubation time at room temperature is 1h, and the incubation time of thrombin with antithrombin III after cross-linking is 1h. Group 4: Antigen coupling conditions: cross-linking agent is Sulfo-EGS, the mass ratio of thrombin to Sulfo-EGS is 10:1, the ratio of thrombin to antithrombin III is 1:1, the cross-linking agent incubation time at room temperature is 0.5h, and the incubation time of thrombin with antithrombin III after cross-linking is 1h. Figure 7From the linear results, it can be seen that the overall signal of group 1 is higher than that of other groups at the same concentration, r>0.99, the result is optimal and meets the requirements.

[0103]

[0104] 3. Minimum detection limit

[0105] The dilution of the zero-concentration calibrator was used as a sample for detection, and the measurement was repeated 20 times to obtain the light signal values ​​of the 20 measurement results. The mean value (M) and standard deviation (SD) were calculated to obtain M+2SD. A two-point regression fitting was performed based on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibrator to obtain a linear equation. The light signal value of M+2SD was substituted into the equation to obtain the corresponding concentration value, which is the minimum detection limit. The results are shown in Table 1.

[0106] 4. Accuracy

[0107] Prepare samples with high and low concentrations using thrombin-antithrombin III complex, repeat the measurement three times, calculate the relative deviation between the average value (denoted as M) and the labeled value, and calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples with high and low concentrations that have been determined by the reference method, repeat the measurement three times for each concentration sample, take the average of the test results, and calculate the relative deviation from the labeled value. Acceptance standard: relative deviation within ±10%.

[0108] B=(MT) / T×100%-------(1)

[0109] B—relative deviation;

[0110] M—average value of measured concentration;

[0111] T—denoted value.

[0112] 5. Precision

[0113] (1) Repeatability evaluation

[0114] The samples with 2 to 3 concentration levels (high level, low level) were tested 10 times each, and the mean value M and standard deviation SD of the 10 measurement results were calculated. The coefficient of variation CV was obtained according to formula (2).

[0115] CV=SD / M×100%----------(2)

[0116] CV—coefficient of variation;

[0117] SD—standard deviation of 10 measurements;

[0118] M—the average value of 10 measurement results.

[0119] (2) Batch-to-batch precision

[0120]

[0121] Where:

[0122] I = total number of days (20, 2 batches per day)

[0123] (Average of two replicates)

[0124] (Average of two replicates)

[0125] A is calculated by taking the square root of the sum of the squares of the differences between the first batch of analysis means and the second batch of analysis means for all days, divided by 2I.

[0126] 6. Interfering substances

[0127] Weigh the interfering substance to be studied. Prepare a stock solution of a certain concentration of the interfering substance. Add bilirubin, triglyceride, hemoglobin and total protein stock solutions to the high and low value plasma used for interference according to the calibrated concentrations. Prepare high concentration interfering substance plasma samples containing bilirubin, triglyceride, hemoglobin and total protein at the concentrations required by the specifications. The control plasma sample is not added with interfering substances. The added volume is controlled within 1 / 20 to avoid the matrix effect of the solvent. Test the high concentration interfering substance sample and the control plasma sample 2-3 times respectively. The mean result of the high concentration interfering substance sample test is recorded as M. The mean result of the control plasma sample test is recorded as T, and the relative deviation B of the measured concentration is calculated according to formula (4).

[0128] B=(MT) / T×100%------------(4)

[0129] Where: Add the interfering substance to the high and low value plasma

[0130] B—relative deviation;

[0131] M—mean value of the concentration of the substance to be tested in samples with high and low concentrations of interfering substances;

[0132] T—mean value of the analyte concentration in control plasma samples.

[0133] The experimental results are shown in Table 1.

[0134] Table 1. Partial performance evaluation results of the thrombin-antithrombin III complex assay kit

[0135]

[0136] Example 4. Clinical performance of the kit of the present invention

[0137] 111 clinical plasma samples were simultaneously tested using the kit of Example 1 of the present invention and an international brand kit (Sysmex kit).

[0138] The detection method of the kit of the present invention (refer to Figure 1 shown), including:

[0139] The sample to be tested and the magnetic microsphere conjugate coated with thrombin monoclonal antibody are added to the reaction tube for the first incubation treatment, the volume ratio of the solution of the magnetic microsphere conjugate coated with thrombin monoclonal antibody to the sample to be tested is 5:1; the time of the first incubation treatment is 5 minutes, and the sample is washed. An enzyme-labeled conjugate labeled with antithrombin III monoclonal antibody is added, and a second incubation treatment is performed to form a double antibody sandwich immune complex, the volume ratio of the enzyme-labeled conjugate labeled with antithrombin III monoclonal antibody to the sample to be tested is 5:1; the time of the second incubation treatment is 5 minutes, and the sample is washed. Substrate solution is added to determine the luminescent signal. The luminescent signal value of the sample to be tested is calculated by the main curve to obtain the concentration of its thrombin-antithrombin III complex.

[0140] The concentration of the above-mentioned samples was also tested using the test kit of Sysmex Corporation (the test was performed according to the instructions of the kit).

[0141] The detection concentration obtained by the kit prepared in the embodiment of the present invention was analyzed and compared with the detection result concentration of the Sysmex kit (instrument model is SYSMEX HISCL-800), and the clinical correlation results are as follows: Figure 8 As shown, the clinical relevance is R 2 =0.902, the result meets the acceptance criteria (acceptance criteria: R 2 >0.9), indicating that the kit of the present invention has a good correlation with the internationally renowned brand kit (Sysmex kit).

[0142] The detection concentration obtained by the kit prepared in Example 1 of the present invention and the detection result concentration of the Sysmex kit (instrument model is SYSMEX HISCL-800) were statistically analyzed for the positive and negative coincidence rate, and the coincidence rate was calculated according to formulas (5)-(7) based on the reference interval (or clinical judgment value).

[0143] Yin-Yang Conformity Rate Statistics Table

[0144]

[0145] Positive coincidence rate = a / (a+c)×100%-----------(5)

[0146] Negative coincidence rate = d / (b+d)×100%-----------(6)

[0147] Total compliance rate = (a+d) / (a+b+c+d)×100%-----------(7)

[0148] The results of the positive and negative coincidence rates are shown in Table 2. The total coincidence rate is 90.99%, and the results meet the acceptance criteria.

[0149] Table 2. Statistics of positive and negative coincidence rates of thrombin-antithrombin III complex assay kit

[0150]

[0151]

[0152] The above experiments show that the present invention provides a method for preparing a thrombin-antithrombin III complex, which solves the problem that there is no commercial thrombin-antithrombin III complex available, and is successfully applied to the development of the thrombin-antithrombin III complex assay kit. The quantitative detection of TAT in the sample is realized, and it has the advantages of high detection sensitivity, high accuracy, good stability, simple operation, high throughput, etc., which can meet the needs of clinical TAT quantitative detection. And compared with imported reagents, the price is relatively low, the cost performance is high, it can alleviate the economic burden of patients, and has certain application value. It is consistent with the expected use of the same variety of imported products of Xisenmeikang that have been listed, and can guide clinical diagnosis and treatment in a timely and accurate manner.

[0153] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A thrombin-antithrombin III complex, characterized in that: The invention is prepared by cross-linking thrombin with a cross-linking agent Sulfo-EGS and then cross-linking with antithrombin III. The mass ratio of thrombin to the cross-linking agent Sulfo-EGS is 10-50:1, and the mass ratio of thrombin to antithrombin III is 1:0.8-1.

0.

2. The thrombin-antithrombin III complex according to claim 1, characterized in that: The mass usage ratio of the thrombin to the cross-linking agent Sulfo-EGS is 10:

1.

3. The thrombin-antithrombin III complex according to claim 1 or 2, characterized in that: The mass dosage ratio of the thrombin to antithrombin III is 1:

1.

4. A method for preparing the thrombin-antithrombin III complex according to claim 1, characterized in that: The following steps are involved: A. replace the stock solution of thrombin with PBS buffer, add Sulfo-EGS, and incubate at room temperature for 0.8h to 1.5h for activation to obtain a mixed solution; B. After replacing the stock solution of antithrombin III with PBS buffer, immediately add all of it to the above mixed solution and incubate at room temperature for 1 hour to 1.5 hours for cross-linking; C. After cross-linking is completed, desalting and purification are performed, and mixing is performed to obtain a solution of thrombin-antithrombin III complex.

5. The method for preparing the thrombin-antithrombin III complex according to claim 4, characterized in that: In step A, the incubation is 1 h to 1.5 h; And / or in step B, the incubation is 1 h to 1.2 h.

6. A thrombin-antithrombin III complex assay kit, characterized in that: The thrombin-antithrombin III complex according to any one of claims 1 to 3 is included as a calibrator.

7. The thrombin-antithrombin III complex assay kit according to claim 6, characterized in that: Also includes at least one of the following: A solution of a magnetic microsphere conjugate coated with a monoclonal antibody to thrombin, A solution of an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody, Substrate liquid.

8. The thrombin-antithrombin III complex assay kit according to claim 7, characterized in that: The magnetic microspheres in the magnetic microsphere conjugate coated with thrombin monoclonal antibody are magnetic microparticles.

9. The thrombin-antithrombin III complex assay kit according to claim 8, characterized in that: The diameter of the magnetic particles is 1000-3000 nanometers.

10. The thrombin-antithrombin III complex assay kit according to claim 7, characterized in that In the solution of the magnetic microsphere conjugate coated with the thrombin monoclonal antibody, the concentration of the magnetic microsphere conjugate coated with the thrombin monoclonal antibody is 0.1-0.3 mg / mL.

11. The thrombin-antithrombin III complex assay kit according to claim 7, characterized in that: The enzyme-labeled conjugate labeled with antithrombin III monoclonal antibody is a conjugate of alkaline phosphatase and antithrombin III monoclonal antibody.

12. The thrombin-antithrombin III complex assay kit according to claim 10, characterized in that: The concentration of the enzyme-labeled conjugate labeled with antithrombin III monoclonal antibody is 0.4-0.6 μg / mL.

13. The thrombin-antithrombin III complex assay kit according to claim 6, characterized in that: The calibrator is prepared by diluting the thrombin-antithrombin III complex with Tris-HCl calibrator diluent, with a concentration of 4 to 20 ng / mL.

14. A method for detecting thrombin-antithrombin III complex for non-diagnostic purposes, characterized in that: The following steps are involved: Obtaining samples to be tested; The thrombin-antithrombin III complex according to any one of claims 1 to 3 is used as a calibrator to prepare a standard curve; the sample to be tested and the magnetic microsphere conjugate coated with the thrombin monoclonal antibody are added to a reaction tube for a first incubation treatment and washing; Add a solution of an enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody, perform a second incubation treatment to form a double antibody sandwich immune complex, and wash; Add substrate solution and measure the luminescent signal; The luminescent signal value of the sample to be tested is calculated using the standard curve to obtain the concentration of the thrombin-antithrombin III complex.

15. The method for detecting thrombin-antithrombin III complex for non-diagnostic purposes according to claim 14, characterized in that: The volume ratio of the solution of the magnetic microsphere conjugate coated with the thrombin monoclonal antibody to the sample to be tested is 4.5-5.5:1; and the time of the first incubation treatment is 4-6 minutes.

16. The method for detecting thrombin-antithrombin III complex for non-diagnostic purposes according to claim 14 or 15, characterized in that: The volume ratio of the solution of the enzyme-labeled conjugate labeled with anti-thrombin III monoclonal antibody to the sample to be tested is 4.5-5.5:1, and the time of the second incubation treatment is 4-6 minutes.

Images