Kit for quantitative detection of thrombin-antithrombin III compound and preparation method thereof

Abstract

The invention belongs to the technical field of immunodetection analysis, and discloses a kit for quantitative detection of a thrombin-antithrombin III compound and a preparation method of the kit. The kit comprises a magnetic bead working solution, an acridinium ester marker working solution, a biotin marker working solution, a TAT calibrator and a TAT quality control product, and quantitative detection of TAT is realized by combining a double-antibody sandwich method with a chemiluminescence technology. The method overcomes the defects that an existing fluorescence immunoassay method is low in repeatability, and an immunoturbidimetry method is high in requirement for specimens and limited in low-concentration detection capacity, and has the advantages of being high in specificity, sensitivity and accuracy and easy, convenient and rapid to operate. The method can be used for clinical auxiliary diagnosis of thrombogenic diseases such as disseminated intravascular coagulation (DIC) and venous thromboembolism (VTE), and provides a basis for blood coagulation state evaluation and clinical decision making.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay technology, and particularly relates to a thrombin-antithrombin III complex quantitative detection kit and a preparation method thereof. BACKGROUND

[0002] Thrombin-antithrombin III complex (TAT) has a molecular weight of about 58.2 kD, is a single-chain glycoprotein, contains 4 glycosylation sites, and the sugar component accounts for about 15%. It is a stable complex formed by the 1:1 combination of thrombin and antithrombin III (AT III). When the vascular endothelium is damaged or the coagulation system is activated, prothrombin is converted into thrombin under the action of coagulation factors, and AT III, as a natural anticoagulant protein, forms TAT by combining with thrombin, thereby terminating the activity of thrombin.

[0003] TAT is mainly distributed in plasma, and its elevated level indicates the generation of a large amount of thrombin in the body, reflecting the excessive activation of the coagulation system, and is commonly seen in hypercoagulable state, acute phase of thrombosis, or high coagulation phase of disseminated intravascular coagulation (DIC). It is a sensitive indicator for dynamic monitoring of coagulation activation. In thrombotic diseases, significant elevation of TAT is closely related to the risk of venous thromboembolism (VTE) and arterial thrombosis (such as myocardial infarction and ischemic stroke), and can be used for diagnosis, efficacy evaluation and recurrence risk prediction; in liver diseases (such as cirrhosis, liver cancer and acute liver failure), the level of TAT is related to the degree of liver function damage and can indicate poor prognosis; in cardiovascular and cerebrovascular diseases (such as atrial fibrillation and cryptogenic stroke), elevated TAT level reflects potential hypercoagulable state and helps etiological diagnosis and risk assessment; in trauma (such as traumatic brain injury) and intensive care (such as sepsis), persistent elevation of TAT is associated with poor prognosis and indicates the risk of secondary damage. In addition, TAT can be used to evaluate the efficacy of thrombolytic therapy and guide anticoagulant therapy to prevent VTE in perioperative management, and its level is affected by gender (women are higher than men) and age (the elderly have elevated levels). Dynamic monitoring of TAT helps to comprehensively evaluate the coagulation state, accurately guide anticoagulant therapy, and timely predict the prognosis of the disease, providing key basis for clinical decision-making.

[0004] The current clinical methods for detecting TAT include fluorescence immunoassay, immunoturbidimetry and chemiluminescence: Although fluorescence immunoassay has high sensitivity and multi-labeling capability, endogenous fluorescent substances in the sample can interfere with the detection results, and the fluorescence signal can decay under long-time light, resulting in poor repeatability of the detection; Immunoturbidimetry is based on the transmission or scattering principle of light, which is easily interfered by substances such as lipoprotein to produce false positive results, and has limited detection capability for low concentration TAT; Chemiluminescence method uses high sensitivity luminescence reaction technology, less affected by sample matrix, wide detection range, high degree of automation, and the accuracy and reliability of the results are better than the first two methods, which has become the mainstream technology for clinical TAT detection in medical institutions at all levels.

[0005] However, the existing chemiluminescence method TAT detection kit still has some optimization space, such as the antibody labeling efficiency of some kits is insufficient, the magnetic bead binding performance is unstable, which leads to the detection sensitivity or repeatability not reaching the ideal level. Therefore, it is of great significance to develop a chemiluminescence method TAT quantitative detection kit with better performance and stable preparation process for improving the quality of clinical detection. SUMMARY

[0006] The present application provides a kind of for thrombin-anti-thrombin III complex quantitative detection kit and preparation method thereof, to solve the problem of low repeatability of fluorescence immunoassay, high requirement of immunoturbidimetry on sample.

[0007] The present application is realized in this way, a kind of for thrombin-anti-thrombin III complex quantitative detection kit, comprising: streptavidin coated magnetic bead working solution; biotin-labeled anti-TAT antibody working solution; acrid ester-labeled anti-TAT antibody working solution; TAT calibrator, including at least two different concentrations of calibration points; TAT quality control product, including at least two different concentrations of quality control points; Optionally, it also includes cleaning fluid, substrate liquid and other auxiliary reagents.

[0008] Preferably, in the magnetic bead working solution, the particle size of the magnetic beads is 2.8-3.2 μm, the concentration is 1.0-3.0 mg / mL, the magnetic beads are coupled with streptavidin on the magnetic beads through active groups (such as carboxyl, amino, etc.), the coating amount of streptavidin is 20 µg / mg, after fixation, the active binding pocket of streptavidin can be combined with biotin through non-covalent force, and the magnetic beads are suspended in a magnetic bead diluent with pH of 7.40±0.1, which contains BSA, sodium casein, and trehalose.

[0009] Preferably, in the biotin-labeled anti-TAT antibody working solution, the antibody concentration is 0.1-1.0 μg / mL, dissolved in a biotin diluent with pH of 7.40±0.1, and containing ANS stabilizer.

[0010] Preferably, in the acrid ester-labeled anti-TAT antibody working solution, the antibody concentration is 0.1-1.0 μg / mL, dissolved in an acrid ester diluent with pH of 6.0±0.1, and the buffer solution contains BSA and trehalose.

[0011] Preferably, the TAT calibrator includes two calibration points with concentrations of 10 ng / mL and 50 ng / mL.

[0012] Preferably, the TAT quality control includes two levels with target values ​​of 10±2 ng / mL (quality control L) and 50±10 ng / mL (quality control M).

[0013] Preferably, the anti-TAT antibody is a mouse-derived monoclonal antibody.

[0014] This invention provides a method for preparing the above-mentioned reagent kit, comprising the following steps: (1) Preparation of magnetic bead working solution: The streptavidin-coated magnetic beads were prepared into a working solution with a concentration of 1.0 to 3.0 mg / mL using magnetic bead diluent; (2) Preparation of biotin-labeled working solution: a. The anti-TAT antibody was reacted with Sulfo-NHS-LC-Biotin in PBS buffer at pH 7.4, and the biotin-labeled antibody was obtained after dialysis purification; b. Dilute the biotin-labeled product obtained in step a with biotin diluent to 0.1–1.0 μg / mL, add ANS solution (used to monitor whether the antibody denatures, aggregates, or produces nonspecific hydrophobic plaques during the labeling process), mix well, and the product is obtained. (3) Preparation of working solution for acridine ester labeling: a. Reaction of anti-TAT antibody with acridine ester in labeling buffer at pH 8.0, followed by chromatography using a gel permeation column (e.g., PD MidiTrap). TM Purify with G-25 and collect the eluent; b. Add antibody preservation solution and glycerol to the elution buffer; c. Dilute the acridine ester labeled product obtained in step b with acridine ester diluent to a concentration of 0.1–1.0 μg / mL to obtain the final product; (4) Preparation of TAT calibrators and quality control samples: Dilute the purified TAT antigen (purchased from Hangzhou Inco Biotechnology Co., Ltd.) to the required concentration with PBS buffer containing 0.5% BSA (pH 7.4±0.1); (5) Dispensing and assembly: Dispense the above components according to the predetermined specifications and assemble them into a kit.

[0015] The present invention also provides the application of the above-described kit in the in vitro detection of thrombin-antithrombin III complex content in human plasma samples for non-diagnostic purposes.

[0016] This invention provides a method for quantitatively detecting thrombin-antithrombin III complexes in a sample, comprising the steps of using the above-mentioned kit, wherein the steps include: a) mixing and incubating the sample, biotin-labeled anti-TAT antibody working solution and acridinium ester-labeled anti-TAT antibody working solution to form a complex; b) adding streptavidin-coated magnetic bead working solution, incubating, and allowing the complex to bind to the magnetic beads; c) performing magnetic separation and washing; d) adding a chemiluminescent substrate and detecting a luminescence signal; e) calculating the content of TAT in the sample according to a calibration curve.

[0017] Compared with the prior art, the embodiments of the present application have the following beneficial effects: High sensitivity and wide linear range: the antibody labeling process, reaction system and buffer formula are optimized in the present application, so that the blank limit (LoB) of the kit is not higher than 0.4 ng / mL, and the linearity is good (r≥0.9900) in the range of 0.4-120 ng / mL, which can accurately detect low and high concentration samples.

[0018] Excellent precision and repeatability: the within-batch CV is not more than 8%, the between-batch CV is not more than 10%, and the recovery rate is between 85%-115%, which ensures the reliability of the detection results.

[0019] Strong anti-interference ability: the performance of the kit is not interfered by high concentrations of bilirubin (20 mg / dL), triglyceride (1400 mg / dL), hemoglobin (500 mg / dL), rheumatoid factor (RF 100 IU / mL) or antinuclear antibody (ANA 1:160).

[0020] Good stability: by introducing ANS, trehalose, glycerol and other stabilizers, the stability of the labeled antibody working solution is significantly improved, which is beneficial to the long-term storage and transportation of the kit.

[0021] No HOOK effect: no HOOK effect is observed at a concentration of up to 1000 ng / mL, which ensures the accuracy of high-value sample detection. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a structural schematic diagram of a thrombin-antithrombin III complex quantitative detection kit provided by the present application; Figure 2 is a whole structural schematic diagram of a thrombin-antithrombin III complex quantitative detection kit provided by the present application. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0024] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0025] Example 1 This invention provides a kit for quantitative detection of thrombin-antithrombin III complex, comprising: Working solution for streptavidin-coated magnetic beads; Biotin-labeled anti-TAT antibody working solution; Acridinium ester-labeled anti-TAT antibody working solution; TAT calibrators include at least two calibration points with different concentrations; TAT quality control materials include at least two quality control points with different concentrations; like Figure 1 As shown, the preparation method of the thrombin-antithrombin III complex quantitative detection kit includes the following steps: (1) Preparation of magnetic bead working solution Magnetic bead dilution solution: Add 11.53g Na2HPO4·12H2O, 2.28g NaH2PO4·2H2O, 8.41g NaCl, 5.01g BSA, 1.50g Casein Na, 1.01g trehalose, and 2.00mL Proclin 300 to 1000mL of purified water. Stir to dissolve and adjust the pH to 7.40±0.1 with HCl / NaOH. Magnetic bead working solution: Take 1~5mg (preferably 3mg in this embodiment) of amino-modified streptavidin magnetic beads with a particle size of 2.8~3.2μm (preferably 3μm in this embodiment), add 1mL of the above magnetic bead diluent, vortex mix well, and a magnetic bead working solution with a concentration of 3.0mg / mL is obtained.

[0026] (2) Preparation of biotin label working solution Biotin labeling buffer: 1000 mL purified water, 2.90 g Na2HPO4·12H2O, 0.30 g NaH2PO4·2H2O, 9.00 g NaCl, pH adjusted to 7.40±0.1; Biotin antibody storage solution: 50 mL purified water, 0.146 g Na2HPO4·12H2O, 0.015 g NaH2PO4·2H2O, 0.448 g NaCl, 1 g BSA.

[0027] Biotin label: Raw materials: 1-5 mg of TAT antibody, 10 mg of Sulfo-NHS-LC-Biotin, 4000 mL of biotin labeling buffer, and glycerol.

[0028] The preparation method of the biotin label is as follows: 1) Dilute the antibody to 1-5 mg / mL with the biotin labeling buffer, mix well after adding the dialysis bag, and dialyze in the biotin labeling buffer at room temperature for 3 hours; 2) Prepare 100 μL of 10 mg / mL Sulfo-NHS-LC-Biotin, add it to the dialysis bag and mix well, and react at room temperature for 1 hour; 3) Dialyze overnight with the biotin labeling buffer; 4) Take the label out of the dialysis bag, add it to a suitable container, and add the biotin labeling buffer to make up the volume to a final concentration of 1-5 mg / mL; 5) Add 1 / 5 volume of biotin antibody storage solution and glycerol. Biotin diluent: 1000 mL purified water, 115.07 g Na2HPO4·12H2O, 22.82 g NaH2PO4·2H2O, 84.20 g NaCl, 50.23 g BSA, and 10 mL Proclin 300, pH adjusted to 7.40±0.1; ANS solution: 50 mg ANS dissolved in 1 mL DMF; Biotin label working solution: Take 0.1-3 μg of biotin label, add 1 mL of biotin diluent and 0.02 mL of ANS solution, and mix well.

[0029] (3) Preparation of acridinium ester label working solution Acridinium ester labeling buffer: 1000 mL purified water, 3.39 g Na2HPO4·12H2O, 0.08 g NaH2PO4·2H2O, 9.01 g NaCl, pH adjusted to 8.0±0.1; Acridinester antibody storage solution: 1000 mL purified water, 2.14 g Na2HPO4·12H2O, 10.33 g NaH2PO4·2H2O, 8.45 g NaCl, 22.22 g BSA, pH adjusted to 6.0±0.1; Acridinester column passing solution: 1000 mL purified water, 2.13 g Na2HPO4·12H2O, 10.21 g NaH2PO4·2H2O, 8.40 g NaCl, pH adjusted to 6.0±0.1; Acridinester dilution solution: 1000 mL purified water, 21.32 g Na2HPO4·12H2O, 102.20 g NaH2PO4·2H2O, 84.21 g NaCl, 50.00 g BSA, 50.01 g trehalose, 10.00 mL Proclin 300, pH adjusted to 6.0±0.1.

[0030] Acridinester label The following materials are used: 1~5 mg TAT antibody, 5 mg acridinester (such as NSP-SA-NHS), 2000 mL acridinester labeling buffer, 50 mL acridinester antibody storage solution, 1000 mL acridinester column passing solution, PD MidiTrap TM G-25 1, glycerol, DMF 2.5 mL.

[0031] The preparation method of the acridinester label is as follows: 1) The desalting column is vertically fixed on the iron stand, the plugs at both ends of the column are removed, the liquid in the column is in a straight line state, and the column is passed with the acridinester column passing solution. After completion, part of the passing liquid is left in the column, and the plugs at both ends of the column body are sealed for standby.

[0032] 2) The antibody is diluted with the acridinester labeling buffer to 1~5 mg / mL. The required antigen and 1000 mL acridinester labeling buffer are added to the dialysis bag, mixed gently, and placed in the acridinester labeling buffer for dialysis at room temperature for 3 hours.

[0033] 3) The liquid in the dialysis bag is taken out and added to a suitable container, and the acridinester and DMF are added and placed in a rotary mixing instrument for reaction at room temperature for 1 hour.

[0034] 4) The plug of the standby desalting column is removed, and after the liquid in the column flows out, the solution after light protection reaction is transferred to the upper end of the desalting column, and the column is separated and purified with the acridinester column passing solution, and the eluate is collected.

[0035] 5) Add 1 / 5 volume of acridinester antibody storage solution and glycerol for standby.

[0036] Acridinium ester label working solution: Take 0.1-1 mL of acridinium ester label, add 1 mL of acridinium ester diluent, mix well.

[0037] (4) Preparation of TAT calibrator and quality control Calibrator and quality control diluent: 0.5% BSA in 0.01M PBS (pH 7.4±0.1); Calibrator concentrate: Dissolve recombinant TAT antigen (purity ≥98%) with 1M NaOH to a final concentration of 5mg / mL; Calibrator intermediate solution: Dilute the concentrate 800-fold with diluent; Dilute the TAT calibrator intermediate solution to 10ng / mL (calibrator 1) and 50ng / mL (calibrator 2) with TAT calibrator quality control diluent; Dilute the TAT calibrator intermediate solution to (10±2) ng / mL (quality control L) and (50±10) ng / mL (quality control M) with TAT calibrator quality control diluent.

[0038] Assembly Assemble the streptavidin magnetic bead working solution, acridinium ester labeled TAT antibody working solution, biotin labeled TAT antibody working solution, TAT calibrator 1, 2, and TAT quality control L, M into a box, and store at 2-8°C.

[0039] Example 2 Kit detection method Use double antibody sandwich method, the specific steps are as follows: Sample addition and incubation: Add 10μL test sample, 100μL biotin label working solution and 100μL acridinium ester label working solution to the reaction tube, incubate at 37°C for 10min; then add 20μL magnetic bead working solution, continue to incubate at 37°C for 10min; Magnetic separation and washing: Place the reaction tube on the magnetic separation rack and stand for 1min, remove the supernatant; add 200μL magnetic bead working solution twice, remove the supernatant after standing for 1min each time; add 200μL magnetic bead working solution for the third time, transfer the solution to a luminescence tube, stand for 1min, then remove the supernatant; Luminescence detection: Place the luminescence tube into the chemiluminescence automatic immune detection system, add 100μL substrate solution 1 (0.09~0.12M nitric acid, 0.3~0.35% hydrogen peroxide) and substrate solution 2 (0.21~0.29M sodium hydroxide, 0.51~0.69% cetrimide) respectively, measure and record the luminescence intensity; the instrument automatically calculates the TAT concentration through the calibration curve.

[0040] Example 3 Kit performance test 1. Accuracy The TAT high value sample was prepared with a diluent to a target concentration of 60 ng / mL. The concentration point was mixed with samples at a concentration of 2-5 ng / mL at a volume ratio of 1:9 to obtain mixed samples, which were detected using the kit. The results are shown in Table 1 below: Table 1: Accuracy results table of thrombin-antithrombin III complex reagent As can be seen from the above table, the recovery rate is within the range of 85%-115%, and the accuracy is qualified.

[0041] 2. Blank limit: The zero concentration calibrator was used as a sample for detection, and 20 repeated measurements were performed. The RLU values (relative luminescence values) of the 20 measurement results were calculated, and the average value (M) and standard deviation (SD) were calculated. The RLU value corresponding to M+2SD was obtained, and a linear equation was obtained by two-point regression fitting according to the concentration-RLU value results between the zero concentration calibrator and the adjacent calibrator. The RLU value of M+2SD was brought into the above equation, and the corresponding concentration value was obtained. The sample concentration corresponding to this value is the blank limit of the kit. The blank limit of the kit is not higher than 0.4 ng / mL.

[0042] 3. Linearity: In the range of 0.4-120 ng / mL, the linear correlation coefficient (r) value is not less than 0.9900.

[0043] Take 1 part of the thrombin-antithrombin III complex high value sample close to the upper limit of the linear interval, and dilute it to 5 concentration samples at a certain proportion. The low concentration sample is close to the lower limit of the linear interval. Repeat the detection of the above samples 2 times, calculate the average value, and calculate the linear correlation coefficient r by least squares method. If the correlation coefficient r is not less than 0.9900, it is considered that the linear setting is reasonable. The verification results are shown in Table 2, and the linear relationship is shown in Figure 2 . Table 2: Linear verification results table of thrombin-antithrombin III complex reagent 4. Reproducibility: The samples with concentrations of (10±2) ng / mL and (50±10) ng / mL were repeatedly detected 10 times respectively, and the average value (M) and standard deviation (SD) of the 10 measurement results were calculated. According to the formula: CV=SD / M×100%, the coefficient of variation (CV) is not more than 8%.

[0044] 5. Batch difference: 3 batches of kits were used to detect (10±2) ng / mL and (50±10) ng / mL samples, respectively, each repeated 10 times, the average value (M) and standard deviation (SD) of 30 measurement results were calculated, according to the formula: CV=SD / Mx100%, the coefficient of variation (CV) was not more than 10%.

[0045] 6. Interfering substances: interfering substances were added to the clinical samples to prepare corresponding concentrations, and the control samples without interfering substances and the samples with interfering substances were detected according to the method in the instruction. The deviation was calculated: deviation=(concentration after adding cross substances-concentration of control group) / concentration of control groupx100%. The deviation was required to be within ±10%, and the detection results showed that it was not affected by jaundice (20mg / dL bilirubin), lipemia (1400mg / dL triglyceride), hemolysis (500mg / dL hemoglobin), rheumatoid factor sample (RF 100 IU / mL), and ANA sample (1:160).

[0046] 7. Hook effect: clinical samples were added with antigen high-value samples to make their concentrations close to 2000ng / mL, and the samples to be detected were obtained. The calibration diluent was used to dilute them to a series of concentrations between 100-2000ng / mL, and each concentration level sample was detected 2 times by 3 batches of reagents on different machine types. The results showed that no hook effect was found at the concentration of 1000ng / mL.

[0047] It should be noted that for the foregoing embodiments, in order to simply describe, they are all expressed as a series of action combinations, but those skilled in the art should know that the present application is not limited by the order of the described actions, because according to the present application, certain steps can be performed in other order or simultaneously. Secondly, those skilled in the art should know that the embodiments described in the specification all belong to preferred embodiments, and the actions and modules involved are not necessarily necessary for the present application.

[0048] In the embodiments provided in the present application, it should be understood that the disclosed device can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the above units is only a logical function division, and there can be another division manner in actual implementation. For example, a plurality of units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the displayed or discussed mutual coupling or communication connection between the interfaces, devices or units can be indirect coupling or communication connection through some interfaces, and can be electrical or other forms.

[0049] The units described as separate components above can or can not be physically separate, and the components shown as units can or can not be physical units, i.e., can be located in one place, or can be distributed to multiple network units. Part or all of the units can be selected to achieve the purposes of the embodiments of the present application according to actual needs.

[0050] The above embodiments are only used to illustrate the technical solutions of the present application, and do not limit the protection scope of the application. Obviously, the described embodiments are only some, but not all of the embodiments of the present application. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still make modifications to the features of the embodiments of the present application according to the situation without conflict, and without creative work, to combine, delete or make other adjustments to the features of the embodiments of the present application according to the situation, so as to obtain different, but essentially not deviating from the concept of the present application. Other technical solutions, which also belong to the scope of protection of the present application.

Claims

1. A kit for quantitative detection of thrombin-antithrombin III complex, characterized in that, include: Working solution for streptavidin-coated magnetic beads; Biotin-labeled working solution; Acridinium ester labeled working solution; Thrombin-Antthrombin III Complex Calibrator; as well as Thrombin-Antithrombin III Complex Control Product.

2. The thrombin-antithrombin III complex quantitative detection kit as described in claim 1, characterized in that, The magnetic beads in the working solution have a particle size of 2.8–3.2 μm and a concentration of 1.0–3.0 mg / mL.

3. The thrombin-antithrombin III complex quantitative detection kit as described in claim 1, characterized in that, The concentration of antibody in the biotin-labeled working solution is 0.1–1.0 μg / mL; the concentration of antibody in the acridinium ester-labeled working solution is 0.1–1.0 μg / mL.

4. The thrombin-antithrombin III complex quantitative detection kit as described in claim 1, characterized in that, The calibrators include two levels with concentrations of 10 ng / mL and 50 ng / mL; the quality control samples include two levels with target values ​​of 10 ± 2 ng / mL and 50 ± 10 ng / mL.

5. The thrombin-antithrombin III complex quantitative detection kit as described in claim 1, characterized in that, The working solution of the biotin label has a buffer system of phosphate buffer at pH 7.40 ± 0.10 and contains ANS stabilizer; the working solution of the acridinium ester label has a buffer system of phosphate buffer at pH 6.0 ± 0.10 and contains trehalose and BSA.

6. The method for preparing the thrombin-antithrombin III complex quantitative detection kit as described in claims 1-5, characterized in that, Includes the following steps: (1) Preparation of streptavidin-coated magnetic bead working solution; (2) Preparation of biotin-labeled working solution; (3) Preparation of working solution for acridine ester labeling; (4) Prepare calibrators and quality control samples of thrombin-antithrombin III complex; (5) Assemble the components obtained in steps (1)-(4) into a kit.

7. The method for preparing the thrombin-antithrombin III complex quantitative detection kit as described in claim 6, characterized in that, In step (2), the antibody was biotin-labeled using Sulfo-NHS-LC-Biotin, and after the labeling reaction, it was purified by dialyzing in phosphate buffer at pH 7.40±0.

10.

8. The method for preparing the thrombin-antithrombin III complex quantitative detection kit as described in claim 7, characterized in that, In step (3), acridinium ester is used to label the antibody. After the labeling reaction, the antibody is purified using a gel chromatography column and then stabilized using an antibody preservation solution containing glycerol.

9. A method for detecting the content of thrombin-antithrombin III complex in a sample in vitro for non-diagnostic purposes, characterized in that, The detection was performed using the kit described in any one of claims 1-5.

10. The use of the kit according to any one of claims 1-5 in the preparation of a product for detecting thrombin-antithrombin III complex.

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