Single reagent, reagent card and method for rapid detection of anti-xa activity

Abstract

The present application relates to a kind of single reagent for detecting anti-Xa activity and corresponding reagent card, the single reagent is made of 2-8g / L low metal chelating ability buffer, 0.1-0.5U / mL activated Xa factor (FXa), 10-30g / L CaCl2, and and protective agent.It provides a kind of new method for detecting anti-Xa activity, and the single reagent and reagent card in it can directly, accurately and quickly detect anti-Xa activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the technology for detecting coagulation factor activity. Specifically, it relates to a detection kit for heparin and low molecular weight heparin anti-Xa activity and its non-disease diagnostic detection method. Background Technology

[0002] Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are commonly used anticoagulants for the treatment or prevention of thromboembolism. UFH is usually the first-line drug in hemodialysis, percutaneous transluminal angioplasty, and cardiopulmonary bypass; however, due to its unstable pharmacokinetics, individual anticoagulant efficacy varies considerably; adverse reactions include bleeding, thrombocytopenia, and long-term use can lead to osteoporosis; the predictability of treatment response is poor, requiring constant dosage adjustments. Therefore, monitoring is recommended when using UFH to achieve better anticoagulation and reduce or avoid complications such as bleeding. LMWH, due to its long half-life and high safety profile, is widely used in the prevention of deep vein thrombosis during orthopedic or general surgery, acute coronary syndrome, acute myocardial infarction, and anticoagulation therapy during pregnancy. However, monitoring is also recommended for obese patients, patients with renal insufficiency, children, and pregnant patients.

[0003] Anti-Xa activity assays are specifically applied to monitor the efficacy of unfractionated heparin, low molecular weight heparin, and novel oral anticoagulants, maximizing their anticoagulant effects while preventing bleeding. Anti-Xa testing directly targets a specific coagulation factor—X—for anticoagulation, offering highly specific action. Compared to the APTT assay, it has fewer interfering factors along the intrinsic coagulation pathway, adheres to international standards, and provides stable and reliable results reflecting the therapeutic range. Over the past 30 years, other anticoagulants, including low molecular weight heparin, fondaparin, direct thrombin inhibitors, and direct Xa anticoagulants, have been gradually approved and used clinically. Most institutions consider anti-Xa activity assays to be more accurate, efficient, and reliable as an anticoagulation monitoring indicator. Low molecular weight heparin (LWMH) is more prevalent than unfractionated heparin, with hundreds of millions of vials used annually. Anti-Xa activity assays are the only method for detecting LWMH and fondaparinux sodium. Therefore, the research, development, and promotion of anti-Xa testing are essential.

[0004] The technology disclosed in CN115219486A is the mainstream anti-Xa activity detection method on the market—the chromogenic substrate method. The principle of the chromogenic substrate method for determining heparin activity is based on the ability of the complex formed by AT III and heparin to neutralize activated Xa factor. In plasma, AT III and heparin form a complex (heparin-ATIII complex). First, a chromogenic substrate is added to the plasma, followed by an excess of Xa factor. When Xa factor is added to the plasma-substrate mixture, two reactions occur simultaneously: first, the substrate hydrolyzes under the action of Xa to release p-nitroaniline (pNA); second, the heparin-AT III complex inhibits Xa factor. When the competitive reaction reaches equilibrium, the amount of pNA is inversely proportional to the heparin concentration in the reaction system. Therefore, the content of heparin or low molecular weight heparin can be calculated by establishing a standard curve. However, the chromogenic substrate method has the following drawbacks: 1) The chromogenic substrate method uses two reagents (chromogenic substrate and Xa factor) and diluent, and the two reagents can react with each other, which may lead to cross-contamination. Therefore, the instrument has high requirements for carrying-through contamination rate.

[0005] 2) The chromogenic substrate needs to be stored away from light, as it is unstable under light or temperature changes and can easily affect the results;

[0006] 3) The chromogenic substrate method mainly detects the correspondence between color and concentration. Therefore, hemolysis, chylous blood, etc. can interfere with the detection, and the sample often cannot be diluted again to reduce the interference.

[0007] 4) Chromogenic substrate methods are often implemented on large coagulation analyzers, which cannot achieve the purpose of timely detection. Heparin and low molecular weight heparin degrade faster, and have higher requirements for timeliness.

[0008] 5) Currently, commercially available anti-Xa test kits are multi-person kits. Once opened, the kits need to be refrigerated and are often used up within half a month or a month.

[0009] CN114965459A discloses a dry chemical competitive inhibition chromogenic substrate method for detecting anti-Xa activity. The method involves collecting fingertip blood from patients, placing it in an anticoagulant tube, diluting it, and finally adding the diluted sample to a dry test strip for detection. The principle of this test strip is as follows: Red blood cells are filtered through the resin fiber layer of the dry chemical test strip. The plasma then enters the middle layer and neutralizes / inhibits excess quantitative Xa. The plasma, carrying the remaining Xa, permeates forward to the chromogenic substrate localization area, where it reacts with p-nitroaniline (PNA) within a specified time to produce a yellow color reaction. When 405nm reflected light illuminates the chromogenic window (color development area), the incident light is reflected by the dark reflector at the bottom layer. The photosensor of the dry chemical detection instrument receives the reflected light and converts it into an electrical signal for discrimination. Simultaneously, 600nm reflected light is used to eliminate interference noise signals. The result is compared with a calibrator test. The instrument automatically generates and memorizes a calibration curve (standard test strips can also be made) to calculate the quantitative anti-Xa activity content, allowing for more accurate measurement of the activity content of heparin-based anticoagulants or substances. However, like existing methods, this method requires the use of a chromogenic substrate, which cannot avoid interference from sample color and the stability of the chromogenic substrate. Summary of the Invention

[0010] Based on this, the purpose of this invention is to provide a single reagent and detection method for direct, rapid, and accurate detection of anti-Xa activity.

[0011] A first aspect of the present invention is to provide a single reagent for detecting anti-Xa activity, the reagent comprising 2-8 g / L of a buffer solution with low metal chelating capacity, 0.1-0.5 U / mL of activating Xa factor (FXa), 10-30 g / L of CaCl2, and a protective agent.

[0012] To improve the high-temperature resistance and storage performance of the reagent, a certain amount of a suitable protective agent is added to the single reagent described in this invention. In some embodiments, the protective agent includes 10-80 g / L of trehalose, 10-50 g / L of glycine, 1-9 g / L of sodium chloride, 1-10 g / L of BSA, and 1-10 g / L of 2-hydroxypropyl-β-cyclodextrin.

[0013] In some embodiments, the buffer solution with low metal chelating capacity has good pH buffering capacity and does not affect the Ca ions in the reagent. The buffer solution with low metal chelating capacity can be one or more of Tris-HCl, Piperazine-1,4-Diethanesulfonic Acid Buffer (PIPES), 3-Modolinium Propanesulfonic Acid (MOPS), and 4-Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES); preferably, the buffer solution with low metal chelating capacity is 2-8 g / L 4-Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES), more preferably 4-6 g / L 4-Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES), and in some embodiments, the pH is preferably 6.0-8.0, and preferably 6.8.

[0014] Factor Xa is a key enzyme in the coagulation cascade, responsible for converting prothrombin to thrombin, which in turn converts fibrinogen to fibrin, forming a clot. This method assesses factor Xa activity by measuring plasma clotting time. In some embodiments, the activated factor Xa concentration is 0.1–0.5 U / mL, preferably 0.1–0.3 U / mL, more preferably 0.18–0.22 U / mL, and most preferably 0.2 U / mL.

[0015] Commercially available reagents mainly consist of a three-reagent system comprising activated Xa factor, a chromogenic substrate of Xa factor, and a diluent. This invention discovers that a single reagent composed of activated Xa factor and CaCl2 can rapidly obtain better detection results. In some embodiments, the CaCl2 concentration is 10–30 g / L, preferably 18–24 g / L, and most preferably 20 g / L. Since the anticoagulant mechanism of sodium citrate is to chelate calcium ions in the blood, preventing blood clotting, adding an appropriate amount of CaCl2 to sodium citrate-anticoagulated blood can directly initiate the reaction and obtain good detection results.

[0016] In some preferred embodiments, the protective agent is 30-60 g / L of trehalose, 15-30 g / L of glycine, 3-7 g / L of sodium chloride, 3-7 g / L of BSA, and 1-4 g / L of 2-hydroxypropyl-β-cyclodextrin.

[0017] In some preferred embodiments, the protective agent is 40-50 g / L of trehalose, 20-30 g / L of glycine, 4-6 g / L of sodium chloride, 4-6 g / L of BSA, and 1-3 g / L of 2-hydroxypropyl-β-cyclodextrin.

[0018] In some preferred embodiments, the protective agent is 45-55 g / L of trehalose, 20-30 g / L of glycine, 4.5-5.5 g / L of sodium chloride, 4.5-5.5 g / L of BSA, and 1.5-2.5 g / L of 2-hydroxypropyl-β-cyclodextrin.

[0019] A second aspect of the present invention is to provide a reagent card for detecting anti-Xa activity, wherein the reagent card is obtained by dispensing any of the above-mentioned single reagents for detecting anti-Xa activity onto a test card for detecting anti-Xa activity and then drying it.

[0020] A third aspect of the present invention is to provide a method for detecting anti-Xa activity, comprising the following steps:

[0021] S1 obtains the test plasma using sodium citrate as an anticoagulant;

[0022] S2 adds the plasma to be tested onto the test card;

[0023] S3 was used to detect the concentrations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) using an optical coagulation analyzer.

[0024] In some of these embodiments, the plasma to be tested is derived from humans, or mammals such as cattle, sheep, dogs, and cats.

[0025] This invention provides a novel method for detecting anti-Xa activity, which can directly, rapidly, and accurately detect anti-Xa activity using a single reagent and reagent card, and has the following advantages.

[0026] 1) This invention abandons the multi-reagent approach and instead develops a single-reagent method for detecting anti-Xa activity. The main detection principle is that the reagent contains appropriate amounts of CaCl2 and activated Xa factor. CaCl2 primarily neutralizes the anticoagulant, allowing blood to form a clot. AT III in the plasma sample forms a complex with heparin (heparin-ATIII complex), which inhibits the activated Xa factor in the reagent. The remaining activated Xa factor activates the blood, ultimately leading to clot formation. Since different concentrations of activated Xa factor have a linear relationship with coagulation time, a standard curve can be established between coagulation time and heparin and low molecular weight heparin concentrations.

[0027] 2) The reagents of this invention can be applied to fully automated coagulation analyzers, but are preferably applied to optical coagulation analyzers OCG-101 and OCG-102. Their main advantages are that the reagent cards have low manufacturing costs, simple processes, and the methodology primarily detects clot formation, unaffected by sample color (such as bilirubin, hemoglobin, triglycerides, etc.).

[0028] 3) The optical coagulation analyzers OCG-101 and OCG-102 to which this invention is applied preferentially use dried reagents, i.e., the reagents are dried at the bottom of the test card. Its main advantage is that the test card can be individually packaged, tested individually, and can be used for immediate detection and storage and transportation at room temperature. Attached Figure Description

[0029] Figure 1 The Bland-Altman analysis graph of unfractionated heparin shows the concentration distribution of unfractionated heparin clinical samples ranging from 0 to 2 U / mL on the horizontal axis, and the difference between the test results of the single reagent described in this invention and the control reagent on the vertical axis. The control reagent is the test result of Haifeng reagent on Sysmex CS-2000i, and the self-developed test result refers to the test result of the single reagent of this invention on OCG-101.

[0030] Figure 2 The Bland-Altman analysis plot of low molecular weight heparin shows the concentration distribution of low molecular weight heparin clinical samples ranging from 0 to 2 U / mL on the horizontal axis, and the difference between the single reagent test results and the control reagent test results on the vertical axis. The control reagent is the test result of Haifeng reagent on Sysmex CS-2000i, and the self-developed test result refers to the detection result of the single reagent of this invention on OCG-101.

[0031] Figure 3 The comparison of unfractionated heparin clinical samples shows that the horizontal axis represents the results of the test using the Haifeng reagent on the Sysmex CS-2000i, and the vertical axis (Wondfo) represents the results of the test using the single reagent of this invention on the OCG-101.

[0032] Figure 4 Clinical sample comparison of low molecular weight heparin: The horizontal axis represents the results of testing with Haifeng reagent on Sysmex CS-2000i. The vertical axis (Wondfo) represents the results of testing with the single reagent of this invention on OCG-101. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0034] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or under conditions recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0035] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0037] The following provides a further description of the reagents and methods for determining anti-Xa activity provided by this invention.

[0038] Example 1

[0039] The experiment was set up with 5 groups:

[0040] Preparation of a single reagent for detecting anti-Xa activity: Add 500 mg of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) to 100 mL of pure water, stir for 10 min until the solid dissolves, and adjust the pH to 6.8 for later use.

[0041] Take 10 mL of the above buffer solution, add 200 mg anhydrous CaCl2, 500 mg trehalose, 250 mg glycine, 50 mg NaCl, 50 mg BSA, and 20 mg 2-hydroxypropyl-β-cyclodextrin. Continue stirring for 30 min until the solid dissolves. Take 5 centrifuge tubes, add 1 mL of the above buffer solution to each tube, and then add 1 U of activated Xa factor (Group 1), 2 U of activated Xa factor (Group 2), 3 U of activated Xa factor (Group 3), 0.4 U of activated Xa factor (Group 4), and 0.5 U of activated Xa factor (Group 5) to the corresponding 5 groups, respectively. Stir for 10 min to mix thoroughly. Aliquot the prepared reagents, transfer 5 μL of reagent to the bottom of the test card, and place the aliquoted test cards in a 37°C oven for 1 h to dry. Attach the gears to the dried reagent cards, and they are ready for use with the semi-automatic blood coagulation analyzer OCG-101 / OCG-102 for detection.

[0042] The test card is readily available. In this invention, the test card used is the OCG-101 / 102 test card sold by Wondfo Biotech. The test card consists of a plastic substrate and gears. The reagent is applied to the reaction area of ​​the substrate. When the sample and reagent react to form a clot, the gears hook the clot up. The instrument records the time from the start of the test to the clot being hooked up, i.e., the coagulation time. The activated Xa factor was purchased from Ark Biosafety Technology (Guangzhou) Co., Ltd.

[0043] Plasma containing sodium citric acid from healthy individuals as an anticoagulant was tested. The mixed plasma was divided into 15 aliquots, each 1.0 mL. Five aliquots were then treated with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of unfractionated heparin, respectively. Another five aliquots were treated with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of low molecular weight heparin, respectively. The remaining five aliquots were treated with 0 ng, 100 ng, 200 ng, 300 ng, and 450 ng of rivaroxaban, respectively. The prepared plasma was shaken well for 10 minutes, and then tested separately. A standard curve of coagulation time versus heparin concentration was plotted. The results are shown in the figure below.

[0044] Table 1. Results of Unfractionated Heparin Test

[0045]

[0046] Table 2 Results of Low Molecular Weight Heparin Test

[0047]

[0048]

[0049] Table 3. Results of rivaroxaban testing

[0050]

[0051] The experimental results show that the five groups have good correlations when testing the concentrations of unfractionated heparin, low molecular weight heparin, and rivaroxaban. Among them, group 2, with an activated factor Xa of 0.2 U / mL, has higher sensitivity. The optimal amount of activated factor Xa is 0.1–0.3 U / mL.

[0052] Example 2

[0053] Two experimental groups were set up: 500 mg of 4-hydroxyethylpiperazine ethyl (HEPES) was added to 100 mL of pure water and stirred for 10 min until the solid dissolved. The pH was then adjusted to 6.8. 10 mL of the above buffer solution was taken, and 0 mg of anhydrous CaCl2 (Group 1), 200 mg of anhydrous CaCl2 (Group 2), 500 mg of trehalose, 250 mg of glycine, 50 mg of NaCl, 50 mg of BSA, and 20 mg of 2-hydroxypropyl-β-cyclodextrin were added. Stirring continued for 30 min until the solid dissolved, then 2.0 U of activated factor Xa was added, and the mixture was stirred for 10 min until homogeneous. The prepared reagent was aliquoted, and 5 μL of the reagent was transferred to the bottom of the test card. The aliquoted test cards were then dried in a 37°C oven for 1 h. The dried reagent cards were then fitted with gears and ready for use with the semi-automatic hemagglutination analyzer OCG-101 / OCG-102 for detection.

[0054] The test was performed using sodium citrate anticoagulated plasma from healthy individuals. The plasma was divided into 10 aliquots, each 1.0 mL. Five aliquots were then treated with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of unfractionated heparin, respectively. The remaining five aliquots were treated with 0.0 U, 0.5 U, 1.0 U, 1.5 U, and 2.0 U of low molecular weight heparin, respectively. The prepared plasma samples were shaken well for 10 minutes, and then tested. A standard curve was plotted as a function of coagulation time and heparin concentration. The results are shown in the table below.

[0055]

[0056] Research has shown that an appropriate amount of CaCl2 must be added to the reagent; otherwise, it cannot neutralize the anticoagulant in the blood collection tube, and the blood will ultimately fail to clot.

[0057] Example 3: Stability Experiment

[0058] Four sets of experiments were conducted: 500 mg of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was added to 100 mL of pure water and stirred for 10 min until the solid dissolved. The pH was then adjusted to 6.8. 10 mL of the above buffer solution was taken, and 200 mg of anhydrous CaCl2, 500 mg of trehalose, and varying amounts of glycine, NaCl, BSA, and 2-hydroxypropyl-β-cyclodextrin were added. Stirring continued for 30 min until the solid dissolved. Then, 1.0 U of activated Xa factor was added, and the mixture was stirred for 10 min until homogeneous. The prepared reagents were dispensed into individual test cards. 5 μL of the reagent was transferred to the bottom of the test card, and the dispensed test cards were dried in a 37°C oven for 1 h. The dried test cards were then fitted with gears and ready for detection using Wondfo Biotech's OCG-101 / OCG-102.

[0059] 3.1 Different groups

[0060]

[0061] The test card with a single reagent was prepared according to the above embodiment. The reagent card was subjected to accelerated aging at 50°C for 2 weeks. The same batch of reference plasma was used for testing during the two weeks. The test was repeated three times at each time point. The mean and standard deviation of the sample were calculated. The results are as follows.

[0062] Group 1: High-temperature stability results

[0063]

[0064] Group 2: Results of High Temperature Stability Test

[0065]

[0066]

[0067] Group 3: Results of High Temperature Stability Test

[0068]

[0069] Group 4: Results of High Temperature Stability Test

[0070]

[0071]

[0072] As can be seen from the table above, after the reagent prepared according to Example 7 was accelerated to 50°C for 14 days, only the deviation of the same sample in Group 2 was within ±10%. This indicates that the reagent has good stability and is suitable for room temperature storage after the addition of glycine, sodium chloride and 2-hydroxypropyl-β-cyclodextrin.

[0073] Example 4: Clinical Comparison Experiment

[0074] The test card with a single reagent (product of the present invention) prepared according to the method in group 2 of Example 1.

[0075] This invention, applied to the OCG-102 instrument, was used to detect 20 clinical samples with different concentrations of standard heparin and 20 clinical samples with different concentrations of low molecular weight heparin. The detection method was as follows: After preheating the test card to 37°C, 20 μL of sample was added, and the corresponding coagulation time was obtained. The heparin concentration in the sample was then determined based on a standard curve of heparin concentration versus coagulation time. The control product was the gold standard – Sysmex CS-2000i and its matching heparin assay kit (chromogenic substrate method). The control product was operated strictly according to its instruction manual, while this invention was operated according to the OCG-102 instruction manual.

[0076] Unfractionated heparin test results (concentration: U / mL)

[0077]

[0078]

[0079] Low molecular weight heparin test results (unit: U / mL)

[0080]

[0081]

[0082] Figure 1 and Figure 3 , Figure 2 and Figure 4The results show that the single reagent, reagent card, and method described in this invention have a better correlation with commercially available reagents. Using the reagent card of this invention, most of the detection data are within the range of commercially available reagents.

[0083] Within ±1.96SD, the test results can provide a valid reference for clinical treatment.

[0084] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A single reagent for detecting anti-Xa activity, characterized in that, The single reagent consists of 2-8 g / L of buffer solution with low metal chelating ability, 0.1-0.5 U / mL of activated Xa factor (FXa), 10-30 g / L of CaCl2, and a protective agent, which includes 10-80 g / L of trehalose, 10-50 g / L of glycine, 1-9 g / L of sodium chloride, 1-10 g / L of BSA, and 1-10 g / L of 2-hydroxypropyl-β-cyclodextrin.

2. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that, The buffer solution is one or more of the following: Tris-HCl, Piperazine-1,4-Diethanesulfonic Acid Buffer (PIPES), 3-Modolinium Propanesulfonic Acid (MOPS), and 4-Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES).

3. The single reagent for detecting anti-Xa activity according to claim 2, characterized in that, The buffer solution is 2-8 g / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES); and / or the pH of the buffer solution is 6.0-8.

0.

4. The single reagent for detecting anti-Xa activity according to claim 3, characterized in that, The buffer solution is 4-6 g / L of 4-hydroxyethylpiperazine ethanesulfonic acid.

5. The single reagent for detecting anti-Xa activity according to claim 3 or 4, characterized in that, The pH of the buffer solution is 6.

8.

6. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that, The concentration of the activated Xa factor is 0.1~0.3 U / mL.

7. The single reagent for detecting anti-Xa activity according to claim 6, characterized in that, The concentration of the activated Xa factor is 0.18-0.22 U / mL.

8. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that, The concentration of CaCl2 is 15~25 g / L.

9. The single reagent for detecting anti-Xa activity according to claim 8, characterized in that, The concentration of CaCl2 is 18~24 g / L.

10. The single reagent for detecting anti-Xa activity according to claim 9, characterized in that, The concentration of CaCl2 is 19-21 g / L.

11. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that, The protective agents are 30-60 g / L trehalose, 15-30 g / L glycine, 3-7 g / L sodium chloride, 3-7 g / L BSA, and 1-4 g / L 2-hydroxypropyl-β-cyclodextrin.

12. The single reagent for detecting anti-Xa activity according to claim 1, characterized in that, The protective agent consists of 45-55 g / L trehalose, 20-30 g / L glycine, 4.5-5.5 g / L sodium chloride, 4.5-5.5 g / L BSA, and 1.5-2.5 g / L 2-hydroxypropyl-β-cyclodextrin.

13. A reagent card for detecting anti-Xa activity, characterized in that, The reagent card is obtained by dispensing the single reagent for detecting anti-Xa activity as described in any one of claims 1-12 onto a test card for detecting anti-Xa activity, and then drying it.

14. The reagent card according to claim 13, characterized in that, The dispensing volume of the single reagent is 4-6 μL.

15. A method for detecting anti-Xa activity, characterized in that, Includes the following steps: S1 Obtain the test plasma using sodium citrate as an anticoagulant; S2. The plasma to be tested is added to the reagent card according to any one of claims 13-14; S3 uses an optical coagulation analyzer to detect the concentrations of unfractionated heparin (UFH) and low molecular weight heparin (LMWH).

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