Arachidonic acid reagent for platelet aggregation function detection and its preparation method

Abstract

This invention relates to the field of biotechnology, and particularly to a reagent for detecting arachidonic acid in platelet aggregation function and its preparation method. This invention provides the application of ethoxyquinoline, dimercaprol, and / or poloxamer 124 in the preparation of a platelet aggregation function detection kit. This invention uses ethoxyquinoline and dimercaprol as a combined antioxidant, improving the stability of the arachidonic acid reagent; the addition of poloxamer 124 and ultrasonic treatment promotes uniform dispersion and stability of the reagent system, thereby improving the sensitivity to anti-platelet aggregation drugs, such as aspirin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an arachidonic acid reagent for detecting platelet aggregation function and its preparation method. Background Technology

[0002] Platelets in the blood play a vital role in hemostasis and coagulation, as well as in repairing damaged blood vessels. They participate in various physiological and pathological processes, including thrombosis, atherosclerosis, cancer metastasis, and inflammatory responses. These processes are closely related to platelet adhesion, release, and aggregation. Detection of platelet aggregation function can be used to assess the diagnosis and treatment of thrombotic and hemorrhagic diseases, determine a patient's responsiveness to antiplatelet therapy, predict thrombotic risk, and thus implement targeted, individualized antiplatelet therapy regimens to reduce the occurrence of thrombotic complications.

[0003] Platelets have various receptors on their surface related to aggregation. Among them, the platelet GP IIb / IIIa membrane receptor is an adhesive glycoprotein located on the platelet surface. Activation of this receptor increases its affinity for soluble fibrinogen, causing platelets to aggregate on bridging fibrin, forming platelet thrombi. Phospholipids in the cell membrane can be catalyzed by phospholipase A2 to release arachidonic acid (AA). AA can then be converted into thromboxane A2 (TXA2) by cyclooxygenase (COX). This substance can indirectly activate the GP IIb / IIIa receptor, leading to platelet aggregation. This process is called the AA pathway of platelet aggregation. This pathway can be inhibited by antiplatelet drugs, such as aspirin. Aspirin works by irreversibly acetylifying the active site of COX, leading to COX inactivation and blocking the conversion of AA to TXA2, thereby inhibiting platelet aggregation.

[0004] Optical turbidimetry is commonly used clinically as the gold standard for platelet aggregation function testing, but this method has drawbacks such as complex operation and poor repeatability. Thromboelastography (TEG) is a novel coagulation function testing method that uses whole blood as the test sample to simulate the in vivo environment and detect the dynamic changes in blood coagulation, determining the speed and intensity of blood clotting. Using this method for platelet aggregation function testing has advantages such as simple operation and the ability to use whole blood. The specific detection principle is as follows: Under excessive heparin anticoagulation, thrombin in the blood is completely inhibited. Fibrin activators contain thrombin-like substances that are insensitive to heparin, which can act on fibrinogen, converting it into fibrin monomers. Subsequently, under the action of coagulation factors, fibrin monomers form a network structure. During this process, platelets are not activated, and the measured MA value mainly reflects the fibrin strength (MA). FWhen fibrin activator is used in combination with arachidonic acid reagent, the total intensity (MA) of fibrin and platelet aggregation can be detected. AA If the patient has taken anti-AA drugs, the result only reflects the intensity of fibrin and uninhibited platelet aggregation. Additionally, testing citrate-anticoagulated whole blood with a kaolin activator can measure the patient's maximum coagulation strength, which is the maximum intensity of fibrin and all platelet aggregation (MA). T According to the following formula I, the platelet inhibition rate (AA%) can be calculated, the platelet function of the patient after medication can be judged, and the inhibitory effect of various antiplatelet drugs can be obtained.

[0005]

[0006] Currently, products on the domestic market related to platelet aggregation function detection via the arachidonic acid (AA) activation pathway, when used with a thromboelastography instrument, can simplify sample preparation and testing procedures. However, they suffer from poor reagent stability, poor repeatability, and insufficient sensitivity to relevant platelet-inhibiting drugs. Among existing products, arachidonic acid reagent (AA reagent) is prone to oxidative rancidity and loss of activity. Furthermore, oily AA tends to aggregate and separate in aqueous reagent systems, further affecting reagent stability and repeatability. The correlation between test results and antiplatelet drug concentrations is poor, lacking sensitivity. Currently, there are few systematic solutions to these problems. For example, some existing reagents lack components relevant to inhibit AA oxidation; while others have not addressed homogeneity issues and lack validation of reagent stability.

[0007] Therefore, providing a platelet aggregation function detection kit based on the AA activation pathway with good stability, precision, and drug sensitivity is of significant practical importance. Summary of the Invention

[0008] In view of this, the composition and its application provided by the present invention are used to prepare a platelet aggregation function detection kit for the AA activation pathway, which has high stability, good precision and good drug sensitivity.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The present invention provides a composition comprising ethoxyquinoline and dimercaprol.

[0011] The present invention also provides for the use of the above composition in any of the following:

[0012] (1) Reduce the oxidative rancidity of arachidonic acid in the reagent;

[0013] (2) Improve the stability of arachidonic acid in the reagent;

[0014] (3) Extend the shelf life of reagents containing arachidonic acid;

[0015] (4) Preparation of reagents containing arachidonic acid;

[0016] (5) Preparation of a platelet aggregation function detection kit;

[0017] The reagents include those in the platelet aggregation function assay kit;

[0018] The platelet aggregation function test kit includes a platelet aggregation function test kit for the arachidonic acid activation pathway.

[0019] In some specific embodiments of the present invention, the above composition further includes poloxamer 124.

[0020] The present invention also provides for the use of the above composition in any of the following:

[0021] (1) Reduce the oxidative rancidity of arachidonic acid in the reagent;

[0022] (2) Improve the stability of arachidonic acid in the reagent;

[0023] (3) Extend the shelf life of reagents containing arachidonic acid;

[0024] (4) Promote the uniform dispersion of reagent systems containing arachidonic acid.

[0025] (5) Improve the sensitivity and / or accuracy of platelet aggregation function detection reagents for antiplatelet aggregation drugs;

[0026] (6) Preparation of reagents containing arachidonic acid;

[0027] (7) Preparation of a platelet aggregation function detection kit;

[0028] The reagents include those in the platelet aggregation function assay kit;

[0029] The antiplatelet aggregation drugs include aspirin;

[0030] The platelet aggregation function test kit includes a platelet aggregation function test kit for the arachidonic acid activation pathway.

[0031] The present invention also provides reagents, including the above-described compositions, and acceptable excipients or adjuvants.

[0032] In some specific embodiments of the present invention, the above-mentioned reagents also include one or more of arachidonic acid, buffer solution, lyophilization protectant or preservative;

[0033] The buffer solution includes Tris buffer;

[0034] The freeze-drying protectant includes trehalose;

[0035] The preservatives include gentamicin.

[0036] In some specific embodiments of the present invention, the arachidonic acid in the above composition or reagent may be replaced by sodium arachidonicate or sodium arachidonicate salt.

[0037] In some specific embodiments of the present invention, the above-mentioned reagents include 1.0% (v / v) arachidonic acid, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaptopropanol, 0.05% (v / v) poloxamer 124, 0.005% (w / v) gentamicin and 20 mM Tris at pH 7.5, as well as acceptable excipients or adjuvants.

[0038] In this invention, w / v refers to the mass-to-volume ratio, expressed in g / mL.

[0039] In some specific embodiments of the present invention, the preparation method of the above-mentioned reagent includes an ultrasonic mixing step.

[0040] In some specific embodiments of the present invention, the preparation method of the above-mentioned reagent includes the following steps:

[0041] Step (a): Mix the buffer solution with the preservative, adjust the pH to 7.5, and obtain solution 1;

[0042] Step (b): Take the solution 1 obtained in step (a) and mix it with the composition as described in claim 3 and the freeze-drying protectant to obtain solution 2;

[0043] Step (c): Arachidonic acid is mixed with solution 2 described in step (b) and ultrasonically mixed to obtain the reagent.

[0044] The present invention also provides a kit comprising the above-described composition or the above-described reagent.

[0045] The present invention also provides a method for detecting platelet aggregation function, including detecting platelet aggregation function based on any of the following:

[0046] (I) The above-mentioned composition;

[0047] (II) The above reagents.

[0048] This invention also provides a method for detecting platelet aggregation function, comprising the step of mixing any one of the following with the sample to be tested:

[0049] (I) The above-mentioned composition;

[0050] (II) The above reagents.

[0051] In some specific embodiments of the present invention, the above-mentioned platelet aggregation function detection method includes the following steps:

[0052] Step (a): Mix the kaolin activator, calcium chloride, and citric acid-modified test sample to obtain a common cup;

[0053] Step (b): Mix the fibrin activator with the heparinized test sample to obtain a fibrin cup;

[0054] Step (c): Mix fibrin activator, AA reagent and heparinized test sample to obtain AA cup;

[0055] Step (d): Perform thromboelastography on the ordinary cup described in step (a), the fibrin cup described in step (b), or the AA cup described in step (c) respectively, and obtain the test results;

[0056] The AA reagent is any one of the following:

[0057] (I) The above-mentioned composition;

[0058] (II) The above reagents.

[0059] The compositions of the present invention and their applications have the following effects:

[0060] This invention proposes, on the one hand, the combined use of ethoxyquinoline and dimercaprol to improve the stability of AA reagent, prevent oxidative rancidity, and extend the reagent's shelf life; on the other hand, by using poloxamer 124 and ultrasonically mixing, the AA reagent system exhibits better homogeneity and stability, improving the AA reagent's sensitivity to antiplatelet drugs, thereby better guiding clinical diagnosis and medication. This invention provides a superior performance standard compared to similar products. Detailed Implementation

[0061] This invention discloses the composition and its application. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0062] Currently, most platelet aggregation function testing kits offer few solutions for addressing the performance issues of AA reagents, such as maintaining reagent stability and homogeneity. To address these problems in existing technologies, this invention aims to develop a platelet aggregation function testing kit for the AA activation pathway, exhibiting good stability, precision, and drug sensitivity.

[0063] The platelet aggregation function test kit (AA) (coagulation method) of this invention is mainly used in conjunction with a thromboelastography instrument. By qualitatively and quantitatively detecting the platelet aggregation function of patients, it can be used to assess the diagnosis and treatment of thrombotic and hemorrhagic diseases, and to determine the clinical efficacy of antiplatelet aggregation drugs.

[0064] This invention proposes using ethoxyquinoline and dimercaprol as a combined antioxidant in arachidonic acid reagent, which can improve the stability of AA reagent. At the same time, the addition of poloxamer 124 and sonication in the reagent promotes uniform dispersion and stability of the reagent system, thereby improving the sensitivity of antiplatelet aggregation drugs, such as aspirin. Thus, a reagent kit product with good stability and precision can be obtained, which can effectively reflect the effect of relevant drugs on the platelet aggregation function of patients.

[0065] The AA reagent is a lyophilized powder containing arachidonic acid (AA), trehalose, gentamicin sulfate, ethoxyquinoline, dimercaprol, poloxamer 124, and Tris: 0.1-2.5% AA, preferably 1.0% (v / v); 0.1-2.0% trehalose, preferably 0.5% (w / v); 0.0005-0.1% gentamicin sulfate, preferably 0.005% (w / v); 0.001-0.1% ethoxyquinoline, preferably 0.01% (v / v); 0.001-0.1% dimercaprol, preferably 0.001% (v / v); 0.001-1.0% poloxamer 124, preferably 0.05% (v / v); 10-100 mM Tris, preferably 20 mM; pH 7.0-8.0, preferably 7.5.

[0066] Preparation method:

[0067] (1) Take the required amount of Tris and gentamicin sulfate according to the formula, add purified water to dissolve them completely, and adjust the pH value to 7.0-8.0;

[0068] (2) Take the amount of trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 according to the formula and add them to the buffer solution in step (1), and stir to dissolve them completely.

[0069] (3) Take the amount of arachidonic acid according to the formula and add it to the solution in step (2), mix well to obtain AA reagent;

[0070] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0071] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0072] The present invention will be further illustrated below with reference to the embodiments:

[0073] Example 1

[0074] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0075] (2) Add trehalose to the buffer solution in step (1) and stir until it is fully dissolved;

[0076] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0077] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0078] The final contents of each component in the AA reagent are as follows: 0.5% (v / v) AA, 0.5% (w / v) trehalose, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0079] Example 2

[0080] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0081] (2) Add trehalose to the buffer solution in step (1) and stir until it is fully dissolved;

[0082] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0083] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0084] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0085] Example 3

[0086] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0087] (2) Take trehalose, ethoxyquinoline, and dimercaprol, add them to the buffer solution in step (1), and stir to dissolve them completely;

[0088] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0089] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0090] The final contents of each component in the AA reagent are as follows: 2.0% (v / v) AA, 0.5% (w / v) trehalose, 0.001% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaprol, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0091] Example 4

[0092] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0093] (2) Add trehalose, ethoxyquinoline, and dimercaprol to the buffer solution in step (1) and stir until fully dissolved;

[0094] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0095] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0096] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaprol, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0097] Example 5

[0098] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0099] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0100] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0101] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0102] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.05% (w / v) ethoxyquinoline, 0.01% (w / v) dimercaptopropanol, 0.01% (v / v) poloxamer 124, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0103] Example 6

[0104] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0105] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0106] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0107] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0108] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 1.0% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaptopropanol, 0.01% (v / v) poloxamer 124, 0.0005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0109] Example 7

[0110] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0111] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0112] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0113] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0114] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaptopropanol, 0.05% (v / v) poloxamer 124, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0115] Example 8

[0116] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0117] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0118] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0119] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0120] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaptopropanol, 0.05% (v / v) poloxamer 124, 0.005% (w / v) gentamicin, and 50 mM Tris (pH 7.5).

[0121] Example 9

[0122] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.0;

[0123] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and poloxamer 124 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0124] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0125] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0126] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaptopropanol, 0.05% (v / v) poloxamer 124, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.0).

[0127] Comparative Example 1

[0128] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0129] (2) Add trehalose and ethoxyquinoline to the buffer solution in step (1) and stir to dissolve them completely;

[0130] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0131] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0132] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0133] Comparative Example 2

[0134] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0135] (2) Add trehalose and dimercaprol to the buffer solution in step (1) and stir to dissolve them completely;

[0136] (3) Add arachidonic acid (AA) to the solution in step (2) and mix well to obtain AA reagent;

[0137] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0138] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.001% (w / v) dimercaprol, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0139] Comparative Example 3

[0140] (1) Take Tris and gentamicin sulfate, add purified water to dissolve them completely, and adjust the pH value to 7.5;

[0141] (2) Take trehalose, ethoxyquinoline, dimercaptopropanol and Triton X-100 and add them to the buffer solution in step (1), and stir to dissolve them completely;

[0142] (3) Add arachidonic acid (AA) to the solution in step (2) and mix it by ultrasonication to obtain AA reagent;

[0143] (4) The AA reagent obtained in step (3) is dispensed into 100 μL bottles and freeze-dried under vacuum to obtain AA reagent lyophilized powder.

[0144] The final contents of each component in the AA reagent are as follows: 1.0% (v / v) AA, 0.5% (w / v) trehalose, 0.01% (w / v) ethoxyquinoline, 0.001% (w / v) dimercaprol, 0.05% (v / v) Triton X-100, 0.005% (w / v) gentamicin, and 20 mM Tris (pH 7.5).

[0145] Example 1

[0146] Blood samples (heparin anticoagulated) from the same person were tested using Examples 1-9 in combination with a self-made fibrin activator, with an imported reagent used as a comparison reagent.

[0147] Table 1: Screening Results of Examples 1-9

[0148]

[0149] As can be seen from Table 1, the relative deviations of the test results of each parameter in Examples 2, 4, and 7 compared with those of the comparison reagent (calculated as shown in Formula II) are relatively small, indicating that the reagent components are effective. Therefore, Examples 2, 4, and 7 are preferred for the AA reagent of the present invention, and the performance differences among the three can be further compared.

[0150]

[0151] Example 2

[0152] Examples 2, 4, and 7 of this invention, as well as Comparative Examples 1 and 2 and the comparison reagents, were all placed in an environment of 2-8°C for 10 days, and blood samples (heparin anticoagulated) from the same person were taken at regular intervals for testing. Specifically, Comparative Example 1 contained only 0.01% ethoxyquinoline as an antioxidant, and Comparative Example 2 contained only 0.001% dimercaprol as an antioxidant; the remaining formulations of both were consistent with Example 4.

[0153] Table 2: Stability Test Results

[0154]

[0155]

[0156] As can be seen from Table 2, the AA reagent of the present invention in Examples 4 and 7 showed good stability within 10 days, and the relative range of the detected MA values ​​was within 10%, which is better than the comparison reagent and Examples 2, 1 and 2.

[0157] Example 2, due to the absence of any added antioxidant components and the lack of a homogeneous reagent system, resulted in rapid reagent inactivation. Comparative Examples 1 and 2, each containing an antioxidant, showed significantly improved stability compared to Example 2, but both underwent complete inactivation at 10 days, similar to the comparative reagent. Example 4 combined two antioxidants, and the reagent retained its activity after 10 days of storage, with a relative range of less than 10%. Example 7 further improved reagent stability by adding poloxamer 124 and undergoing ultrasonic mixing, with a relative range less than that of Example 4.

[0158] Example 3

[0159] Blood samples from the same individual (anticoagulated with heparin and containing different concentrations of aspirin) were tested using Examples 4, 7, and Comparative Example 3 in combination with a self-made fibrin activator, with an imported reagent used as a comparison reagent. In Comparative Example 3, 0.05% Triton X-100 was used as the surfactant, and the remaining composition and ultrasonic treatment method were the same as in Example 7.

[0160] Table 3: Linear Range Detection Results

[0161]

[0162] As can be seen from Table 3, Example 7 of the AA reagent of the present invention, which is a formulation with added poloxamer 124 and ultrasonically mixed, has a good correlation with the concentration of the antiplatelet aggregation drug aspirin, with a correlation coefficient r of 0.995. It has high sensitivity and is superior to the comparison reagent, Example 4 without the above treatment, and Comparative Example 3 using Triton X-100 as a surfactant. Therefore, Example 7 of the AA reagent of the present invention is preferred, which can accurately evaluate the effect of the drug taken by the patient and guide the clinical dosage.

[0163] Example of effect 4

[0164] Using the same batch of platelet aggregation function test kits (AA route) (AA reagent preparation method is shown in Example 7), blood samples from the same person (including sodium citrate and heparin anticoagulated samples, with a certain amount of aspirin added) were tested 10 times. The test method is as follows:

[0165] 1. Allow the test reagents to warm to room temperature. Add 200 μL and 100 μL of distilled water to the fibrin activator and AA reagent bottles respectively, and shake to mix thoroughly.

[0166] 2. Open the instrument application software, enter the patient information, and select the corresponding test type.

[0167] 3. Load sample cups onto the three channels of the thromboelastography instrument.

[0168] 4. Add 1 mL of citric acidified blood to the kaolin activator tube and mix by inverting the tube 5 times.

[0169] 5. Add 20 μL of calcium chloride to the bottom of the sample cup in channel 1, add 340 μL of the blood sample activated by the above kaolin activator to the cup, and press the button to start the test.

[0170] 6. Take 10 μL of the reconstituted fibrin activator and add it to the bottom of the sample cups in channels 2 and 3, respectively.

[0171] 7. Add 10 μL of the reconstituted AA reagent to the bottom of the sample cup in channel 3.

[0172] 8. Add 360 μL of heparinized blood to the sample cups in channels 2 and 3 respectively, and mix by blowing and aspirating 3 times. After adding the sample, quickly press the button to start the test.

[0173] 9. After the test is completed, click "Stop" on the test interface to end the test, remove the sample cup, and dispose of it as medical waste.

[0174] 10. Select the test results of the three cups in the data interface, click "AA Inhibition Rate" to obtain the platelet inhibition rate.

[0175] Table 4: Precision test results of the reagent kit

[0176]

[0177] As can be seen from Table 4, the CV of the MA value obtained by the kit of the present invention (using the AA reagent of Example 7) is all within 5%, and the CV of the platelet inhibition rate is also within 5%, indicating that the platelet aggregation function detection kit of the present invention has good detection precision.

[0178] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a composition comprising ethoxyquinoline, dimercaprol, and poloxamer 124 in any of the following: (1) Reduce the oxidative rancidity of arachidonic acid in the reagent; (2) Improve the stability of arachidonic acid in the reagent; (3) Extend the shelf life of reagents containing arachidonic acid; (4) Promote the uniform dispersion of reagent systems containing arachidonic acid; (5) Improve the sensitivity and / or accuracy of platelet aggregation function test reagents for antiplatelet aggregation drugs; (6) Preparation of reagents containing arachidonic acid; (7) Preparation of a platelet aggregation function detection kit; The reagents include those in the platelet aggregation function assay kit; The antiplatelet aggregation drugs include aspirin; The platelet aggregation function test kit includes a platelet aggregation function test kit for the arachidonic acid activation pathway.

2. Arachidonic acid reagent, characterized in that, It includes 0.01 g / mL ethoxyquinoline, 0.001 g / mL dimercaprol, 0.05 g / mL poloxamer 124, as well as arachidonic acid, buffer, lyophilization protectant and preservative.

3. The arachidonic acid reagent as described in claim 2, characterized in that, The buffer solution includes Tris buffer; The freeze-drying protectant includes trehalose; The preservatives include gentamicin.

4. The arachidonic acid reagent as described in claim 3, characterized in that, It includes 1.0 g / mL arachidonic acid, 0.5 g / mL trehalose, 0.01 g / mL ethoxyquinoline, 0.001 g / mL dimercaptopropanol, 0.05 g / mL poloxamer 124, 0.005 g / mL gentamicin and 20 mM Tris at pH 7.5, as well as acceptable excipients or adjuvants.

5. The arachidonic acid reagent as described in claim 2, characterized in that, The preparation method of the arachidonic acid reagent includes the following steps: Step (a): Mix the buffer solution with the preservative, adjust the pH to 7.5, and obtain solution 1; Step (b): Take the solution 1 described in step (a) and mix it with ethoxyquinoline, dimercaptopropanol, poloxamer 124 and the lyophilization protectant to obtain solution 2; Step (c): Arachidonic acid is mixed with solution 2 described in step (b) and ultrasonically mixed to obtain the arachidonic acid reagent.

6. A reagent kit, characterized in that, It includes the arachidonic acid reagent according to any one of claims 2 to 5, and acceptable excipients or auxiliaries.

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