CN117106856A

CN117106856A - anti-Xa activity assay kit and preparation method thereof

Info

Publication number: CN117106856A
Application number: CN202310859738.2A
Authority: CN
Inventors: 鲁翌; 刘介
Original assignee: Wuhan Thalys Biological Technology Co ltd
Current assignee: Wuhan Thalys Biological Technology Co ltd
Priority date: 2023-07-13
Filing date: 2023-07-13
Publication date: 2023-11-24

Abstract

The application discloses an anti-Xa activity assay kit. It comprises an R1 reagent and an R2 reagent; the R1 reagent comprises: a chromogenic substrate, a buffer system, an anti-interference component and a preservative; the R2 reagent includes: rabbit factor Xa, buffer system, protein protectant and preservative. The application overcomes the defects of poor accuracy and high cost in the prior art; the method has the advantages of high accuracy, wide linear range, good repeatability and stability and low preparation cost.

Description

anti-Xa activity assay kit and preparation method thereof

Technical Field

The application relates to the technical field of in-vitro diagnosis, in particular to an anti-Xa activity determination kit and application thereof. More specifically, it is a heparin (salt) or low molecular weight heparin (salt) potency anti-Xa activity determination kit and its preparation method.

Background

Common heparin (UFH), low molecular heparin (LMWH), heparin derivatives and other Xa inhibitor drugs are mainly used for the prevention and treatment of thromboembolic diseases. The common point of these drugs is the anticoagulation effect of inhibiting Xa activity, except that different anticoagulants contain different contents or concentrations of Xa-resistant activity, wherein, common heparin and low molecular heparin are the most widely used anticoagulants at present.

Factor Xa is a serine protease that is located upstream of the cascade of blood coagulation reactions. Factor Xa is well centered in the intrinsic and extrinsic coagulation pathways. Factor Xa inhibitors therefore can block both endogenous and exogenous coagulation.

Heparin has complex pharmacokinetic and pharmacodynamic properties, resulting in a narrow therapeutic concentration range and extreme individual variability, requiring frequent laboratory monitoring and dose adjustments. The Activated Partial Thromboplastin Time (APTT) assay is a traditional method of monitoring UFH, which, although simple, rapid and inexpensive to operate, is poorly accurate, difficult to standardize, and varies widely from laboratory to laboratory, and has not achieved a uniform consensus on the relevant clinical therapeutic scope. At present, the measurement result is stable, the result can be standardized, and the anti-Xa (blood coagulation factor) activity measurement kit with the consensus treatment range becomes a better choice for UFH monitoring, but the anti-Xa activity measurement kit has high cost and complex operation.

Due to the drawbacks/deficiencies of the prior art: (1) The APTT detection method cannot ensure the accuracy of the detection result, has large error and no unified judgment standard, and has large subjectivity and poor accuracy according to the measurement result; (2) The existing imported anti-Xa activity determination kit (chromogenic substrate method) has poor anti-interference performance and stability and high cost.

Therefore, it is necessary to develop a high accuracy, low cost heparin (salt) or low molecular weight heparin (salt) potency anti-Xa activity assay kit and method.

Disclosure of Invention

The first object of the present application is to overcome the shortcomings of the background art, and to provide an anti-Xa activity assay kit (chromogenic substrate method), which is an anti-Xa activity assay kit of heparin (salt) or low molecular weight heparin (salt) titer, and has the advantages of high accuracy, wide linear range, good repeatability and stability, and low preparation cost, and can save 60% -70% of the cost (i.e., the cost of the prepared kit is only 30% -40% of the cost of the existing imported kit) compared with the cost of the existing imported anti-Xa activity assay kit (chromogenic substrate method). According to the application, heparin (or other similar substances) in plasma and Antithrombin (AT) form a complex, excessive added factor Xa is inhibited, the residual factor Xa acts with a specific substrate thereof, the absorption degree of paranitroaniline (pNA) is measured AT the wavelength of 405nm, the generated amount of pNA is inversely proportional to the concentration of heparin (or other similar substances) in the plasma to be detected, and the accuracy and stability of a detection result are ensured; the method solves the problems of poor detection accuracy of APTT, poor anti-interference performance and stability of an anti-Xa activity detection kit (chromogenic substrate method) and high cost in the prior market.

A second object of the application is to provide a method for preparing an anti-Xa activity assay kit.

In order to achieve the first object of the present application, the present application has the following technical solutions: an anti-Xa activity assay kit, characterized in that: including R1 and R2 agents;

the R1 reagent mainly comprises: a chromogenic substrate, a buffer system, an anti-interference component and a preservative;

the R2 reagent mainly comprises: rabbit factor Xa, buffer system, protein protectant and preservative.

In the above technical scheme, the buffer system in the R1 reagent and the R2 reagent is at least one selected from HEPES, tris or phosphate buffer salt solution; the concentration of the buffer system in the R1 reagent and the R2 reagent is 10mM-100mM (nmol/L, abbreviation: nM), and the pH is 7.2-7.6;

the preservative in the R1 reagent and the R2 reagent is at least one selected from sodium azide, proclin300 and Prionex; the addition of the preservative for the R1 reagent and the R2 reagent does not affect the reagent test, only plays a role in inhibiting or killing bacteria or fungi and the like in the reagent, and ensures the stability of the quality of the reagent.

In the technical scheme, the preservative in the R1 reagent and the R2 reagent is ProClin300 with the concentration of 0.1-0.5 g/L.

In the above technical scheme, the optimal working concentration of the buffer system in the R1 reagent and the R2 reagent is 50nM;

the optimum working concentration of the preservative ProClin300 in the R1 reagent and the R2 reagent is 0.2ml/L.

In the technical scheme, the anti-interference component of the R1 reagent mainly comprises nitrite and a nonionic surfactant Tween-20, wherein the concentration of the nitrite is as follows: 1.0-5.0g/L, tween-20 concentration is: 1.0-4.0ml/L;

the chromogenic substrate in the R1 reagent is Z-D-Arg-Gly-Arg-pNA.2HCI (S-2765) with the concentration of: 1-3nM.

In the above technical scheme, the optimal working concentration of nitrite is: 3.0g/L; the optimal working concentration of tween-20 was: 2.0ml/L;

the optimal working concentration of chromogenic substrate S-2765 in the R1 reagent is: 2nM.

In the above technical scheme, the R2 reagent protein protectant is mainly gelatin, glycerol, polyethylene glycol 600 (PEG 600), sodium chloride, and the like, wherein the gelatin concentration is: 0.5-1.5g/L, and the glycerol concentration is as follows: the concentration of 4.0-12.0ml/L, PEG600 is: 10.0-30.0ml/L, sodium chloride concentration is: 1.0-10.0 g/L;

the concentration of rabbit factor Xa (active protein) in the R2 reagent is: 220.0-440.0IU/mL.

In the above technical scheme, the optimal working concentration of gelatin in the R2 reagent protein protectant is: the optimal working concentration of the glycerol is 1.0 g/L: the optimal working concentration of 8.0ml/L, PEG600 is: the optimal working concentration of 20.0ml/L sodium chloride is: 5.0g/L;

the optimal working concentration of active protein rabbit factor Xa in the R2 reagent is as follows: 330.0IU/mL.

The above reagent or solution is purified water having a conductivity of 1. Mu.s/cm (25 ℃ C.).

In order to achieve the second object of the present application, the present application has the following technical scheme: the preparation method of the anti-Xa activity assay kit is characterized by comprising the following steps of: comprises the following steps of the method,

step one: taking raw and auxiliary materials;

step two: preparing an R1 reagent and an R2 reagent according to the production process flow and the quality control point; cleaning and drying the auxiliary materials, and performing treatments such as bottle drying and cover drying;

r1 reagent comprises 50mmol/L Hepes buffer, 2.0ml/L Tween-20, 3.0g/L nitrite, 2.0mg/L S-2765, 0.2ml/L Proclin300;

the preparation method of the R1 reagent comprises the following steps:

(1) Preparing a buffer solution: preparing a 50mmol/L buffer system (1L R1 reagent is prepared, and the explanation is given), weighing 11.9g Hepes reagent, adding 900ml purified water, stirring at uniform speed, fully dissolving, and using 5mol/L NaOH;

adjusting the pH to 7.4+/-0.2;

(2) Weighing tween-20: 2.0ml and nitrite: 3.0g, stirring at uniform speed until the mixture is fully dissolved;

(3) Weighing S-2765:1.5mg (specification: 11.0 mg. Apprxeq.15 nM), and because of the relatively small amount of addition, a high concentration stock solution can be used (for example, 150mg dissolved in 10ml of purified water, 0.1ml added to 1L, namely, concentration: 1.5 mg/L);

(4) Adding 0.2ml Proclin300 preservative and fully stirring;

(5) The volume is fixed to 1L by purified water, namely the R1 reagent;

the preparation method of the R2 reagent comprises the following steps:

r2 reagent comprises 50mmol/L Hepes buffer, 1g/L gelatin, 8ml/L glycerol, 20ml/L polyethylene glycol 600, 5g/L sodium chloride, 330IU factor Xa, 0.2ml/L Proclin300;

adjusting the pH to 7.4+/-0.2;

(2) Preparing a protein protective agent: weighing 1g of gelatin and 5g of sodium chloride, weighing 8ml of glycerin and 20ml of polyethylene glycol 600, and uniformly stirring 0.330.0IU of Xa factor until the materials are fully dissolved;

(3) Adding 0.2ml Proclin300 preservative and fully stirring;

step three: debugging the prepared R1 reagent and R2 reagent respectively, checking, and sub-packaging in penicillin bottles after the checking is qualified; if the test is not qualified, reworking for debugging again;

step four: packaging the packaged product, checking, sealing, labeling, and packaging in instruction book;

step five: and (5) packaging and inspecting the packaged product, and warehousing the packaged product serving as a finished product of the Xa resistance activity measuring kit after the packaged product is qualified.

The application has the following beneficial effects:

according to the assay kit for the Xa activity, the stability of the activity of the rabbit Xa is enhanced by using the protein protectant, and the chromogenic substrate S-2765 is used as a reagent raw material, so that the assay kit has the advantages of wide linear range, strong anti-interference capability, good repeatability and good stability when being matched, and can realize replacement of an imported Xa activity assay kit (compared with the price of the existing imported Xa activity assay kit (chromogenic substrate method)), the cost of the application can be saved by 60% -70% (namely, the price of the prepared kit is only 30% -40% of the price of the existing imported kit), the requirement of clinical examination can be fully met, the cost of purchasing detection reagents in hospitals can be reduced, and the detection cost of patients can be reduced.

Drawings

FIG. 1 is a scaled correlation linear regression of an embodiment of the present application;

FIG. 2 is a correlation linear regression of a test linear range according to an embodiment of the present application;

FIG. 3 is a correlation linear regression of the test linear range of comparative example 3 of the present application;

fig. 4 is a correlation linear regression of the test linear range of comparative example 4 of the present application.

FIG. 5 is a flow chart showing the preparation of the anti-Xa activity assay kit of the present application.

Detailed Description

The following detailed description of the application is, therefore, not to be taken in a limiting sense, but is made merely by way of example. While making the advantages of the present application clearer and more readily understood by way of illustration.

The application provides an anti-Xa activity assay kit (chromogenic substrate method), which is characterized in that heparin and Antithrombin (AT) in a sample form a compound, the compound can inhibit the activity of Xa factor in the reagent, the residual Xa factor can decompose chromogenic substrate S-2765 to release Paranitroaniline (PNA), the latter has an absorption peak AT 405nm wavelength, and the reaction kinetics of the paranitroaniline is inversely proportional to the UFH level in the sample. Therefore, heparin activity can be calculated from the amount of p-nitrobenzene produced. S-2765 is a chemically synthesized activated factor X (FXa) specific substrate with the molecular formula: Z-D-Arg-Gly-Arg-pNA.2HCI, the molecular weight is: 714.6Da. The FXa-series rabbit plasma factor X is obtained by purifying a product activated by a Russell viper factor X activator by an ion exchange and gel filtration chromatography method. FXa activates prothrombin in vivo along with other clotting factors, producing thrombin, triggering a clotting cascade.

The kit is an all-liquid reagent, and has the advantages of convenient use, low cost, wide linear range and good stability. Can realize the replacement of the imported Xa-resisting activity determination kit, fully meet the requirement of clinical examination, simultaneously reduce the cost of purchasing detection reagents in hospitals and reduce the detection cost of patients.

The method of the application mainly utilizes the absorbance change of the measured chromogenic substance to calculate the content of the measured substance. Generally, PNA is selected as the chromogenic material, free PNA is yellow, the measurement wavelength is 405nm, and the compound connected with PNA is colorless, and the chromogenic substrate S-2765 is selected for the test. Nitrite as a reducing agent can increase the anti-hemoglobin capacity; nonionic surfactants such as polyethylene oxide fatty alcohols, polyethylene oxide-propylene oxide fatty alcohols, polyethylene glycol alkyl ethers, sodium dodecylbenzenesulfonate, and mixed fatty acid glycerides; through the effects of emulsification, washing and the like of the surfactant, the interfering substances of the hemolyzed sample are removed, and meanwhile, the influence of total bilirubin is eliminated. The application optimizes and obtains an accurate and dry-resistant component.

Since factor Xa is mainly a protein, maintaining the activity of the protein is mainly to maintain the stability of the spatial structure of the protein. Factors influencing the activity of proteins are mainly the pH of the solution, the concentration of salts and sugars, the action of microorganisms, the oxidation of air, etc. On the one hand, amino acids are very good antioxidants; various sugars can play a role in protecting the activity of proteins, and the ionic integrator has multiple functions, such as oxidation resistance and corrosion resistance, and can also prevent some ion deposition; the glycerol and the sorbitol can keep the surface of the antigen-antibody moist, so that the antigen-antibody cannot be deactivated due to water loss. Other polymers of water-soluble hetero proteins or chemically inert high molecular substances (such as PEG) can form a protective film on the surface of the protein due to their large molecular weight, so that the protein structure is prevented from being damaged. On the other hand, as the preservation time is prolonged, organic matters such as amino acids, saccharides and the like in antigen-antibody and stabilizer are easily corroded by microorganisms in air and water, so that a certain preservative is added in the application.

In order to verify the optimal buffer system, stability and anti-interference effect of the kit, the application is verified in examples and comparative examples, and the detection of the kit adopts a fully automatic coagulation analyzer XRM produced by German BE company, and the application is described in detail below with reference to specific examples and comparative examples:

examples

The anti-Xa activity assay kit comprises an R1 reagent and an R2 reagent, wherein the compositions of the reagents are shown in the table 1:

table 1 example R1 reagent and R2 reagent formulations

The preparation method of the anti-Xa activity assay kit comprises the following steps,

step one: taking raw and auxiliary materials;

the preparation method of the R1 reagent comprises the following steps:

the pH was adjusted to 7.4.+ -. 0.2.

(2) Weighing tween-20: 2.0ml and nitrite: 3.0g, stirring at a constant speed until the mixture is fully dissolved.

(3) Weighing S-2765:1.5mg, and a high concentration stock solution (for example, 150mg dissolved in 10ml of purified water, 0.1ml added to 1L, namely, 1.5 mg/L) can be used because of the small amount added.

(4) 0.2ml of Proclin300 preservative was added and stirred well.

(5) And (3) using purified water to fix the volume to 1L to obtain the R1 reagent.

The preparation method of the R2 reagent comprises the following steps:

(1) R2 reagent comprises 50mmol/L Hepes buffer, 1g/L gelatin, 8ml/L glycerol, 20ml/L polyethylene glycol 600, 5g/L sodium chloride, 330IU factor Xa, 0.2ml/LProclin 300;

(2) Preparing a buffer solution: preparing a 50mmol/L buffer system (1L R1 reagent is prepared, and the explanation is given), weighing 11.9g Hepes reagent, adding 900ml purified water, stirring at uniform speed, fully dissolving, and using 5mol/L NaOH;

adjusting the pH to 7.4+/-0.2;

(3) Preparing a protein protective agent: weighing 1g of gelatin and 5g of sodium chloride, weighing 8ml of glycerin and 20ml of polyethylene glycol 600, and uniformly stirring 0.330.0IU of Xa factor until the materials are fully dissolved;

(4) Adding 0.2ml Proclin300 preservative and fully stirring;

step five: and (5) packaging and inspecting the packaged product, and warehousing the packaged product as a finished product of the Xa resistance activity determination kit after the packaged product is qualified by inspection (shown in figure 5).

Comparative example 1

An anti-Xa activity assay kit in comparative example 1, comprising an R1 reagent and an R2 reagent, wherein the buffer system of the R1 reagent and the R2 reagent is 10mmol/L, and other components are the same as the examples. The preparation method of this comparative example is the same as that of example.

Comparative example 2

An anti-Xa activity assay kit in comparative example 2, comprising an R1 reagent and an R2 reagent, wherein the buffer system of the R1 reagent and the R2 reagent is 100mmol/L, and other components are the same as the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 3

An anti-Xa activity assay kit in comparative example 3, comprising an R1 reagent and an R2 reagent, wherein the concentration of the R1 reagent S-2765 is as follows: factor Xa concentration in 1.0mg/L and R2 reagent was: 220IU/L, other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 4

An anti-Xa activity assay kit in comparative example 4, comprising an R1 reagent and an R2 reagent, wherein the concentration of the R1 reagent S-2765 is: the Xa factor concentration in the 3.0mg/L and R2 reagent is: 440IU/L, other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 5

An anti-Xa activity assay kit in comparative example 5, comprising an R1 reagent and an R2 reagent, wherein the concentration of Tween-20 in the R1 reagent is as follows: 1ml/L; the nitrite concentration was: 1g/L, the other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 6

An anti-Xa activity assay kit in comparative example 6, comprising an R1 reagent and an R2 reagent, wherein the concentration of Tween-20 in the R1 reagent is as follows: 3ml/L; the nitrite concentration was: 5g/L, the other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 7

An anti-Xa activity assay kit in comparative example 7, comprising an R1 reagent and an R2 reagent, wherein the concentration of gelatin in the R2 reagent is as follows: 0.5g/L; the glycerol concentration was: 4ml/L; the concentration of the polyethylene glycol 600 is 10.0ml/L; the sodium chloride concentration is: 1.0g/L, the other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 8

An anti-Xa activity assay kit in comparative example 8, comprising an R1 reagent and an R2 reagent, wherein the concentration of gelatin in the R2 reagent is as follows: 1.5g/L; the glycerol concentration was: 12ml/L; the concentration of polyethylene glycol 600 is 30.0ml/L; the sodium chloride concentration is: 10.0g/L, the other components remain the same as in the examples. The preparation method of the anti-Xa activity assay kit of the comparative example is the same as that of the example.

Comparative example 9

An anti-Xa activity assay kit in comparative example 9, comprising an R1 reagent and an R2 reagent, wherein the R1 reagent and the R2 reagent do not contain a Proclin300 preservative, and the other components are the same as in the examples.

The results of the tests of examples and comparative examples 1 to 9 are shown below:

(1) Scaling and results thereof

Absorbance change rate detection was performed for formulation examples and comparative examples 1 to 9. The detection sample is heparin calibrator with theoretical concentration of 0, 0.8 and 2.0 IU/ml. The absorbance reference ranges for the calibrator are shown in Table 2 (heparin calibrator brand: wo Fen; lot number: N0714558; expiration date: 2024.08.01).

The detection instrument may BE selected from various brands of coagulometer such as STAR-MAX instrument of Stago, CS5100 instrument of Hizikia, T0P750 instrument of America Wo Fen, XRM instrument of BE, germany, etc. The specific parameters may BE suitably adjusted for different instruments and the detection data for example of the XRM instrument of BE is shown in Table 3. The calibration result meets the requirements: linear regression equation R for standard curve ² And the measurement result of the calibrator is within the reference range of absorbance of the calibrator.

Table 2 absorbance reference ranges for calibrator

Calibrator heparin concentration	Absorbance (. DELTA.OD/min)
		0IU/ml	0.78-0.88
0.8IU/ml	0.38-0.48
		2.0IU/ml	0.10-0.20

Table 3 examples and comparative examples heparin calibrators

From the test results linear regression analysis of Table 3, the linear regression equations of the calibrations of examples and comparative examples 3-9 satisfy R ² 0.98. Gtoreq., satisfactory (wherein the correlation linear regression of the test linear range of the present embodiment is shown in FIG. 2); whereas comparative example 1 and comparative example 2, R of the calibrator linear regression equation ² <0.98, which is not satisfactory. From the calibrator absorbance reference range results, comparative examples 1-3 were not within the reference range. Specifically, for each comparative example, the low buffer system prepared in comparative example 1 and the high buffer system prepared in comparative example 2 both have an influence on the measurement of the calibrator, and the buffer system of the concentration of the example is selected as the optimal buffer system according to the common sense of the buffer capability of the buffer system and the test comparison result. As a result of the measurement, R in comparative example 3 was found to be different in the concentrations of the chromogenic substrates S-2765 and Xa in comparative example 3 and comparative example 4 ² = 0.9972, R in comparative example 4 ² 0.9868 are satisfactory (wherein the correlation linear regression of the test linear range of comparative example 3 is shown in fig. 3, the correlation linear regression of the test linear range of comparative example 4 is shown in fig. 4), whereas the measurement of the absorbance of the calibrator in comparative example 3 is higher than the reference range, which means that the concentrations of chromogenic substrate S-2765 and factor Xa are low, the reaction is insufficient, whereas the measurement of the absorbance of the calibrator in comparative example 4 is within the reference range, the chromogenic substrate S-2765 and factor Xa in comparative example 4 can meet the test requirements, and the concentrations of the examples are optimal concentrations in view of comprehensive practical and economic considerations. Linear regression equation R satisfying the standard curve in comparative examples 5 to 9 ² Not less than 0.98, and the measurement result of the calibrator is within the reference range of absorbance of the calibrator, which indicates the anti-interference components and protein in the reagentThe protective agent and the preservative have less influence on the absorbance of the calibrator. Comprehensive analysis, anti-Xa activity assay reagent calibration optimal concentration as in the example preparation.

(2) Tamper-resistant test

The reagents prepared in the examples were simultaneously tested with the reagents prepared in comparative examples 5 and 6, and the control samples (normal mixed plasma, plus a small amount of heparin) and the control samples were added to different concentrations of the interferent samples (containing 150mg/dL, 300mg/dL, 450mg/dL human hemoglobin (Hb), 10mg/dL, 15mg/dL, 20mg/DL Bilirubin (DB), 200mg/dL, 400mg/dL, 600mg/dL, 800mg/dL Triglycerides (TG)), the interferent sample test results were compared with the control sample test results, the recovery was calculated, and the detection instrument was exemplified by the XRM instrument of BE, the detection results are shown in Table 4:

TABLE 4 anti-tamper test results

The data in table 4 are the comparative data for the examples, comparative example 5, comparative example 6 test interferent sample and the comparative sample (non-interferent sample), the examples, comparative example 5, comparative example 6 test comparative sample differ, the sample is unchanged, but the test reagents are different, recovery = (interferent sample-comparative sample)/comparative sample 100%.

From the analysis of the anti-interference test results in table 4, the anti-interference capability of the example is obviously better than that of the comparative example 5, but the difference between the anti-interference capability of the example and that of the comparative example 6 is not very obvious, the difference between the example and the comparative example 5 is different from the difference between the anti-interference capability of the comparative example 6 and the concentrations of tween-20 and nitrite in the R1 reagent, and from the recovery rate results, it can be seen that (the ratio of the sample with the interference agent to the sample without the interference agent) the anti-interference agent (tween-20 and nitrite) is added to enhance the anti-interference capability of the reagent, but the anti-interference capability is not continuously increased along with the increase of the concentration of the anti-interference agent, namely, the anti-interference capability enters the plateau, and does not always rise, so that the optimal concentrations of the anti-interference components tween-20 and nitrite of the R1 reagent in the anti-Xa activity determination kit are prepared as in the example, and the cost is reduced while the expected anti-interference capability is achieved.

(3) Stability test

The experimental example performs long-term stability and acceleration stability tests.

The R1 reagent and the R2 reagent in the examples and the comparative examples 7 and 8 are stored for 12 months in a light-proof environment at 2-8 ℃, and the stability of each group in 12 months is tracked; meanwhile, each group is taken out for detection after thermal acceleration and destruction at 37 ℃ for different days, and heparin calibrator with theoretical concentration of 0, 0.8 and 2.0IU/ml (heparin calibrator brand: wo Fen; batch number: N0714558; efficacy period: 2024.08.01) are detected. Taking the XRM instrument of BE as an example, the test results are shown in Table 5 for the long-term stability test results and Table 6 for the accelerated stability test results, and the Relative Deviation (RD) is compared with the first test (time: 0), requiring a change in the relative deviation of less than 10%. The test results are shown in tables 5 and 6:

TABLE 5 accelerated stability test results

TABLE 6 results of long term stability test

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Note that: s1, i.e. calibrator S1, heparin concentration: 0IU/mL; s2, i.e. calibrator S2, heparin concentration: 0.8IU/mL; s3, i.e. calibrator S3, heparin concentration: 2.0IU/mL; absorbance units: delta OD/min. As can be seen from Table 5, under the preservation condition of 37 ℃, the relative deviation change of examples and comparative example 8 is less than 10% for 9 days, the relative deviation change of comparative example 7 is less than 10% for 6 days, and the relative deviation change of 9 days is more than 10%, i.e., the stability of examples can be preserved for 9 days at 37 ℃, and the stability of examples and comparative example 8 is better than that of comparative example 7.

As can be seen from Table 6, the comparative examples 8 and 8 showed a change of less than 10% in 12 months relative deviation under the preservation condition of 2-8deg.C, whereas the comparative example 7 showed a change of less than 10% in 6 months relative deviation and a change of more than 10% in 9 months relative deviation, i.e., the examples and comparative example 8 showed a long-term stability of 12 months under the preservation condition of 2-8deg.C, the comparative example 7 showed a 6 months, and the examples and comparative example 8 showed a better stability than the comparative example 7. I.e., 2-8deg.C, the examples can be stored for 12 months. From the accelerated stability and long-term stability tests, the difference of the components in the preparation of the examples and the comparative examples 7 and 8 is that the concentrations of gelatin, glycerol, polyethylene glycol 600 (PEG 600) and sodium chloride protein protectant in the R2 reagent are different; therefore, the protein protectants such as gelatin, glycerol, polyethylene glycol 600 (PEG 600), sodium chloride and the like can obviously increase the stability of the reagent, and the preservation time of the reagent is longer. Comparative example 8 protein protectant concentration was higher than in the examples, but there was no significant difference in stability correlation. Comprehensive analysis, the optimal concentrations of the protein protectants gelatin, glycerol, polyethylene glycol 600 (PEG 600) and sodium chloride in the R2 reagent are prepared as in the examples.

(4) Linear range

Diluting a high-concentration sample approaching the upper limit of a linear range into at least 5 samples with different concentrations by using a physiological saline multiple ratio, repeatedly measuring each concentration for 3 times, taking the theoretical concentration as (x), taking the average value of measured results as (y), solving a linear regression equation, calculating a correlation coefficient (r) of linear regression, requiring the linear correlation coefficient r to be more than or equal to 0.9900, and testing the results as shown in table 7:

table 7 test linear ranges for examples, comparative example 3 and comparative example 4

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From the test results analysis of Table 7, the lines of examples and comparative example 4Correlation coefficient R ² Not less than 0.99, the reagents of the illustrative examples all meet the above linear range requirements; whereas the linear range R of comparative example 3 ² 0.9794, the linear range is far narrower, that is, the absorbance of the test result is larger than the theoretical value when the heparin is high in concentration, and the reagent preparation proves that the concentrations of chromogenic substrates S-2765 and R2 in the R1 reagent and the rabbit factor Xa are lower than those in the examples, so that the high-concentration heparin sample cannot be fully reacted; comparative example 4 linear regression equation analysis and linear range were similar to those of the examples, but the concentrations of chromogenic substrate S-2765 and factor Xa in the R1 reagent of comparative example 4 were higher than those of the examples, indicating that the chromogenic substrate and factor Xa were added in excess and were not fully utilized. Comprehensive analysis, the optimal concentrations of chromogenic substrate S-2765 and R2 reagent rabbit factor Xa in the R1 reagent in the anti-Xa activity assay kit are prepared as in the examples.

(5) Calibration curve drawing

When a calibration curve is drawn, the calibrator is dissolved. The test results of the reagent prepared in the embodiment show the corresponding relation between absorbance and heparin concentration, the higher the heparin concentration is, the lower the absorbance degree is, and conversely, the lower the heparin concentration is, the higher the absorbance degree is, as shown in fig. 1, which is a calibration curve of the calibration material concentration and the absorbance logarithm of the heparin in the embodiment.

And drawing a corresponding standard curve by taking absorbance as an ordinate (Y axis) and the corresponding heparin calibrator concentration as an abscissa (X axis), and calculating to obtain a regression equation of the heparin concentration calibration curve.

From the calibration curve, the quantitative relationship between the concentration of the calibrator and the measurement result of the reagent of the example is good, and the reagent R1 and the reagent R2 prepared by the example can meet the test requirement.

In conclusion, the components and the preparation method of the R1 reagent and the R2 reagent are adopted, and the Xa-resistant activity determination kit is used for calibration, anti-interference performance, stability and linear range test, so that the embodiment of the application is proved to be the optimal choice.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Other non-illustrated parts are known in the art.

Claims

1. An anti-Xa activity assay kit, characterized in that: including R1 and R2 agents;

the R1 reagent comprises: a chromogenic substrate, a buffer system, an anti-interference component and a preservative;

the R2 reagent comprises: rabbit factor Xa, buffer system, protein protectant and preservative.

2. The anti-Xa activity assay kit according to claim 1, wherein: the buffer system in the R1 reagent and the R2 reagent is at least one of HEPES, tris or phosphate buffer salt solution; the concentration of the buffer system in the R1 reagent and the R2 reagent is 10mM-100mM, and the pH value is 7.2-7.6;

the preservative in the R1 reagent and the R2 reagent is at least one selected from sodium azide, proclin300 and Prionex.

3. The anti-Xa activity assay kit according to claim 2, wherein: the preservative in the R1 reagent and the R2 reagent is ProClin300 with the concentration of 0.1-0.5 g/L.

4. The kit for assaying anti-Xa activity according to claim 3, wherein: the concentration of the buffer system in the R1 reagent and the R2 reagent is 50nM;

the concentration of the preservative ProClin300 in the R1 reagent and the R2 reagent is 0.2ml/L.

5. The kit for assaying anti-Xa activity according to claim 4, wherein: the anti-interference component of the R1 reagent comprises nitrite and a nonionic surfactant Tween-20, wherein the concentration of the nitrite is as follows: 1.0-5.0g/L, tween-20 concentration is: 1.0-4.0ml/L;

the chromogenic substrate in the R1 reagent is S-2765 with the concentration: 1-3nM.

6. The kit for assaying anti-Xa activity according to claim 5, wherein: the nitrite concentration was: 3.0g/L; the concentration of tween-20 was: 2.0ml/L;

the concentration of chromogenic substrate S-2765 in the R1 reagent is: 2nM.

7. The kit for assaying anti-Xa activity according to claim 6, wherein: the R2 reagent protein protective agent comprises gelatin, glycerol, polyethylene glycol 600 and sodium chloride, wherein the gelatin has the concentration of: 0.5-1.5g/L, and the glycerol concentration is as follows: 4.0-12.0ml/L, polyethylene glycol 600 concentration is: 10.0-30.0ml/L, sodium chloride concentration is: 1.0-10.0 g/L;

the concentration of rabbit factor Xa in the R2 reagent is: 220.0-440.0IU/mL.

8. The anti-Xa activity assay kit according to claim 7, wherein: the concentration of gelatin in the R2 reagent protein protectant is: 1.0g/L, the concentration of glycerol is: the concentration of 8.0ml/L, PEG600 is: the concentration of 20.0ml/L sodium chloride is: 5.0g/L;

the concentration of active protein rabbit factor Xa in the R2 reagent is as follows: 330.0IU/mL.

9. The method of preparing an anti-Xa activity assay kit according to any one of claims 1-8, wherein: comprises the following steps of the method,

step one: taking raw and auxiliary materials;

step two: preparing an R1 reagent and an R2 reagent according to the production process flow and the quality control point; cleaning and drying the auxiliary materials, and performing bottle and cover drying treatment;

the preparation method of the R1 reagent comprises the following steps:

(1) Preparing a buffer solution: preparing a 50mmol/L buffer system, weighing 11.9g Hepes reagent, adding 900ml purified water, stirring at uniform speed, fully dissolving, and using 5mol/L NaOH;

adjusting the pH to 7.4+/-0.2;

(3) Weighing S-2765:1.5mg, since the addition amount is relatively small, a high concentration stock solution can be used;

(4) Adding 0.2ml Proclin300 preservative and fully stirring;

(5) The volume is fixed to 1L by purified water, namely the R1 reagent;

the preparation method of the R2 reagent comprises the following steps:

adjusting the pH to 7.4+/-0.2;

(3) Adding 0.2ml Proclin300 preservative and fully stirring;