CN115219486B

CN115219486B - Detection kit for anti-Xa activity of heparin and low molecular heparin and non-disease diagnosis detection method thereof

Info

Publication number: CN115219486B
Application number: CN202210803714.0A
Authority: CN
Inventors: 丁重辉; 杨娟
Original assignee: Beijing Succeeder Technology Inc
Current assignee: Beijing Succeeder Technology Inc
Priority date: 2022-07-09
Filing date: 2022-07-09
Publication date: 2023-05-30
Anticipated expiration: 2042-07-09
Also published as: CN115219486A

Abstract

The application relates to the technical field of biological factor activity detection, and in particular discloses a detection kit for anti-Xa activity of heparin and low-molecular heparin and a non-disease diagnosis detection method thereof. The detection kit comprises a sample diluent, a chromogenic substrate solution and an activating factor mixed solution; the activating factor mixed solution comprises the following components: 5-10nkat/mL of activating factor X, 50-200 mu g/mL of dextran sulfate, 0.06-0.1wt% of activating factor preservative and 1.8-2.4wt% of buffer reagent; the using method comprises the following steps: mixing the sample diluent with a sample to be detected, adding a chromogenic substrate solution, and then culturing at 35-37 ℃; and then adding the activating factor mixed solution, uniformly mixing, and detecting the signal intensity of the chromogenic substrate. The detection kit has the advantages of accurate detection and strong anti-interference capability.

Description

Detection kit for anti-Xa activity of heparin and low molecular heparin and non-disease diagnosis detection method thereof

Technical Field

The application relates to the technical field of biological factor activity detection, in particular to a detection kit for anti-Xa activity of heparin and low-molecular heparin and a non-disease diagnosis detection method thereof.

Background

Heparin was first discovered from the liver and then: heparin is widely contained in various animal organs and tissues. Currently, heparin is commonly used as an extract from the mucosa of bovine lung or porcine small intestine. The physiological effects of heparin include anticoagulation and blood lipid reduction. Heparin, whether in vivo or in vitro, has a strong anticoagulation effect as an anticoagulant, and thus is widely used as an anticoagulant in clinic.

Heparin itself has no anticoagulation, but has strong affinity with AT III in plasma, and heparin-AT III (1:1 complex) can be formed quickly after the heparin-AT III contacts, and the binding makes the AT III have obvious configuration change and exposes its central peptide (RCL), so that the capacity of AT III to bind Xa and IIa is greatly improved, and the activity of AT III after heparin binding can be improved by about 1000 times, so that the coagulation effect can be produced.

The principle of determining heparin activity by a chromogenic substrate method is as follows: heparin activity was determined based on the ability of the heparin-AT III complex formed by AT III and heparin to neutralize factor Xa. AT III in plasma and heparin in plasma are capable of forming heparin-AT III complexes; in the detection, chromogenic substrate is added into plasma, then excessive Xa factor is added, and when Xa factor is added into the plasma-chromogenic substrate mixed solution, two reactions simultaneously occur, namely: the chromogenic substrate hydrolyzes under the action of factor Xa to release paranitroaniline (pNA), and the heparin-AT III complex has an inhibitory effect on the activity of factor Xa, and when the competing reactions reach equilibrium, the amount of pNA is inversely proportional to the heparin concentration in the reaction system.

Wherein, the clinical significance of the anti-Xa activity monitoring is as follows: the dosage adjustment of the medicine is convenient, so that a patient can enter the treatment range more quickly, and the anticoagulation treatment target can be realized more accurately; the medication is prevented from being insufficient, and the occurrence probability of venous thrombosis is reduced; preventing overdose and reducing bleeding risk; preparing in the perioperative period, and reducing bleeding and thrombosis complications in the perioperative period; theoretically, heparin should be monitored for efficacy until therapeutic doses are reached for each administration. Other anti-Xa drugs also need to be monitored in life threatening situations in special populations.

Most of the products capable of realizing the detection of the Xa-resistant activity of heparin and low-molecular heparin are foreign kit products, and specific reagents contained in the kit are unknown, so that the detection of the project is more dependent on imported products. In addition, when the related detection kit is used at present, the detection sample is often a plasma sample, and the plasma sample contains hemoglobin, bilirubin, chyle and the like, and all substances can cause interference to the detection result; when the content of heparin or low molecular weight heparin in the plasma sample is too low, the interference effect is more obvious, so that the detection accuracy of the detection products is influenced. Therefore, it is necessary to provide a detection kit for heparin and low molecular heparin with high anti-interference capability and accurate detection results.

Disclosure of Invention

In order to improve the anti-interference capability and detection accuracy of the related detection kit, the application provides a detection kit for anti-Xa activity of heparin and low-molecular heparin and a non-disease diagnosis detection method thereof.

In a first aspect, the present application provides a kit for detecting anti-Xa activity of heparin and low molecular heparin, which adopts the following technical scheme:

a detection kit for anti-Xa activity of heparin and low molecular heparin comprises a sample diluent, a chromogenic substrate solution and an activating factor mixed solution; the activating factor mixed solution comprises the following components: activator X5-10 nkat/mL, dextran sulfate 50-200 μg/mL, preservative 0.06-0.1wt% and buffer agent 1.8-2.4wt%.

Through adopting above-mentioned technical scheme, this application through reasonable in design's activation factor mixed solution, especially wherein dextran sulfate (dextran sulfate is DS) the interpolation can reduce heparin antagonist such as platelet factor 4 (PF 4) to the influence of testing result (PF 4 can restrain heparin activity) to the effect of improvement detection accuracy is realized. The mixed solution of the activating factors of the components is combined with the interaction of the chromogenic substrate solution and the sample diluent, so that the finally prepared detection kit has excellent detection accuracy and anti-interference capability. The method comprises the following steps: after the kit is used, the correlation of the kit in a linear range of 0-2 IU/mL is better when heparin and low-molecular-weight heparin are detected; excellent repeatability: the horizontal 1CV is not more than 10%, and the horizontal 2CV is not more than 8%; higher accuracy and better anti-bilirubin F, anti-hemoglobin, anti-bilirubin C and anti-chyle effects: anti-bilirubin F <26.9mg/dL, anti-hemoglobin <407.2mg/dL, anti-bilirubin C <20.9mg/dL, anti-chyle <4592FTU.

Optionally, the preservative for the activating factor is at least one selected from Proclin 300, sodium benzoate, potassium sorbate and sodium azide.

Optionally, the chromogenic substrate solution comprises the following components based on the weight of the chromogenic substrate solution: 0.7-1.2mg/mL of chromogenic substrate and 0.06-0.1wt% of preservative for chromogenic substrate.

Optionally, the preservative for a chromogenic substrate is at least one selected from Proclin 300, sodium benzoate, potassium sorbate and sodium azide.

Optionally, the chromogenic substrate contained in the chromogenic substrate solution is Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA.

By adopting the above technical scheme, the reaction rate of the chromogenic substrate Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA (i.e. S-2732) selected in the application and other reagents in the application is slower than that of other chromogenic substrates such as chromogenic substrates S-5288, S-2765, PA2705 or CBS 02.44. The heparin content is different, the reaction rate is different, and the heparin concentration can have good linear relation with OD/min during detection, so that the detection purpose of the application can be realized. However, the other chromogenic substrates listed above have a relatively high reaction rate after addition, and it is difficult to achieve accurate detection because the heparin concentration and OD/min have a good linear relationship in a relatively large heparin concentration range during detection, and thus the detection sensitivity is relatively low.

Optionally, the buffer reagent comprises, based on the weight of the activator mixture: 0.512-0.703wt% of tris (hydroxymethyl) aminomethane, 0.22-0.32wt% of disodium ethylenediamine tetraacetate, 0.996-1.216wt% of sodium chloride and 0.08-0.12wt% of polyethylene glycol.

Optionally, the activator mixture further comprises 18-25wt% of a protein protectant based on the weight of the activator mixture.

Through adopting above-mentioned technical scheme, the selection of protein protection agent, further protection activating factor X for this protein stability improves, guarantees that the detection kit of this application is stable in stipulating the validity period, and then guarantees the test accuracy.

Optionally, the protein protectant comprises, based on the weight of the activator mixture: bovine serum albumin 0.8-1.2wt%, glycerin 18-23wt%.

By adopting the technical scheme, compared with other protein protective agents, the other protein protective agents can be, for example, single PEG6000, single mannitol, single sucrose, single DMSO, single glycerol and the like, and the bovine serum albumin and the glycerol are used as the composite protein protective agent, so that the effect of effectively protecting the activating factor X for a longer time is achieved, and the problem of poor detection results caused by activity loss of the activating factor X is effectively relieved.

Optionally, the activating factor mixed solution is prepared by a method comprising the following steps: adding dextran sulfate, an activating factor preservative and a buffer agent into water to obtain an initial mixed solution, wherein the pH value of the initial mixed solution is 8.3-8.5; and adding the activating factor X into the initial mixed solution, and uniformly mixing to obtain the activating factor mixed solution.

Optionally, the polyethylene glycol is selected from at least one of polyethylene glycol 6000, polyethylene glycol 4000 and polyethylene glycol 8000.

Optionally, the polyethylene glycol is polyethylene glycol 6000.

Alternatively, the activator X is derived from bovine, porcine or human.

Alternatively, the activator X is of bovine origin.

In a second aspect, the present application provides a method for diagnosing and detecting a non-disease in the above-mentioned kit, which adopts the following technical scheme:

the non-disease diagnosis detection method of the kit comprises the following steps:

mixing the sample diluent with a sample to be detected, adding a chromogenic substrate solution, and then culturing at 35-37 ℃; and then adding the activating factor mixed solution, uniformly mixing, and detecting the signal intensity of the chromogenic substrate.

By adopting the technical scheme, the application method selected in the application needs to be noted that: after the sample diluent and the sample to be detected are mixed, a chromogenic substrate is added for incubation for a certain time, and then an activating factor mixed solution is added; rather than adding the activating factor mixture for incubation for a period of time before adding the chromogenic substrate. The latter has high requirements on incubation time, and the incubation time needs to be strictly controlled, and clinical tests are generally carried out on a plurality of items at the same time, so that incubation overtime is easy to occur, and the deviation of test results is caused. The technical scheme mainly considers the addition sequence of the chromogenic substrate and the activating factor X, and the prolonged incubation time has no obvious influence on the result; by adopting the technical scheme, the accuracy of the detection result can be further improved.

Alternatively, the incubation time after addition of the chromogenic substrate solution is 30-240s.

In summary, the present application has the following beneficial effects:

1. according to the method, the reasonable-design activating factor mixed solution, particularly the dextran sulfate added into the activating factor mixed solution is combined with a specific chromogenic substrate and a reagent adding sequence, so that the effects of improving the detection accuracy and improving the anti-interference capability of the detection reagent are achieved.

2. Specific protein protectants are selected for use in the present application: bovine serum albumin 0.8-1.2wt% and glycerin 18-23wt% to further improve the protection effect on the activating factor X and to improve the detection accuracy.

Drawings

FIG. 1 is a standard curve of heparin and low molecular heparin of the present application; among them, FIG. 1 (A) is a standard curve of heparin, and FIG. 1 (B) is a standard curve of low-molecular heparin.

Detailed Description

The present application is described in further detail below with reference to the drawings and examples.

Examples

The activating factor X of the present application, also known as factor Xa or FXa; detection principle of the detection kit of the application: the detection kit for Xa-resistant activity is detected by a chromogenic substrate method, and the principle is to determine the heparin activity based on the capability of a complex formed by AT III and heparin to neutralize activated Xa factor. The method comprises the steps of adding a chromogenic substrate into plasma firstly, adding excessive Xa factor, and simultaneously carrying out two reactions when Xa factor is added into a plasma-substrate mixed solution, wherein the two reactions are carried out on the substrate under the action of Xa to release paranitroaniline (pNA), the other is inhibition of the heparin-AT III complex on Xa factor, and after the competing reaction reaches equilibrium, the amount of pNA is inversely proportional to the concentration of heparin in a reaction system, so that the content of heparin or low-molecular heparin is calculated by establishing a standard curve.

Example 1

A detection kit for anti-Xa activity of heparin and low molecular heparin contains a sample diluent, a chromogenic substrate solution and an activating factor mixed solution.

Wherein, the components contained in the sample diluent are: sodium chloride and preservatives; the preparation method of the sample diluent comprises the following steps: dissolving sodium chloride in purified water, and adding preservative to make the mass fraction of sodium chloride in the sample diluent be 0.9% and the mass fraction of preservative be 0.08%.

The chromogenic substrate liquid comprises the following components: a chromogenic substrate and a preservative; the preparation method of the chromogenic substrate liquid comprises the following steps: the chromogenic substrate S-2732 powder (i.e., suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA HCl) was dissolved in purified water to prepare a chromogenic substrate initial solution having a final concentration of 0.85mg/mL, and then 0.08wt% of preservative was added (i.e., the mass fraction of preservative in the chromogenic substrate solution was 0.08%).

The preparation method of the activating factor mixed solution comprises the following steps: dissolving 0.606wt% of Tris (hydroxymethyl) aminomethane (Tris), 0.28wt% of disodium ethylenediamine tetraacetate (EDTA), 1.023wt% of sodium chloride, 0.1wt% of PEG6000, 1wt% of bovine serum albumin, 20wt% of glycerol, 100 mug/mL of dextran sulfate and 0.08wt% of preservative in purified water, and magnetically stirring and uniformly mixing to obtain an initial mixed solution; then adding hydrochloric acid to adjust the pH value to 8.40, and adding FXa 7.5nkat/mL to obtain the activating factor mixed solution.

The non-disease diagnosis detection method of the detection kit for the Xa resisting activity of the heparin and the low molecular heparin comprises the following steps:

mixing 40 mu L of a sample diluent and 10 mu L of a sample to be detected, wherein the sample to be detected is a plasma sample; then adding 100 mu L of chromogenic substrate solution, then culturing for 60s at 37 ℃, and cleaning a sample needle once; then adding 100 mu L of the activating factor mixed solution, uniformly mixing, and detecting the signal intensity of the chromogenic substrate for 30-60s.

1. Method for making standard curve of detection kit in use

The method comprises the following steps:

1. the preparation method of the detection kit for the Xa resisting activity of heparin and low molecular heparin comprises the following steps: the detection kit for the anti-Xa activity of heparin and low molecular heparin comprises a sample diluent, a chromogenic substrate solution and an activating factor mixed solution.

The specific preparation method of the sample diluent comprises the following steps: sodium chloride was taken and dissolved in purified water so that the sodium chloride content was 0.9wt%, and then 0.08wt% of preservative was added.

The chromogenic substrate in the chromogenic substrate solution is S-2732, and the specific preparation method comprises the following steps: the chromogenic substrate S-2732 powder was dissolved in purified water and formulated into a chromogenic substrate solution having a final chromogenic substrate S-2732 concentration of 0.85mg/mL and a preservative content of 0.08wt%.

The activating factor in the activating factor mixed solution is activating factor X (namely FXa), and the specific preparation method comprises the following steps: 0.606wt% of Tris (hydroxymethyl) aminomethane (Tris), 0.28wt% of disodium ethylenediamine tetraacetate (EDTA), 1.023wt% of sodium chloride, 0.1wt% of PEG6000, 1wt% of bovine serum albumin, 100 mug/mL of dextran sulfate, 0.08wt% of preservative and 20wt% of glycerol are dissolved in purified water, and the mixture is stirred and mixed by magnetic force, then hydrochloric acid is added to adjust the pH to 8.40, and FXa 7.5nkat/mL is added.

2. The specific preparation method of heparin or low molecular heparin standard substance comprises the following steps:

freeze-drying heparin standard: and adding 0.05wt% sodium benzoate and 1.5wt% sucrose into the plasma sample, respectively adding heparin international standard substances (07/328) with different concentrations, so that the theoretical final concentration of the heparin international standard substances is 0IU/mL, 1IU/mL and 2IU/mL respectively, and subpackaging and freeze-drying with 1.1 mL/bottle after uniform mixing.

Lyophilization of low molecular heparin standards: adding 0.05wt% sodium benzoate and 1.5wt% sucrose into the plasma sample, respectively adding low molecular heparin international standard substances (11/176) with different concentrations to ensure that the theoretical final concentration of the low molecular heparin is respectively 0IU/mL, 1IU/mL and 2IU/mL, and sub-packaging and freeze-drying with 1.1 mL/bottle after uniform mixing.

And (3) re-dissolving a standard substance: accurately adding purified water into 1 mL/bottle heparin standard or 1 mL/bottle low molecular heparin standard, re-dissolving, slightly mixing, standing for 10min, and balancing to room temperature.

3. Test instrument: SF-8200 full-automatic coagulation tester.

4. Establishment of an anti-Xa activity detection standard curve:

the method comprises the following specific steps:

calibrating heparin standard substances of 0IU/mL, 1IU/mL and 2IU/mL by using the detection kit for anti-Xa activity of heparin and low molecular heparin, firstly taking 40 mu L of sample diluent, then taking 10 mu L of plasma sample, adding the 10 mu L of plasma sample into a test cup, adding 100 mu L of chromogenic substrate solution, uniformly mixing, and incubating for 60s at 37 ℃; after the sample needle is washed, 100 mu L of the activating factor mixed solution is added, and then the absorbance is measured at 405nm for 30-60s.

Calibrating low molecular heparin standard substances of 0IU/mL, 1IU/mL and 2IU/mL by using the detection kit for the anti-Xa activity of heparin and low molecular heparin, firstly taking 40 mu L of sample diluent, then taking 10 mu L of plasma sample, adding the 10 mu L of plasma sample into a test cup, adding 100 mu L of chromogenic substrate solution, uniformly mixing, and incubating at 37 ℃ for 60s; after the sample needle is washed, 100 mu L of the activating factor mixed solution is added, and then the absorbance is measured at 405nm for 30-60s.

And (3) according to the heparin concentration and the corresponding test OD, preparing a standard curve by taking the OD/min logarithmic value as an abscissa and the heparin content as an ordinate. And (5) establishing a standard curve of the low-molecular heparin. See fig. 1, heparin standard curve formula: y= -0.3541x-0.589, r ² =1.0, low molecular heparin standard curve formula: y= -7.291x-1.233, r ² ＝1.0。

5. Sample testing: automatic analysis and test are carried out according to the steps of the table 1, and heparin or low molecular heparin content is obtained through conversion.

TABLE 1 reaction system

2. Component determination of the kit of the present application

(1) Determination of chromogenic substrate in chromogenic substrate solution

Five different chromogenic substrates were tested in total in the development of the kit, the information of each chromogenic substrate is shown in Table 2, the purity of the four substrates is not less than 95%, wherein Km (Michaelis constant, mi constant) of S-2732 is maximum, which indicates that the affinity between the chromogenic substrate and the substrate is minimum.

For determining the optimal chromogenic substrates and the optimal reaction system, five chromogenic substrates are respectively tested on an SF-8200 full-automatic coagulometer, the same activating factor Xa is selected, and 4 different reaction systems are adopted, and the measurement results are shown in Table 3.

TABLE 2 chromogenic substrate information summary table

Table 3 summary of test results

Note that:

reaction system 1: incubation time 60s, loading sequence: firstly adding an activating factor X and then adding a chromogenic substrate;

reaction system 2: incubation time 240s, loading sequence: firstly adding an activating factor X and then adding a chromogenic substrate;

reaction system 3: incubation time 60s, loading sequence: firstly adding a chromogenic substrate and then adding an activating factor X;

reaction system 4: incubation time 240s, loading sequence: the chromogenic substrate is added first and then the activating factor X is added.

Selection of the reaction system: wherein, the reaction system 1 and the reaction system 2 adopt a sample adding sequence of adding the activating factor X and then adding the chromogenic substrate, and the incubation time is respectively 60s and 240s; the results show that the OD/min of the measurement is obviously reduced along with the prolonged incubation time, and the detection results at different times are obviously different. The sample adding sequence has higher requirements on incubation time, the incubation time needs to be strictly controlled, and a plurality of items are generally tested simultaneously during clinical detection, so that the condition of incubation overtime easily occurs, thereby causing deviation of a test result and inaccurate test result. The reaction system 3 and the reaction system 4 adopt a sample adding sequence of adding a chromogenic substrate and then adding an activating factor X, the incubation time is respectively 60s and 240s, and the result shows that the incubation time has no obvious influence on the result, so that the kit of the application adopts the reaction system 3.

Selection of chromogenic substrates: the result of the reaction system 3 shows that when the concentration of the heparin is 0IU/mL, each chromogenic substrate reacts with the activating factor X, wherein the reaction rate of the chromogenic substrate S-2732 is slower, and when the heparin content is different, the reaction rates are different; the reaction rate of the rest chromogenic substrates is too high, so that the sensitivity of the kit and the detection method is reduced, and therefore, the chromogenic substrates are not suitable for the sample adding sequence of adding the chromogenic substrates and then adding the activating factor X, namely, the reagent and the method have lower adaptation degree; therefore, the kit adopts a chromogenic substrate S-2732.

(2) Determination of protein protectants for activating factor X

The results of table 4 show that by adding different protein protectants to enhance FXa stability: when 1.5wt% PEG6000, 2g/L mannitol, 2g/L sucrose, 1.5wt% DMSO or 1.5wt% glycerol and the like are independently used as protein protective agents, the protein protective agents have no obvious protective effect on FXa, the concentration of the heparin is gradually increased along with the time, and the absolute deviation and the relative deviation are not satisfactory. When 1wt% BSA, 20wt% glycerol and 1wt% BSA+20wt% glycerol are used as the composite protein protective agent, the composite protein protective agent has obvious protective effect on FXa, wherein the 1wt% BSA+20wt% glycerol is more preferable, the absolute deviation of the measured heparin concentration at 0IU/mL is not more than +/-0.1, and the relative deviation of (0, 2) is not more than +/-15 percent, so that the composite protein protective agent is satisfactory.

TABLE 4 protection effect of different protein protectants on activation factor X at different heparin concentrations

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The requirements are: the absolute deviation of 0IU/mL is not more than + -0.1, (0, 2) is not more than + -15%, and the absolute deviation in the table is the relative deviation of the absolute deviation at the corresponding heparin concentration.

(3) Determination of dextran sulfate

Platelet factor 4 (PF 4) is easy to bind and neutralize heparin and combine with heparin to form PF 4/heparin binding protein, because PF4 prevents the formation of heparin and AT complex, thereby affecting accuracy, and the addition of Dextran Sulfate (DS) can reduce the influence of heparin antagonists such as PF4, etc., improving accuracy. The result of setting different DS concentrations within the range of 0-200 mug/mL shows that the absolute deviation of DS concentration 0.01 mug/mL and 1 mug/mL from the heparin concentration 0IU/mL measured without DS is smaller than 0.05, (0, 2) is smaller than 15%, and the DS addition amount is insufficient and cannot reduce the influence of heparin antagonist such as platelet factor 4 (PF 4) and the like, so that the concentration is not usable, while the absolute deviation of 100 mug/mL and 200 mug/mL from the heparin concentration 0IU/mL measured without DS is not smaller than 0.05, (0, 2) is larger than 15%, the DS can play a role of reducing the influence of heparin antagonist such as platelet factor 4 (PF 4) and the like, and the difference of the result of 100 mug/mL and 200 mug/mL is smaller, so that the DS addition amount adopts 100 mug/mL in the application.

TABLE 5 addition of different DS concentrations

The requirements are: the absolute deviation of 0IU/mL is not less than 0.05, and the (0, 2) relative deviation is more than 15 percent.

3. Analytical performance assessment of the kit of the present application

(1) Linear range

High concentration samples (i.e., 2IU/mL concentration) near the upper limit of the test interval were diluted to 6 concentrations in the ratios of 4/5, 2/5, 1/10, and 0, respectively, with normal plasma, and each diluted concentration was tested 3 times, and the average value of the measurement results was obtained. And (3) taking the dilution concentration as an independent variable, taking the average value of the measurement result as a dependent variable to calculate a linear regression equation, and calculating a correlation coefficient of linear regression: the final calculation gave heparin r=0.9993, low molecular heparin r=0.9987, and the specific results are shown in table 6. The result shows that the kit provided by the application has better correlation in the linear range of 0-2 IU/mL.

Table 6 Linear range of detection kit for anti-Xa activity of heparin and low molecular heparin

(2) Blank limit

The sample dilutions were tested with the reagents, the test was repeated 20 times as in example 1, the mean (x) and Standard Deviation (SD) of the 20 test results were calculated, the blank was calculated as x+2sd, and the results are shown in table 7, x+2sd= -0.03, less than 0.01.

Table 7 blank for detection kit of anti-Xa activity of heparin and low molecular heparin

(3) Repeatability of

Under repetitive conditions, a bottle of reagent was taken, and the test was repeated 10 times with quality control level 1 and level 2, respectively, as in the examples, and the average value (x), standard Deviation (SD) and Coefficient of Variation (CV) of the measured values of the quality control at different levels were calculated. The results are shown in Table 8, and the results show that the kit provided by the application has good repeatability, wherein the level 1CV is not more than 10%, and the level 2CV is not more than 8%.

Table 8 repeatability of test kit for anti-Xa activity of heparin and low molecular heparin

(4) Accuracy of

The reagent was tested by the method of the example using heparin/low molecular heparin international standard diluted to a certain concentration, repeated 3 times, and the relative deviation was calculated. If the relative deviation of the results of 3 times is not more than +/-15%, the test result is judged to be qualified. The results are shown in Table 9, and the results show that the repeatability of the kit is better, and the CV (constant velocity) of the kit is not more than +/-15% when 3 repeated tests are carried out on the international standard of heparin/low molecular heparin (1 IU/mL).

Table 9 accuracy of detection kit for anti-Xa Activity of heparin and Low molecular heparin

(5) Anti-interference capability

The samples of the interferents (heparin 0, 1 IU/mL) were prepared from bilirubin F, hemoglobin, bilirubin C, and chyle at different concentration ratios, and the interferent samples were tested using the reagents according to the methods of the examples. The results are shown in Table 10; the results show that the kit has good specificity, and the anti-bilirubin F is less than 26.9mg/dL, the anti-hemoglobin is less than 407.2mg/dL, the anti-bilirubin C is less than 20.9mg/dL, and the anti-chyle is less than 4592FTU.

Table 10 heparin and low molecular heparin anti-Xa activity detection kit anti-interference agent test

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3. Comparison of the Performance of the kit of the present application with the existing kit products

The performance of the detection kit for the anti-Xa activity of heparin and low-molecular heparin of different manufacturers is compared, the detection kit for the anti-Xa activity of heparin and low-molecular heparin of each manufacturer is used for sample preparation, then the sample preparation is carried out, the sample preparation is tested in an SF-8200 automatic coagulometer, and the reaction systems of the detection kits for the anti-Xa activity of heparin and low-molecular heparin of different manufacturers are shown in Table 11; the results of comparison of the heparin and low molecular heparin anti-Xa activity detection kits from different manufacturers are shown in Table 12.

The reaction systems of the detection kits for the anti-Xa activity of heparin and low molecular heparin of different manufacturers are as follows.

TABLE 11 reaction systems of detection kits for anti-Xa Activity of heparin and Low molecular heparin from different manufacturers

Reagent(s)	Self-grinding reagent	Company B	Company C	Company D
					Sample (mu L)	10	25	10	25
Sample diluent (mu L)	40	75	/	75
					Chromogenic substrate solution (μL)	100	100	100	100(FXa)
Incubation time(s)	60	240	180	240
					Mixed solution of activating factors	100	100	75	100 (bottom)
Test time(s)	30～60	30～60	20～80	30～60
					Total volume (mu L)	250	300	185	300

Note that: A. the B, C reagent adopts a sample adding sequence of adding the substrate and then adding the FXa, and the D reagent adopts a sample adding sequence of adding the FXa and then adding the substrate.

The comparison of heparin and low molecular heparin anti-Xa activity detection kits from different manufacturers is as follows.

Table 12 comparison of different kits

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As can be seen from the data results in Table 8, when the kit is used for detecting the Xa-resistant activity of heparin and low-molecular heparin, the linear range of detection on heparin and low-molecular heparin is large (0-2.0 IU/mL), and the kit has excellent anti-interference ability on hemoglobin, bilirubin F, bilirubin C and chyle, and the anti-interference concentration on hemoglobin is 407.2mg/dL at the highest.

The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims

1. A method for the non-disease diagnostic detection of heparin and low molecular heparin anti-Xa activity, comprising the steps of:

mixing the sample diluent with a sample to be detected, adding a chromogenic substrate solution, and then culturing at 35-37 ℃; then adding the activating factor mixed solution, uniformly mixing, and detecting the signal intensity of the chromogenic substrate;

the preparation method of the sample diluent comprises the following steps: dissolving sodium chloride in purified water, and adding a preservative to enable the mass fraction of the sodium chloride to be 0.9% and the mass fraction of the preservative to be 0.08% in the sample diluent;

the chromogenic substrate liquid is used as a reference, and comprises the following components: 0.85/mg/mL of a chromogenic substrate, 0.08wt% of a preservative, wherein the chromogenic substrate is Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA;

taking the activating factor mixed solution as a reference, the activating factor mixed solution comprises the following components: 7.5nkat/mL of activating factor X, 0.606wt% of tris (hydroxymethyl) aminomethane, 0.28wt% of disodium ethylenediamine tetraacetate, 1.023wt% of sodium chloride, 0.1wt% of PEG6000, 1wt% of bovine serum albumin, 20wt% of glycerol, 100 mug/mL of dextran sulfate and 0.08wt% of preservative.

2. The method for diagnosing and detecting a non-disease according to claim 1, wherein the activating factor mixture is prepared by a method comprising the steps of: adding dextran sulfate, preservative, tris (hydroxymethyl) aminomethane, disodium ethylenediamine tetraacetate, sodium chloride, PEG6000, bovine serum albumin and glycerol into water to obtain an initial mixed solution, so that the pH value of the initial mixed solution is 8.3-8.5; and adding the activating factor X into the initial mixed solution, and uniformly mixing to obtain the activating factor mixed solution.

3. The method of claim 1, wherein the activator X is bovine, porcine or human.