CN115219486A

CN115219486A - Detection kit for anti-Xa activity of heparin and low molecular heparin and non-disease diagnosis detection method thereof

Info

Publication number: CN115219486A
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: 2022-10-21
Anticipated expiration: 2042-07-09
Also published as: CN115219486B

Abstract

The application relates to the technical field of biological factor activity detection, and particularly 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 mug/mL of dextran sulfate, 0.06-0.1wt% of preservative for activating factor 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 an 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 found: heparin is widely contained in organs and tissues of various animals. Currently, heparin is usually extracted from the mucosa of the small intestine of cattle or pigs. The physiological effects of heparin include anticoagulation and blood lipid lowering. Heparin, as an anticoagulant drug, has a strong anticoagulant effect both in vivo and in vitro, and thus is widely used as an anticoagulant in clinical practice.

Although heparin itself has no anticoagulation effect, it has strong affinity with AT III in plasma, and after the two are contacted, heparin-AT III (1:1 complex) can be quickly formed, and the binding can make AT III produce obvious configuration change, and expose its central peptide segment (RCL), so that the capability of AT III for binding Xa and IIa can be greatly raised, and the activity of AT III after being bound with heparin can be raised by about 1000 times, so that the coagulation action can be produced.

The principle of the chromogenic substrate method for determining the heparin activity 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, firstly, the chromogenic substrate is added into the plasma, then the excessive Xa factor is added, when the Xa factor is added into the plasma-chromogenic substrate mixed solution, two reactions occur simultaneously, which are respectively: the chromogenic substrate is hydrolyzed under the action of the Xa factor to release p-nitroaniline (namely pNA), the heparin-AT III compound has an inhibition effect on the activity of the Xa factor, and when the two competing reactions reach equilibrium, the amount of pNA is inversely proportional to the concentration of heparin in the reaction system.

Among these, the clinical significance of monitoring of anti-Xa activity is: the dosage of the medicine is convenient to adjust, so that the patient can enter a treatment range more quickly, and the anticoagulation treatment target is realized more accurately; the medicine insufficiency is prevented, and the occurrence probability of venous thrombosis is reduced; excessive medicine is prevented, and bleeding risk is reduced; preparing in a perioperative period, and reducing bleeding and thrombosis complications in the perioperative period; theoretically, heparin should be monitored for efficacy at each administration until a therapeutic dose is reached. Other anti-Xa agents, in life threatening situations in particular populations, also need to be monitored.

Since most of the current products capable of detecting the anti-Xa activity of heparin and low molecular weight heparin are foreign kit products, and specific reagents contained in the kit are unknown, the detection of the item is more dependent on imported products. In addition, when the related detection kit is used at present, a detection sample is often a plasma sample which contains hemoglobin, bilirubin, chyle and the like, and all the substances can bring interference to a 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 product is influenced. Therefore, it is necessary to provide a detection kit for anti-Xa activity of heparin and low molecular weight heparin, which has high anti-interference ability and accurate detection result.

Disclosure of Invention

In order to improve the anti-interference capability and detection accuracy of related detection kits, 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 detection kit for anti-Xa activity of heparin and low molecular weight 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 activated factor mixed solution; the activating factor mixed solution comprises the following components: 5-10nkat/mL of activating factor X, 50-200 mug/mL of dextran sulfate, 0.06-0.1wt% of preservative for activating factor and 1.8-2.4wt% of buffer reagent.

Through adopting above-mentioned technical scheme, this application can reduce the influence of heparin antagonists such as platelet factor 4 (being PF 4) to the testing result (PF 4 can restrain heparin activity) through the addition of reasonable in design's activation factor mixed liquid, especially dextran sulfate (dextran sulfate is DS) wherein to realize improving the effect of detection accuracy. The detection kit prepared finally has excellent detection accuracy and anti-interference capability by adopting the activating factor mixed solution of the components and combining the mutual matching action of the chromogenic substrate solution and the sample diluent. The method specifically comprises the following steps: after the kit is used, when the kit is used for detecting heparin and low molecular weight heparin, the correlation of the kit in a linear range of 0-2 IU/mL is good; the repeatability is excellent: level 1CV is no greater than 10%, level 2CV is no greater than 8%; the accuracy is higher and anti bilirubin F, anti hemoglobin, anti bilirubin C and anti chyle effect are better: anti-bilirubin F <26.9mg/dL, anti-hemoglobin <407.2mg/dL, anti-bilirubin C <20.9mg/dL, anti-chyle <4592FTU.

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

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

Optionally, the preservative for 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 employing the above-described protocol, the reaction rate of the chromogenic substrate selected for use in the present application, suc-Ile-Glu (γ -Pip) -Gly-Arg-pNA (i.e., S-2732), and other reagents of the present application, is relatively slow compared to 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 and OD/min have a good linear relation in the detection process, so that the detection purpose of the application can be realized. However, the other chromogenic substrates listed above have a high reaction rate after addition, and it is difficult to achieve accurate detection and low detection sensitivity because the heparin concentration and OD/min are in a good linear relationship in a wide heparin concentration range during detection.

Optionally, based on the weight of the activated factor mixed solution, the buffer reagent includes: 0.512-0.703wt% of trihydroxymethyl aminomethane, 0.22-0.32wt% of disodium ethylenediamine tetraacetic acid, 0.996-1.216wt% of sodium chloride and 0.08-0.12wt% of polyethylene glycol.

Optionally, the activated factor mixed solution further includes 18-25wt% of a protein protective agent, based on the weight of the activated factor mixed solution.

By adopting the technical scheme, the activation factor X is further protected by selecting the protein protective agent, so that the stability of the protein is improved, the stability of the detection kit in the specified expiration date is ensured, and the test accuracy is further ensured.

Optionally, based on the weight of the activated factor mixed solution, the protein protectant includes: 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, or single mannitol, or single sucrose, or single DMSO or single glycerol, and the like.

Optionally, the activated factor mixed solution is prepared by a method comprising the following steps: adding dextran sulfate, preservative for activating factors and buffer reagent into water to obtain initial mixed solution, wherein the pH value of the initial mixed solution is 8.3-8.5; and adding an activated factor X into the initial mixed solution, and uniformly mixing to obtain the activated 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 activating factor X is derived from bovine, porcine or human.

Alternatively, the activating factor X is bovine in origin.

In a second aspect, the present application provides a non-disease diagnosis detection method of the above 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 an activating factor mixed solution, uniformly mixing, and detecting the signal intensity of the chromogenic substrate.

By adopting the technical scheme, the application method selected by the application needs attention as follows: after mixing the sample diluent and the sample to be detected, adding a chromogenic substrate, incubating for a certain time, and adding an activating factor mixed solution; instead, the mixed solution of the activating factor is added for incubation for a certain time, and then the chromogenic substrate is added. The latter has higher requirement on the incubation time, needs to strictly control the incubation time, and is generally used for simultaneously testing multiple projects clinically, so that the incubation timeout is easy to occur, and the test result is deviated. The technical scheme of the application mainly considers the addition sequence of the chromogenic substrate and the activating factor X, and the extension of the incubation time has no obvious influence on the result; by adopting the technical scheme, the accuracy of the detection result can be further improved.

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

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

1. the method has the advantages that the reasonable activating factor mixed liquid, especially dextran sulfate added therein, is designed, and specific chromogenic substrates and reagent adding sequences are combined, so that the effects of improving the detection accuracy and improving the anti-interference capability of detection reagents are realized.

2. Particular protein protecting agents are selected for use in the present application: bovine serum albumin 0.8-1.2wt% and glycerol 18-23wt% to further improve the protection effect on the activated factor X and improve the detection accuracy.

Drawings

FIG. 1 is a standard curve of heparin and low molecular weight heparin according to the present application; wherein, FIG. 1 (A) is a standard curve of heparin, and FIG. 1 (B) is a standard curve of low molecular weight heparin.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples.

Examples

The activating factor X, also referred to as factor Xa or FXa; the detection principle of the detection kit of the application is as follows: the detection kit for the anti-Xa activity is used for detection by the chromogenic substrate method, which is based on the principle that the complex formed by AT III and heparin neutralizes activated factor Xa to determine the heparin activity. The method comprises the steps of firstly adding a chromogenic substrate into plasma and then adding excessive factor Xa into the plasma, wherein when the factor Xa is added into a plasma-substrate mixed solution, two reactions occur simultaneously, wherein the substrate plays a role in hydrolyzing paranitroaniline (pNA) under the action of Xa, the heparin-AT III compound inhibits the factor Xa, and after a competitive reaction reaches an equilibrium, the amount of pNA is inversely proportional to the heparin concentration 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 the anti-Xa activity of heparin and low-molecular-weight heparin contains a sample diluent, a chromogenic substrate solution and an activated factor mixed solution.

Wherein, the components contained in the sample diluent are as follows: sodium chloride and preservatives; the preparation method of the sample diluent comprises the following steps: dissolving sodium chloride in purified water, and adding a preservative to ensure that the mass fraction of the sodium chloride in the sample diluent is 0.9 percent and the mass fraction of the preservative is 0.08 percent.

The chromogenic substrate solution comprises the following components: a chromogenic substrate and a preservative; the preparation method of the chromogenic substrate solution comprises the following steps: the chromogenic substrate S-2732 powder (namely Suc-Ile-Glu (gamma-Pip) -Gly-Arg-pNA HCl) is dissolved by purified water and prepared into a chromogenic substrate initial solution with the final concentration of 0.85mg/mL, and then 0.08wt% of preservative is added (namely the mass fraction of the preservative in the chromogenic substrate solution is 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 Ethylene Diamine Tetraacetic Acid (EDTA), 1.023wt% of sodium chloride, 0.1wt% of PEG6000, 1wt% of bovine serum albumin, 20wt% of glycerol, 100 mu g/mL of dextran sulfate and 0.08wt% of preservative in purified water, and uniformly mixing by magnetic stirring to obtain an initial mixed solution; then hydrochloric acid is added to adjust the pH value to 8.40, and FXa 7.5nkat/mL is added to prepare an activated factor mixed solution.

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

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

1. Method for making standard curve when detection kit is used

The method comprises the following steps:

1. the preparation method of the detection kit for the anti-Xa activity of heparin and low-molecular-weight heparin comprises the following steps: the kit for detecting the anti-Xa activity of the heparin and the low molecular heparin comprises a sample diluent, a chromogenic substrate solution and an activated 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 content of sodium chloride was 0.9wt%, and 0.08wt% of a preservative was added.

The chromogenic substrate in the chromogenic substrate solution is S-2732, and the specific preparation method comprises the following steps: and dissolving the chromogenic substrate S-2732 powder with purified water, and preparing the chromogenic substrate solution with the chromogenic substrate S-2732 of which the final concentration is 0.85mg/mL and the preservative content is 0.08wt%.

The activated factor in the activated factor mixed solution is activated factor X (namely FXa), and the specific preparation method comprises the following steps: dissolving 0.606wt% of Tris (hydroxymethyl) aminomethane (Tris), 0.28wt% of Ethylene Diamine Tetraacetic Acid (EDTA), 1.023wt% of sodium chloride, 0.1wt% of PEG6000, 1wt% of bovine serum albumin, 100 mu g/mL of dextran sulfate, 0.08wt% of preservative and 20wt% of glycerol in purified water, uniformly mixing by magnetic stirring, adding hydrochloric acid to adjust the pH value to 8.40, and then adding FXa 7.5nkat/mL.

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

freeze-drying a heparin standard product: adding 0.05wt% of sodium benzoate and 1.5wt% of sucrose into the plasma sample, then respectively adding heparin international standard substances (07/328) with different concentrations to ensure that the theoretical final concentrations of the heparin international standard substances are 0IU/mL, 1IU/mL and 2IU/mL respectively, and subpackaging and freeze-drying by 1.1 mL/bottle after uniformly mixing.

Freeze-drying of low-molecular heparin standard: adding 0.05wt% of sodium benzoate and 1.5wt% of sucrose into the plasma sample, then respectively adding low molecular heparin international standard substances (11/176) with different concentrations to ensure that the theoretical final concentrations of the low molecular heparin are respectively 0IU/mL, 1IU/mL and 2IU/mL, and subpackaging and freeze-drying by 1.1 mL/bottle after uniformly mixing.

Redissolving the standard product: accurately adding purified water into a heparin standard substance with the volume of 1 mL/bottle or a low molecular heparin standard substance with the volume of 1 mL/bottle for redissolving, gently mixing uniformly, and standing for 10min to balance to room temperature for later use.

3. The test instrument: SF-8200 full-automatic blood coagulation tester.

4. Preparation of an anti-Xa activity test 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 the anti-Xa activity of the heparin and the low molecular weight heparin, firstly sampling 40 mu L of the diluent, then taking 10 mu L of a plasma sample and adding the plasma sample into a test cup together, then adding 100 mu L of chromogenic substrate liquid and uniformly mixing, and incubating for 60s at 37 ℃; then, after the sample needle is cleaned, 100 mu L of activating factor mixed liquor is added, and then the absorbance is measured at 405nm for 30-60s.

The low molecular heparin standard substances of 0IU/mL, 1IU/mL and 2IU/mL are calibrated by using the detection kit for the anti-Xa activity of the heparin and the low molecular heparin, 40 mu L of the diluent is sampled, 10 mu L of the plasma sample is added into a test cup together, 100 mu L of the chromogenic substrate liquid is added and mixed evenly, and the mixture is incubated for 60s at 37 ℃; then, after the sample needle is cleaned, 100 mu L of activating factor mixed liquor is added, and then the absorbance is measured at 405nm for 30-60s.

And (4) according to the heparin concentration and the corresponding test OD, taking the OD/min logarithm value as an abscissa and the heparin content as an ordinate, and making a standard curve. The standard curve of the low molecular heparin is formulated in the same way. Referring to fig. 1, the standard curve formula for heparin is: y = -0.3541 x-0.589 ² =1.0, the standard curve formula of low molecular heparin is: y = -7.291x-1.233 ² ＝1.0。

5. And (3) sample testing: the automatic analysis and test are carried out according to the steps in the table 1, and the content of the heparin or the low molecular heparin is obtained through conversion.

TABLE 1 reaction System

2. Compositional determination of kits of the present application

(1) Determination of chromogenic substrate in chromogenic substrate solution

Five different chromogenic substrates are selected and tested during the development of the kit, the information of each chromogenic substrate is shown in table 2, the purities of the four substrates are all more than or equal to 95%, wherein the Km (Michaelis constant) of S-2732 is the largest, and the affinity between the substrate and the substrate is the smallest.

In order to determine the optimal chromogenic substrate and the optimal reaction system, five chromogenic substrates are respectively tested on an SF-8200 full-automatic coagulator, the same activated factor Xa is selected, 4 different reaction systems are selected, and the determination results are shown in Table 3.

TABLE 2 summary of chromogenic substrate information

TABLE 3 summary of test results

Note:

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

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

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

reaction system 4: incubation time 240s, loading order: the chromogenic substrate is added first, followed by the activating factor X.

Selection of the reaction system: wherein, the reaction system 1 and the reaction system 2 adopt a sample adding sequence of firstly adding the activated factor X and then adding the chromogenic substrate, and the incubation time is respectively 60s and 240s; the result shows that the OD/min of the measurement is obviously reduced along with the prolonging of the incubation time, and the detection results at different times have obvious difference. This application of sample order is higher to the incubation time requirement, need strictly control the incubation time, and generally test simultaneously for multinomial item during clinical detection, consequently takes place to incubate the condition overtime easily to arouse that the test result has the deviation, the testing result is inaccurate. The reaction system 3 and the reaction system 4 adopt a sample adding sequence of firstly adding the chromogenic substrate and then adding the activated 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 reaction system 3 is selected as the kit.

Selection of chromogenic substrate: the result of the reaction system 3 shows that when the content of the heparin is different, each chromogenic substrate reacts with the activated factor X at 0IU/mL, wherein the reaction rate of the chromogenic substrate S-2732 is slower, and the reaction rates are different when the content of the heparin is different; the reaction rate of other 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 activated factor X, namely, the reagent has low suitability for the method; therefore, the kit of the application selects the chromogenic substrate S-2732.

(2) Determination of protein protective agent for activating factor X

The results of table 4 show that by adding different protein protectants to enhance the stability of FXa: when PEG6000, 2g/L mannitol, 2g/L sucrose, 1.5wt% DMSO, 1.5wt% glycerol or the like is used alone as a protein protectant, the protein protectant has no significant protective effect on FXa, and the measured heparin concentration gradually increases with time, and the absolute deviation and the relative deviation do not meet the requirements. While it is satisfactory that the present reagent employs, as the complex protein protecting agent, 1wt% BSA, 20wt% glycerol and 1wt% BSA +20wt% glycerol as the total content, a significant protecting effect against FXa, where 1wt% BSA +20wt% glycerol is more preferable, and the relative deviation at the measured heparin concentration of 0IU/mL does not exceed. + -. 0.1, (0,2 ] does not exceed. + -. 15%, so that the present reagent employs 1wt% BSA +20wt% glycerol as the FXa protecting agent.

TABLE 4 protective Effect of different protein protectants on activating factor X at different heparin concentrations

The method comprises the following steps: the absolute deviation of 0IU/mL is not more than plus or minus 0.1, (0,2 ] the relative deviation is not more than plus or minus 15%, and the absolute deviation in the table is the relative deviation of the absolute deviation under the corresponding heparin concentration.

(3) Determination of dextran sulfate

Platelet factor 4 (PF 4) is easy to combine and neutralize heparin, and combines with heparin to form PF 4/heparin binding protein, PF4 hinders the formation of heparin and AT complex, and then influences accuracy, and the addition of Dextran Sulfate (DS) can reduce the influence of heparin antagonists such as PF4, and improves accuracy. The present application sets different DS concentrations in the range of 0-200 mug/mL respectively, and the results show that DS concentrations of 0.01 mug/mL and 1 mug/mL are less than 0.05 in absolute deviation from the heparin concentration of 0IU/mL measured when no DS is added, (0,2 ] is less than 15% in relative deviation, which cannot reduce the influence of heparin antagonists such as platelet factor 4 (PF 4) because the DS addition amount is insufficient, and therefore the concentrations are unusable, whereas 100 mug/mL and 200 mug/mL are not less than 0.05 in absolute deviation from the heparin concentration of 0IU/mL measured when no DS is added, (0,2 ] is more than 15% in relative deviation, and the DS can play a role in reducing the influence of heparin antagonists such as platelet factor 4 (PF 4), and the difference between 100 mug/mL and 200 mug/mL is small, therefore the DS addition amount of 100 mug/mL is used in the present application.

TABLE 5 addition of different DS concentrations

The method comprises the following steps: the absolute deviation of 0IU/mL is not less than 0.05, (0,2 ] the relative deviation is more than 15%.

3. Evaluation of assay Performance of kits of the present application

(1) Linear range of

High concentration samples (i.e., 2 IU/mL) near the upper limit of the test interval were diluted to 6 concentrations at ratios of 4/5, 2/5, 1/10, and 0, respectively, with normal plasma, and each diluted concentration was tested 3 times to obtain the mean value of the measurement results. And (3) solving a linear regression equation by taking the dilution concentration as an independent variable and the mean value of the measurement result as a dependent variable, and calculating a correlation coefficient of linear regression: finally, the final calculation results show that the r =0.9993 of heparin and the r =0.9987 of low molecular heparin, 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 detection kit Linear Range for anti-Xa Activity of heparin and Low molecular heparin

(2) Margin limit

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

TABLE 7 detection kit blank limits for anti-Xa activity of heparin and low molecular weight heparins

(3) Repeatability of

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

TABLE 8 repeatability of the detection kit for anti-Xa activity of heparin and low molecular weight heparins

(4) Accuracy of

The reagents were tested with the heparin/low molecular heparin international standard diluted to a certain concentration according to the method of example, and the detection was repeated 3 times to calculate the relative deviation. And if the relative deviation of the results of 3 times does not exceed +/-15%, judging that the result is qualified. The results are shown in Table 9, and show that the kit has good repeatability, and CV does not exceed +/-15% when the kit is used for 3 times of repeated tests on heparin/low molecular heparin international standard (1 IU/mL).

TABLE 9 detection kit accuracy of anti-Xa activity of heparin and low molecular heparin

(5) Anti-interference capability

Bilirubin F, hemoglobin, bilirubin C and chyle are respectively prepared into interferent samples (heparin 0, 1 IU/mL) with different concentration ratios, and the interferent samples are tested by using the reagents according to the method of the embodiment. The results are shown in Table 10; the result shows that the kit has good specificity, 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 detection kit for anti-Xa activity of heparin and low molecular weight heparin

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

The performance of the detection kits for the anti-Xa activity of heparin and low molecular heparin of different manufacturers is compared, the detection kits for the anti-Xa activity of heparin and low molecular heparin of various manufacturers are used for sample preparation and then are tested in an SF-8200 automatic blood coagulation instrument, 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 the test kits for the anti-Xa activity of heparin and low molecular weight heparin from different manufacturers are shown in Table 12.

The reaction system of the detection kit for the anti-Xa activity of heparin and low molecular heparin of different manufacturers is as follows.

TABLE 11 reaction systems of detection kits for anti-Xa activity of heparin and low molecular weight heparin from different manufacturers

Reagent	Self-grinding reagent	Company B	C Corp Ltd	Company D
					Sample (μ L)	10	25	10	25
Sample Diluent (μ L)	40	75	/	75
					Chromogenic substrate solution (. Mu.L)	100	100	100	100(FXa)
Incubation time(s)	60	240	180	240
					Activating factor mixed liquor	100	100	75	100 (bottom)
Test time(s)	30～60	30～60	20～80	30～60
					Total volume (μ L)	250	300	185	300

Note: A. b, C reagents all used a sample addition sequence of adding substrate first and then FXa, and D reagent used a sample addition sequence of adding FXa first and then adding substrate.

Different manufacturers of heparin and low molecular weight heparin anti Xa activity of the test kit comparison.

TABLE 12 comparison of different kits

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

The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.

Claims

1. A detection kit for anti-Xa activity of heparin and low molecular heparin is characterized by comprising a sample diluent, a chromogenic substrate solution and an activated factor mixed solution; the activating factor mixed solution comprises the following components: 5-10nkat/mL of activating factor X, 50-200 mug/mL of dextran sulfate, 0.06-0.1wt% of preservative for activating factor and 1.8-2.4wt% of buffer reagent.

2. The detection kit according to claim 1, wherein the chromogenic substrate solution comprises the following components by weight based on the chromogenic substrate solution: 0.7-1.2mg/mL chromophoric substrate and 0.06-0.1wt% preservative for chromophoric substrate.

3. The detection kit according to claim 2, wherein the chromogenic substrate contained in the chromogenic substrate solution is Suc-Ile-Glu (γ -Pip) -Gly-Arg-pNA.

4. The detection kit according to claim 1, wherein the buffer reagent comprises, based on the weight of the activator mixture: 0.512-0.703wt% of trihydroxymethyl aminomethane, 0.22-0.32wt% of disodium ethylenediamine tetraacetic acid, 0.996-1.216wt% of sodium chloride and 0.08-0.12wt% of polyethylene glycol.

5. The detection kit according to claim 1, wherein the activated factor mixture further comprises 18 to 25wt% of a protein protecting agent, based on the weight of the activated factor mixture.

6. The detection kit according to claim 5, wherein the protein protective agent comprises, based on the weight of the mixture of activating factors: bovine serum albumin 0.8-1.2wt%, glycerin 18-23wt%.

7. The detection kit according to any one of claims 1 to 6, wherein the activator cocktail is prepared by a method comprising the steps of: adding dextran sulfate, preservative for activating factors and buffer reagent into water to obtain initial mixed solution, wherein the pH value of the initial mixed solution is 8.3-8.5; adding an activated factor X into the initial mixed solution, and uniformly mixing to obtain the activated factor mixed solution.

8. The detection kit according to claim 1, wherein the polyethylene glycol is at least one selected from the group consisting of polyethylene glycol 6000, polyethylene glycol 4000 and polyethylene glycol 8000.

9. The test kit according to claim 1, wherein the activated factor X is derived from bovine, porcine or human.

10. The non-disease diagnostic test method of the test kit of any one of claims 1 to 9, comprising the steps of: