阅读说明：本技术 一种抗凝血酶ⅲ活性测定试剂盒及其制备方法 (Antithrombin III activity determination kit and preparation method thereof ) 是由 邹红建 胡军 隋延锋 于 2020-12-28 设计创作，主要内容包括：本发明涉及临床检验领域,特别涉及一种抗凝血酶Ⅲ活性测定试剂盒及其制备方法。该试剂盒的制备原料包括稀释缓冲液、凝血酶试剂和发色底物试剂；稀释缓冲液为含有苯扎溴铵的生理盐水；凝血酶试剂包括凝血酶、肝素钠、海藻糖、氯化钠和缓冲液；发色底物试剂包括发色底物、乳糖、海藻糖和缓冲液。本发明中稀释缓冲液添加苯扎溴铵,能改变血浆中纤维蛋白原的结构或状态,避免纤维蛋白原干扰凝血酶裂解底物的反应速率,提高检测抗凝血酶III活性的准确性。(The invention relates to the field of clinical examination, and particularly relates to an antithrombin III activity determination kit and a preparation method thereof. The preparation raw materials of the kit comprise a dilution buffer solution, a thrombin reagent and a chromogenic substrate reagent; the dilution buffer is normal saline containing benzalkonium bromide; the thrombin reagent comprises thrombin, heparin sodium, trehalose, sodium chloride and buffer; the chromogenic substrate reagent comprises a chromogenic substrate, lactose, trehalose and a buffer. In the invention, the benzalkonium bromide is added into the dilution buffer solution, so that the structure or the state of fibrinogen in plasma can be changed, the fibrinogen is prevented from interfering the reaction rate of a thrombin cracking substrate, and the accuracy of detecting the activity of antithrombin III is improved.)

1. An antithrombin III activity determination kit is characterized in that preparation raw materials of the kit comprise a dilution buffer solution, a thrombin reagent and a chromogenic substrate reagent;

the dilution buffer solution is normal saline containing benzalkonium bromide;

the thrombin reagent comprises thrombin, heparin sodium, trehalose, sodium chloride and buffer;

the chromogenic substrate reagent comprises a chromogenic substrate, a preservative, lactose, trehalose and a buffer.

2. The kit according to claim 1, wherein the concentration of benzalkonium bromide in the dilution buffer is between 0.01 w/v% and 0.2 w/v%.

3. The kit of claim 1, wherein the thrombin reagent comprises the following components: 3-6U/mL of thrombin, 2-5U/mL of heparin sodium, 8-16 w/v% of trehalose, 0.9-3 w/v% of sodium chloride and the balance of buffer solution.

4. The kit of any one of claims 1 to 3, wherein the thrombin reagent further comprises lysine hydrochloride, protein stabilizer II and

5. the kit according to claim 4, wherein the thrombin reagent comprises the following components: 3-6U/mL of thrombin, 2-5U/mL of heparin sodium, 8-16 w/v% of trehalose, 0.9-3 w/v% of sodium chloride, 0.05-0.5 w/v% of lysine hydrochloride, 0.05-0.8 v/v% of protein stabilizer II,0.1-2 v/v% and the balance of buffer solution.

6. The kit of claim 1, wherein the thrombin is bovine thrombin.

7. The kit of claim 1, wherein the concentration of each component in the chromogenic substrate reagent is: 0.4-2 mmol/L of chromogenic substrate, 0.06-0.2 w/v% of preservative, 2-8 w/v% of lactose, 4-10 w/v% of trehalose and the balance of buffer solution.

8. The kit according to claim 7, characterized in that the chromogenic substrate is selected from the group consisting of H-D-Phe-Pip-Arg-pNA.2HCl, CH₃One or more of OCO-Gly-Pro-Arg-pNA. AcOH and H-D-CHG-Ala-Arg-pNA. AcOH.

9. The kit according to claim 1, wherein the buffer is 25-100 mmol/L, pH 7.4.4-8.4 Tris-HCl buffer.

10. The method for producing a kit according to any one of claims 1 to 9, wherein the dilution buffer, the thrombin reagent and the chromogenic substrate reagent are prepared separately, and the thrombin reagent and the chromogenic substrate reagent are lyophilized.

Technical Field

The invention relates to the field of clinical examination, and particularly relates to an antithrombin III activity determination kit and a preparation method thereof.

Background

The promotion and inhibition of blood coagulation in normal human body are in dynamic equilibrium. Antithrombin III (hereinafter referred to as AT-III), which is the most important anticoagulant substance in the human body, controls the processes of blood coagulation and fibrinolysis to maintain the coagulation balance of the body, and thus changes in the activity of AT-III are important indicators for diagnosing Disseminated Intravascular Coagulation (DIC), liver cirrhosis, sepsis, thrombotic diseases (myocardial infarction, venous thrombosis, etc.), congenital AT-III deficiency, hemophilia, and aplastic anemia.

AT-III is a globulin protein, synthesized in the liver. Studies have shown that low levels or activity of antithrombin III are both inherited and acquired. The congenital genetic deficiency of antithrombin III in blood is divided into type I and type II, and the type I antithrombin III deficiency is characterized by the reduction of antithrombin III synthesis and the reduction of antithrombin III content and activity in blood. The lack of antithrombin III form II is characterized by a normal synthesis of antithrombin III and a normal level of antithrombin III in the blood, but shows a reduced activity of antithrombin III due to a partial loss of function of antithrombin III. There are various causes of low content or activity of acquired antithrombin iii, such as decreased synthesis of antithrombin iii caused by liver disease; antithrombin iii loss by renal disease; loss of antithrombin iii by drugs such as heparin, and consumption of antithrombin iii by disseminated intravascular coagulation. Antithrombin iii deficiency, whether inherited or acquired, carries a risk of thrombosis to the patient, putting a difficult to predict threat to their health and life. The detection of antithrombin III activity is of great significance in the clinical diagnosis of congenital antithrombin III defects or abnormalities.

At present, two methods are used for detecting antithrombin III, one is to detect the content of antithrombin III antigen, such as an immunoassay method, the method is specific and sensitive, but the operation is complicated and time-consuming, and the deficiency of antithrombin III II cannot be detected; the other method is to detect the activity of antithrombin III, such as a coagulation method and a chromogenic substrate method, wherein the coagulation method is to determine the activity of AT-III in a sample through plasma coagulation time, and because the influenced factors are more, the specificity and the accuracy are poorer, and the operation is complicated. At present, in a kit sold in the market, a substrate chromogenic method is a general method for determining antithrombin III activity in blood plasma, and the method has the characteristics of high sensitivity, good accuracy, short detection time and the like, can be suitable for various automatic analytical instruments, and is widely applied clinically.

Principle of substrate chromophoric method: antithrombin III (AT-III) is a multifunctional serine protease inhibitor, an inhibitor of thrombin (FIIa) and activated proteases of factors VII, XI, X and the like. In the presence of an excess of serine protease, antithrombin iii in plasma forms with serine protease 1:1, the remaining protease which does not form a complex with antithrombin iii acts on a specific chromogenic substrate, the substrate is cleaved to produce a 4-nitroanilide (4-nitroanilide, pNA) chromogenic group, the concentration of 4-nitroanilide and the change in its absorbance at 405nm are positively correlated, and the amount of 4-nitroanilide produced is inversely proportional to the antithrombin iii activity. Thus, by measuring the change in absorbance at 405nm, the level of activity of antithrombin III in the plasma can be calculated.

US patent No. 5546007 and Chinese patent No. CN102690862A disclose a kit for detecting antithrombin III activity by thrombin-based chromogenic substrate method, wherein the two methods respectively optimize ion concentration and screen heparin derivatives to reduce the influence of heparin cofactor II (HC II) on the interference of antithrombin III activity determination, but thrombin and chromogenic substrate are unstable in aqueous solution and are not suitable for long-term storage. In addition, chinese patent CN106153612A discloses a kit for determining antithrombin iii activity by using an activated factor x (fxa) -based chromogenic substrate method, which avoids interference of heparin cofactor and anticoagulant factors such as hirudin, argatroban, dabigatran and the like in a sample. However, the activated factor X (FXa) is extremely easy to inactivate and expensive, and the manufacturing cost of the kit is increased.

The chromogenic methods of substrates, whether they are based on thrombin or on activated factor X (FXa), require the presence of an excess of thrombin or FXa, and, according to the blood coagulation cascade theory, other coagulation factors present in the plasma are more or less able to consume or inactivate the activity of thrombin and activated factor X, thus interfering with the detection of antithrombin III activity.

The current chromogenic substrate method for detecting antithrombin III activity still needs to be improved.

Disclosure of Invention

In view of this, the invention provides an antithrombin III activity determination kit and a preparation method thereof. According to the invention, by optimizing conditions and screening specific additives, the antithrombin III activity detection kit based on the interaction of thrombin and a chromogenic substrate is provided, and the kit has the advantages of good stability, high sensitivity, strong anti-interference performance, small batch difference, convenience in use and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an antithrombin III activity determination kit, wherein the preparation raw materials of the kit comprise a dilution buffer solution, a thrombin reagent and a chromogenic substrate reagent;

the dilution buffer is normal saline containing benzalkonium bromide;

the thrombin reagent comprises thrombin, heparin sodium, trehalose, sodium chloride and buffer;

the chromogenic substrate reagent comprises a chromogenic substrate, lactose, trehalose and a buffer.

The heparin cofactor II or antithrombin III in the plasma sample can capture the catalytic active site of thrombin, thereby inhibiting the thrombin from catalyzing the fibrinogen to be converted into fibrin and playing the role of anticoagulation. However, based on the principle of the interaction of thrombin with its chromogenic substrate, heparin inhibits thrombin activity by enhancing antithrombin III activity to form a 1:1 antithrombin III-thrombin complex, with the remaining thrombin acting on the substrate to dissociate the chromogenic group. In contrast to the substrate, a large amount of fibrinogen is present in the plasma sample, which likewise interacts with thrombin, thereby consuming the activity of thrombin, leading to higher results in the determination of antithrombin III activity.

In order to avoid the influence of fibrinogen in a plasma sample on the detection of antithrombin III activity, the dilution buffer solution disclosed by the invention can dilute the sample, reduce the interference of trace other coagulation factors (such as HC II) which can influence the thrombin activity in the sample, reduce the inhibition effect on thrombin, reduce the concentration of thrombin and a substrate in a kit and save the production cost; on the other hand, the dilution buffer contains a surfactant benzalkonium bromide, and can form a complex with fibrinogen to change the conformation of the fibrinogen, so that the catalytic active site of thrombin cannot be captured, and the influence of interference on the detection of the activity of antithrombin III is eliminated. And has no influence on the activity of the thrombin, thereby improving the accuracy of detecting the activity of the antithrombin III.

Preferably, the concentration of benzalkonium bromide in the dilution buffer is 0.01 w/v% to 0.2 w/v%.

Preferably, the concentration of each component in the thrombin reagent is: 3-6U/mL of thrombin, 2-5U/mL of heparin sodium, 8-16 w/v% of trehalose, 0.9-3 w/v% of sodium chloride and the balance of buffer solution.

Preferably, the thrombin reagent further comprises lysine hydrochloride, protein stabilizer II and

preferably, the concentration of each component in the thrombin reagent is: 3-6U/mL of thrombin, 2-5U/mL of heparin sodium, 8-16 w/v% of trehalose, 0.9-3 w/v% of sodium chloride, 0.05-0.5 w/v% of lysine hydrochloride, 0.05-0.8 v/v% of protein stabilizer II,0.1-2 v/v% and the balance of buffer solution.

Preferably, the thrombin is bovine thrombin.

Preferably, the concentration of each component in the chromogenic substrate reagent is: 0.4-2 mmol/L of chromogenic substrate, 0.06-0.2 w/v% of thimerosal sodium, 2-8 w/v% of lactose, 4-10 w/v% of trehalose and the balance of buffer solution.

Preferably, the preservative is thimerosal sodium.

Preferably, the chromogenic substrate is selected from the group consisting of H-D-Phe-Pip-Arg-pNA.2HCl (S2238), CH₃One or more of OCO-Gly-Pro-Arg-pNA. AcOH, H-D-CHG-Ala-Arg-pNA. AcOH (PA 2493).

Preferably, the buffer is Tris-HCl buffer of 25-100 mmol/L, pH 7.4.4-8.4.

The invention also provides a preparation method of the kit, which comprises the steps of respectively preparing a dilution buffer solution, a thrombin reagent and a chromogenic substrate reagent, and freeze-drying the thrombin reagent and the chromogenic substrate reagent.

The invention provides an antithrombin III activity determination kit and a preparation method thereof. The preparation raw materials of the kit comprise a dilution buffer solution, a thrombin reagent and a chromogenic substrate reagent; the dilution buffer is normal saline containing benzalkonium bromide; the thrombin reagent comprises thrombin, heparin sodium, trehalose, sodium chloride and buffer; the chromogenic substrate reagent comprises a chromogenic substrate, lactose, trehalose and a buffer. The invention has the following advantages:

in the invention, the benzalkonium bromide is added into the dilution buffer solution, so that the structure or state of fibrinogen in plasma can be changed, the fibrinogen is prevented from interfering the reaction rate of a thrombin cracking substrate, and the accuracy of detecting the activity of antithrombin III is improved;

in the invention, additives of lysine hydrochloride, protein stabilizer II andnot only can improve the stability of the thrombin aqueous solution, but also can enhance the precision for detecting the activity of the antithrombin III;

aiming at the problems that the thrombin and the chromogenic substrate are generally unstable in an aqueous solution and cannot be stored for a long time, the stability of the thrombin and the chromogenic substrate in the aqueous solution can be effectively improved by adopting a specific additive, and the thrombin and the chromogenic substrate exist in a dry powder type appearance form, so that the stability of the thrombin and the chromogenic substrate can be better enhanced;

experiments show that the AT-III activity determination kit has excellent performance, is stable for 18 months AT the temperature of 2-8 ℃, has relative deviation of-4.8% -5.2%, and has precision CV value: 6.3 percent; the consistency of the difference between bottles is good, and the CV value is as follows: 3.6 percent; linear range [ 3.6%, 130%]Correlation of R with²>0.99; the kit can be stably maintained for 24h at 37 ℃ after redissolution, can be stably stored for 55 days at 25 ℃ and can be stably stored for at least 90 days at 2-8 ℃; the stability is high.

Drawings

FIG. 1-1 Effect of different concentrations of surfactant on fibrinogen;

FIGS. 1-2 calibration curves for example 1 and example 2;

FIG. 2 is a standard curve of antithrombin III activity;

FIG. 3 correlation of antithrombin III activity assay kit;

FIG. 4 linearity of antithrombin III activity detection kit;

FIG. 5 is a model difference diagram of an antithrombin III activity detection kit.

Detailed Description

The invention discloses an antithrombin III activity determination kit and a preparation method thereof, and a person skilled in the art can realize the determination by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The antithrombin III activity determination kit and the preparation method thereof provided by the invention can be purchased from the market. Wherein, the protein stabilizer II is purchased from England Biotech limited, Huzhou.

The invention is further illustrated by the following examples:

example 1

Preparation of dilution buffer: 0.075g of benzalkonium bromide was weighed out and dissolved in 100mL of physiological saline.

Preparation of thrombin reagent: the components of the medicine are 4.5U/mL bovine thrombin, 3.5U/mL heparin sodium, 12% (w/v) trehalose, 0.25% (w/v) lysine hydrochloride, 2% (w/v) sodium chloride, 0.4% (v/v) protein stabilizer II and 1% (v/v)60mmol/L Tris-hydrochloric acid buffer solution (pH8.0).

Preparation of substrate reagent: preparing 75mmol/L Tris-HCl buffer solution containing 0.1% (w/v) thimerosal, 5% (w/v) lactose and 7% (w/v) trehalose, and having a pH value of 8.0; finally, the thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 1.2 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 2

Preparation of dilution buffer: 100mL of physiological saline.

Preparation of thrombin reagent: the components of the medicine are 4.5U/mL bovine thrombin, 3.5U/mL heparin sodium, 12% (w/v) trehalose, 0.25% (w/v) lysine hydrochloride, 2% (w/v) sodium chloride, 0.4% (v/v) protein stabilizer II and 1% (v/v)60mmol/L Tris-hydrochloric acid buffer solution (pH8.0).

Preparation of substrate reagent: preparing 75mmol/L Tris-HCl buffer solution containing 0.1% (w/v) thimerosal, 5% (w/v) lactose and 7% (w/v) trehalose, and having a pH value of 8.0; finally, the thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 1.2 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 3

Preparation of dilution buffer: 0.075g of benzalkonium bromide was weighed out and dissolved in 100mL of physiological saline.

Preparation of thrombin reagent: the components of the medicine are 4.5U/mL bovine thrombin, 3.5U/mL heparin sodium, 12% (w/v) trehalose, 0.25% (w/v) sodium chloride, 60mmol/L trihydroxymethyl aminomethane-hydrochloric acid buffer solution with the pH value of 8.0.

Preparation of substrate reagent: preparing 75mmol/L Tris-HCl buffer solution containing 0.1% (w/v) thimerosal, 5% (w/v) lactose and 7% (w/v) trehalose, and having a pH value of 8.0; finally, the thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 1.2 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 4

Preparation of dilution buffer: 0.025g of benzalkonium bromide was weighed out and dissolved in 100mL of physiological saline.

Preparation of thrombin reagent: the components of the medicine are 3U/mL bovine thrombin, 2U/mL heparin sodium, 8% (w/v) trehalose, 0.05% (w/v) lysine hydrochloride, 0.9% (w/v) sodium chloride, 0.05% (v/v) protein stabilizer II and 0.1% (v/v)25mmol/L Tris-hydrochloric acid buffer solution (pH7.4).

Preparation of substrate reagent: preparing 50mmol/L Tris-HCl buffer solution containing 0.06% (w/v) thimerosal, 2% (w/v) lactose and 4% (w/v) trehalose, and having a pH value of 7.4; finally, the thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 0.4 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 5

Preparation of dilution buffer: 0.05g of benzalkonium bromide was weighed out and dissolved in 100mL of physiological saline.

Preparation of thrombin reagent: the components of the medicine are 6U/mL bovine thrombin, 5U/mL heparin sodium, 16% (w/v) trehalose, 0.5% (w/v) lysine hydrochloride, 3% (w/v) sodium chloride, 0.8% (v/v) protein stabilizer II and 2% (v/v)100mmol/L Tris-hydrochloric acid buffer solution (pH8.4).

Preparation of substrate reagent: preparing 100mmol/L Tris-HCl buffer solution containing 0.2% (w/v) thimerosal, 8% (w/v) lactose and 10% (w/v) trehalose, and having a pH value of 8.4; finally, thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 2 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 6

Preparation of dilution buffer: 0.1g of benzalkonium bromide was weighed out and dissolved in 100mL of physiological saline.

Preparation of thrombin reagent: the ratio of the components is 5.0U/mL bovine thrombin, 2.7U/mL heparin sodium, 10.3% (w/v) trehalose, 0.17% (w/v) lysine hydrochloride, 2.1% (w/v) sodium chloride, 0.30% (v/v) protein stabilizer II, 0.7% (v/v)39mmol/L Tris-hydrochloric acid buffer solution (pH8.1).

Preparation of substrate reagent: preparing 68mmol/L Tris-HCl buffer solution containing 0.12% (w/v) thimerosal, 5.5% (w/v) lactose and 6% (w/v) trehalose, and having a pH value of 7.8; finally, the thrombin substrate PA2493 was added and mixed well with stirring to a concentration of 1.5 mmol/L.

After the thrombin reagent and the substrate reagent are prepared, the thrombin reagent and the substrate reagent are subpackaged and freeze-dried to prepare freeze-dried powder.

Example 7 procedures for antithrombin III Activity determination

The antithrombin III activity detection kit is redissolved, a TECO1800 full-automatic blood coagulation analyzer is adopted, and parameter setting is carried out according to an instruction provided by a manufacturer: mixing a sample (a calibrator, a quality control product or plasma) and a dilution buffer solution reagent according to a ratio of 1:40, taking out a mixed solution with a certain volume, adding a thrombin reagent with the same volume, mixing and incubating for 2min, adding a thrombin substrate reagent, mixing and incubating for 25s, and reading the signal intensity (absorbance OD (optical Density) value) of a chromogenic substrate in a sample to be detected under the irradiation of a wavelength of 405 nm; and finally, calculating the signal intensity and comparing the signal intensity with a standard curve of a standard substance with known concentration to obtain the activity of antithrombin III in the blood.

Test example 1 evaluation of the Performance of the antithrombin III Activity detection kit of the present invention

1. Effect of the interferent fibrinogen

A series of fibrinogen-containing concentrations (266mg/mL, 26% and 13%) were also obtained by diluting a calibration stock, a general-purpose product of TECO, Germany, with a known AT-III activity of 104%, in equal proportions with physiological saline and containing 266mg/mL fibrinogen,133 mg/mL, 66.5mg/mL and 33.25mg/mL) samples were prepared, a series of concentrations of surfactant were prepared, the kit of example 1, example 2, example 4, example 5 and example 6 was used to measure the absorbance OD values corresponding to the activity of AT-iii of the above samples, each sample was repeatedly tested 3 times to obtain an average value, a calibration curve was drawn for each example, the results are shown in fig. 1-1, and the calibration curve R was plotted as the concentration of surfactant increased²More towards 1.

The values measured in example 1 and example 2 were selected to plot the results of the calibration curves of the examples as shown in FIGS. 1-2, and based on the calibration curves of this example, the reagents of example 1 and example 2 were used to measure fixed-value plasma, and each sample was tested in duplicate for 3 averaging, the results of which are shown in Table 1.

Table 1 example 1 and example 2 assay of fixed value plasma

Example 1 Linear R of calibration curves, illustrated by FIGS. 1-1 and 1-2²The concentration of fibrinogen in the sample is reduced along with the increase of the dilution factor, the absorbance of the sample is consistent with that of the sample in example 1 and example 2, namely the dilution factor of the sample can be optimized and the fibrinogen surfactant is added into the dilution, the fibrinogen influence on the reaction rate of thrombin cleavage substrates can be eliminated, and the accuracy of the kit for detecting the activity of the plasma sample AT-III is improved, as can be seen from Table 1, the relative deviation of the results of detecting normal and abnormal plasma AT-III activities in example 1 is 0.4% and-2.54%, respectively, and is obviously better than that of example 2. Therefore, the surfactant can ensure that the AT-III activity of the kit for detecting the reagent kit is not interfered by fibrinogen.

2. Effect of stabilized Thrombin compositions on Thrombin stability

Dividing the kits of example 1 and example 3 into three groups, respectively, redissolving, placing at 2-8 ℃, 25 ℃ and 37 ℃ for a certain time interval, detecting normal quality control plasma and abnormal quality control plasma by the same or a group of operators on the same instrument according to the operation method of antithrombin III activity determination, recording the change of the absorbance OD value of the detection result, and judging whether the thrombin activity of the kit is reduced or not, as shown in Table 2:

TABLE 2-1 stability of antithrombin III Activity assay kit at 2 deg.C-8 deg.C

TABLE 2-2 stability of antithrombin III Activity assay kit at 25 deg.C

TABLE 2-3 stability of antithrombin III Activity assay kit at 37 deg.C

The results in Table 2 show that the thrombin activity after reconstitution of the reagents of example 1 is not significantly lost for at least 90 days at 2 ℃ to 8 ℃ or more, respectively; can be stably stored for 55 days at 25 ℃; the stability of thrombin, which is remarkably superior to that of the kit of example 3, is maintained stably for 20 hours at 37 ℃, indicating that the composition provided by the present invention (lysine hydrochloride, protein stabilizer II and) Can maintain the stability of thrombin for a long time.

Test example 2 evaluation of analytical Properties of the kit of the present invention

1. Example 1 plotting of calibration Standard Curve

The calibrator was diluted with physiological saline at a range of concentrations (104%, 52%, 26%, 13%) and the OD values of the calibrator solutions were measured on a TECO1800 coagulation analyzer using the antithrombin III activity assay kit described in example 1, and standard curves were plotted with the OD values as ordinate and the corresponding antithrombin III activity values as abscissa, as shown in fig. 2.

As shown in figure 2, the kit of the invention is based on a thrombin chromogenic substrate method, and the detected absorbance OD value and the corresponding antithrombin III activity show a good linear relation, wherein the linear relation is defined as a function that Y is-0.0029X +0.0375, and R is²＝0.9985。

2. Example 1 measurement of precision

According to the operation method of antithrombin III activity determination, the same or a group of operators perform on the same instrument, the normal quality control plasma and the abnormal quality control plasma are repeatedly determined for 10 times, and the mean, standard deviation and coefficient of variation CV of the determination values are calculated as follows:

TABLE 3 precision of antithrombin III Activity test kit

The coefficient of variation, CV, is generally used to measure the precision of an assay, with smaller CV values indicating better precision of the assay results. The results in Table 3 show that the CV value of the kit is 3.25 percent when the kit is used for detecting the activity of the normal quality control plasma antithrombin III; the CV is 5.91 percent when the activity of the plasma antithrombin III is abnormally controlled, which shows that the kit has excellent precision.

3. Example 1 detection of inter-batch Difference

Taking out the three batches of the kit, redissolving, carrying out detection on the activities of the normal quality control plasma and the abnormal quality control plasma AT-III for 10 times by the same or a group of operators on the same instrument according to the operation method for measuring the activity of the antithrombin III, and calculating the average number, the standard deviation and the coefficient of variation CV of the measured value as follows:

TABLE 4-1 detection of Interlot differences in Normal quality control plasma by antithrombin III Activity

TABLE 4-2 batch-to-batch differences in the detection of abnormal quality-controlled plasma by the antithrombin III Activity detection kit

The coefficient of variation, CV, is generally used to measure the precision of an assay, with smaller CV values indicating better precision of the assay results. The results in table 4 show that the precision (CV%) values of the normal value quality control plasma and the abnormal value quality control plasma detected by the kit of the invention are 4.69% and 6.38%, respectively, which indicates that the kit of the invention has excellent batch-to-batch difference.

4. Example 1 in-batch Difference detection

The 10 boxes of the kit of the invention in the same batch are re-dissolved, and the determination is carried out on the normal quality control plasma and the abnormal quality control plasma respectively by the same or a group of operators on the same instrument according to the operation method of the antithrombin III activity determination, namely: optionally, one of the cartridges was tested on the quality control plasma 10 times, and each cartridge was tested on the quality control plasma 1 time, and the mean, standard deviation and coefficient of variation CV of the test values were calculated as follows:

TABLE 5-1 kit for detecting antithrombin III Activity for the Intra-batch Difference in quality control of Normal plasma

TABLE 5-2 kit for detecting antithrombin III Activity for the detection of abnormal values in the quality control of plasma batch differences

The results in table 5 show that the precision (CV%) values of the normal value quality control plasma and the abnormal value quality control plasma detected by the kit of the invention are 2.96% and 7.81%, respectively, which indicates that the kit of the invention has excellent batch difference.

5. Example 1 detection of correlation with commercially available imported kits

The kit of the present invention and the commercially available kit were used for the detection of AT-III activity in 170 fresh plasma samples (including normal and abnormal samples) on a TECO1800 full-automatic biochemical analyzer, respectively, and correlation analysis was performed on the measurement values (see the results in the figure, X-axis represents the measurement value of the commercially available kit A, and Y-axis represents the measurement value of the kit of the present invention).

FIG. 3 shows that the linear equation relating the kit of the present invention and the commercially available kit is: y is 0.949x + 5.5011, correlation coefficient: r²Although the overall correlation coefficient is poor due to slightly large deviation of individual normal value samples and less abnormal value plasma samples, the detection result basically conforms to the negative and positive results, and the result shows that the kit of the invention has good correlation with the commercial kit A.

6. Example 1 detection of the Linear Range

AT-III samples with 144% activity were diluted as follows: 129.6%, 115.2%, 100.8%, 86.4%, 72%, 57.6%, 43.2%, 28.8%, 14.4%, 7.2% and 3.6%, and the test was repeated 4 times for each diluted sample, and the measurement results were averaged. The dilution is used as independent variable, and the mean value of the measured results is used as dependent variable to calculate linear regression equation and related coefficient R, as shown in figure 4.

As shown in fig. 4, the linear regression equation y is 136.5x +3.3683, and R is 0.992, so the linear range of the reagent of the present invention: 3.6 to 140 percent.

7. Example 1 detection of anti-interference of the kit

4 portions of mixed plasma were taken, one portion was used as control, and the other three portions were added with 3 potential interferents: bilirubin (Db), hemoglobin (Hb), and triglyceride (chyle) were prepared in two or three concentration gradients for each interferent, and plasma sample AT-iii activity was measured in 4 replicates using the kit of example 1, and averaged, as shown in table 6:

TABLE 6 anti-interference of antithrombin III Activity detection kit

As shown in Table 6, in each plasma sample containing 30mg/dL bilirubin, 200mg/dL hemoglobin and 5000 mg/dL chyle, the relative deviation of the activity of the kit for detecting the plasma sample AT-III in comparison with that of a blank plasma sample is 1.81 percent, 0.55 percent and 3.15 percent respectively, namely, no inhibition effect exists, and the kit has good specificity.

8. Example 1 detection of model Difference in kits

The kit of example 1 was used to perform the calibration on TOP 700 and TECO1800, respectively, and 22 plasma samples were tested according to the calibration curve on the respective instruments, and the results of the testing are shown in table 7 for the range of AT-iii activity [ 66%, 128% ]. In order to analyze the model difference of the kit for detecting the activity of the plasma sample AT-III on the two instruments, a model difference curve chart 5 is drawn by taking the result of AT-III activity detection of a TOP 700 instrument as the abscissa and the result of TECO1800 instrument as the ordinate.

TABLE 7 model differences of antithrombin III Activity detection kits

Table 7 shows that the relative deviation of the activity of the kit of the invention in the respective TOP 700 and TECO1800 instruments for AT-III activity in plasma samples is substantially within + -5%, and that FIG. 5 reflects the correlation R of the activity of the kit of the invention in the respective TOP 700 and TECO1800 instruments for AT-III activity in plasma samples²Since 0.9765 represents the difference in model, the kit of the present invention is small.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.