CN117265064A

CN117265064A - Preparation method of inhibitor detection kit

Info

Publication number: CN117265064A
Application number: CN202311205484.9A
Authority: CN
Inventors: 杨建鑫; 李夏; 张娟丽; 李伟甲; 赵晓培; 杨会
Original assignee: Zhengzhou Puwan Medical Technology Co ltd
Current assignee: Zhengzhou Puwan Medical Technology Co ltd
Priority date: 2023-09-15
Filing date: 2023-09-15
Publication date: 2023-12-22

Abstract

The embodiment of the disclosure discloses a preparation method of an inhibitor detection kit. One embodiment of the method comprises the following steps: the kaolin and sodium chloride are subjected to constant volume and uniform mixing to obtain a first reagent stock solution; constant volume and uniform mixing are carried out on the calcium chloride and the sodium chloride, so as to obtain a second reagent stock solution; dissolving tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid to obtain a buffer solution; adding bovine serum albumin, trehalose and sodium chloride into a buffer solution to obtain a freeze-drying protection solution; adding coagulation factor serine protease into the freeze-drying protection liquid to obtain a stock solution; subpackaging and freeze-drying the stock solution to obtain a trypsin-like serine protease cup; and forming the inhibitor detection kit by the first reagent stock solution after split charging, the second reagent stock solution after split charging, the common cup and the trypsin-like serine protease cup. The embodiment improves the accuracy of the detection result, reduces the condition of excessive or insufficient dosage and shortens the detection time.

Description

Preparation method of inhibitor detection kit

Technical Field

The embodiment of the disclosure relates to the technical field of medical instruments, in particular to a preparation method of an inhibitor detection kit.

Background

The prepared inhibitor detection kit can be used for detecting the inhibitor drug residues in blood samples, and can be used for determining whether the concentration of the residual drug is within a normal range. Currently, in detecting inhibitor drug residues in blood samples, the following methods are generally adopted: inhibitor drug residues in blood samples are detected by conventional coagulation experiments, high performance liquid chromatography or antitrypsin-like serine protease activity assays.

However, the inventors found that when the inhibitor drug residue is detected in the above manner, there are often the following technical problems:

firstly, when the conventional blood coagulation experiment is used for detection, the detection is easily influenced by the type of the medicine, the type of the reagent and the operation mode of the experiment, so that the accuracy of the obtained detection result is lower, and the accuracy of the determined administration dosage is lower, so that the dosage is too much or too little; when the high performance liquid chromatography method is used for detection, the detection steps are more, and an additional detection machine is needed, so that the detection time is longer; when the antitrypsin-like serine protease activity is used for detection, the accuracy of the detection result is higher only when the proteins of the whole blood coagulation waterfall are complete, but the conditions of the whole blood coagulation waterfall are fewer when the proteins of the whole blood coagulation waterfall are complete, so that the accuracy of the detection result is lower, the detection stability is poor and the dosage is excessive or too little.

Second, when preparing the detection kit, only the concentration of the liquid in the detection kit is tested repeatedly, so that the comprehensiveness of the test on the detection kit is low, the detection accuracy of the prepared detection kit is low, and the prepared detection kit is poor in practicality.

Thirdly, detect the inhibitor medicine residue in the blood sample, need use the sampler to get one person from many people a cup and detect, the precision of sampler is inconsistent, causes the error of detecting great, and the operation step of sampling repeatedly is comparatively loaded down with trivial details, and the time consuming of detecting is longer.

The above information disclosed in this background section is only for enhancement of understanding of the background of the inventive concept and, therefore, may contain information that does not form the prior art that is already known to those of ordinary skill in the art in this country.

Disclosure of Invention

The disclosure is in part intended to introduce concepts in a simplified form that are further described below in the detailed description. The disclosure is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Some embodiments of the present disclosure propose methods of preparing inhibitor detection kits to address one or more of the technical problems mentioned in the background section above.

In a first aspect, some embodiments of the present disclosure provide a method of preparing an inhibitor detection kit, the method comprising: carrying out constant volume and uniform mixing treatment on kaolin and sodium chloride to obtain a first reagent stock solution; performing constant volume and uniform mixing treatment on the calcium chloride and the sodium chloride to obtain a second reagent stock solution; dissolving tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid to obtain a buffer solution; adding bovine serum albumin, trehalose and sodium chloride into the buffer solution to obtain a freeze-drying protection solution; adding coagulation factor serine protease into the freeze-drying protection liquid to obtain a stock solution; sub-packaging the first reagent stock solution and the second reagent stock solution; subpackaging and freeze-drying the stock solution to obtain a trypsin-like serine protease cup; and forming an inhibitor detection kit by the first reagent stock solution after split charging treatment, the second reagent stock solution after split charging treatment, the common cup and the trypsin-like serine protease cup.

The above embodiments of the present disclosure have the following advantageous effects: according to the preparation method of the inhibitor detection kit disclosed by the embodiment of the invention, the accuracy of detecting the inhibitor drug residues in the blood sample is improved, the condition of excessive or insufficient dosage is reduced, the detection time consumption is shortened, and the stability of detecting the inhibitor drug residues is improved. Specifically, the accuracy of the detection of the inhibitor drug residue is low, the stability of the detection of the inhibitor drug residue is poor, the dosage is too large or too small, and the time consumption of the detection of the inhibitor drug residue is long because: when the conventional blood coagulation experiment is used for detection, the type of the medicine, the type of the reagent and the experimental operation mode are easily affected, so that the accuracy of the obtained detection result is low, the accuracy of determining the taken medicine dosage is low, and the medicine dosage is excessive or too low; when the high performance liquid chromatography method is used for detection, the detection steps are more, and an additional detection machine is needed, so that the detection time is longer; when the antitrypsin-like serine protease activity is used for detection, the accuracy of the detection result is higher only when the proteins of the whole blood coagulation waterfall are complete, but the conditions of the whole blood coagulation waterfall are fewer when the proteins of the whole blood coagulation waterfall are complete, so that the accuracy of the detection result is lower, the detection stability is poor and the dosage is excessive or too little. According to the preparation method of the inhibitor detection kit, firstly, kaolin and sodium chloride are subjected to volume fixing and uniform mixing treatment to obtain a first reagent stock solution. Thus, a first reagent stock solution can be obtained, which can be used for preparing an inhibitor detection kit. Then, the calcium chloride and the sodium chloride are subjected to constant volume and uniform mixing treatment, and a second reagent stock solution is obtained. Thus, the method can be used for preparing an inhibitor detection kit. And then, carrying out dissolution treatment on the tris (hydroxymethyl) aminomethane and the ethylenediamine tetraacetic acid to obtain a buffer solution. Thus, can be used for preparing freeze-drying protective liquid. Then, bovine serum albumin, trehalose and sodium chloride were added to the above buffer to obtain a freeze-dried protective solution. Thus, it can be used for preparing stock solutions. Next, the coagulation factor serine protease was added to the above-mentioned lyoprotectant to obtain a stock solution. Thus, it can be used for preparing trypsin-like serine proteinase cups. Then, the first reagent stock solution and the second reagent stock solution are subjected to a split charging process. Thus, the method can be used for preparing an inhibitor detection kit. And secondly, sub-packaging and freeze-drying the stock solution to obtain the trypsin-like serine protease cup. Thus, the method can be used for further preparing an inhibitor detection kit. And finally, forming the inhibitor detection kit by the first reagent stock solution after the split charging treatment, the second reagent stock solution after the split charging treatment, the common cup and the trypsin-like serine protease cup. Thus, the prepared inhibitor detection kit can be obtained. Also, because the inhibitor detection kit only detects trypsin-like serine protease, and the conventional hemagglutination experiment is a universal clotting detection, the conventional hemagglutination experiment has different detection results for different drug types, so that the detection of the inhibitor detection kit is less influenced by the drug types, the accuracy of the detection result is improved, and the condition of excessive or insufficient dosage is reduced. And because the thrombus elastography instrument is used for detection, an additional detection machine is not required, and only the hemagglutination time is detected, so that the detection steps are reduced, the detection time consumption is shortened, and the detection resource consumption is reduced. In addition, the inhibitor detection kit of the scheme only needs to detect the protein in the first half section of the blood coagulation waterfall under the condition that the inhibitor drug residues are determined through the blood coagulation time, so that even if the protein integrity of the whole blood coagulation waterfall is lower, the influence on the accuracy of a detection result is smaller. Thereby improving the accuracy of the detection result and reducing the situation of excessive or insufficient dosage. Therefore, the accuracy of the detection result is improved, the time consumption of detection is shortened, and the situation of excessive or insufficient dosage is reduced.

Drawings

The above and other features, advantages, and aspects of embodiments of the present disclosure will become more apparent by reference to the following detailed description when taken in conjunction with the accompanying drawings. The same or similar reference numbers will be used throughout the drawings to refer to the same or like elements. It should be understood that the figures are schematic and that elements and components are not necessarily drawn to scale.

Fig. 1 is a flow chart of some embodiments of methods of preparing an inhibitor detection kit according to the present disclosure.

Detailed Description

Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the drawings and embodiments of the present disclosure are for illustration purposes only and are not intended to limit the scope of the present disclosure.

It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings. Embodiments of the present disclosure and features of embodiments may be combined with each other without conflict.

It should be noted that the terms "first," "second," and the like in this disclosure are merely used to distinguish between different devices, modules, or units and are not used to define an order or interdependence of functions performed by the devices, modules, or units.

It should be noted that references to "one", "a plurality" and "a plurality" in this disclosure are intended to be illustrative rather than limiting, and those of ordinary skill in the art will appreciate that "one or more" is intended to be understood as "one or more" unless the context clearly indicates otherwise.

The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

Fig. 1 illustrates a flow chart 100 of some embodiments of a method of preparing an inhibitor detection kit according to the present disclosure. The preparation method of the inhibitor detection kit comprises the following steps:

and 101, carrying out constant volume and uniform mixing treatment on kaolin and sodium chloride to obtain a first reagent stock solution.

In some embodiments, kaolin and sodium chloride may be sized and homogenized to provide a first reagent stock solution.

In some alternative implementations of some embodiments, the kaolin and sodium chloride may be sized and homogenized to provide a first reagent stock solution by:

In the first step, the kaolin and the sodium chloride are subjected to constant volume treatment by purified water. Wherein, the concentration of the kaolin after the constant volume treatment can be 3.0mg/mL, and the concentration of the sodium chloride after the constant volume treatment can be 0.9%. In practice, the kaolin and sodium chloride may be placed in the same empty volumetric flask, and purified water may be added to perform the constant volume treatment.

And secondly, uniformly mixing the kaolin subjected to constant volume treatment with sodium chloride to obtain a first reagent stock solution. In practice, the kaolin and the sodium chloride subjected to the constant volume treatment can be uniformly mixed upside down, so that the kaolin and the sodium chloride subjected to the constant volume treatment are uniformly mixed.

And 102, carrying out constant volume and uniform mixing treatment on the calcium chloride and the sodium chloride to obtain a second reagent stock solution.

In some embodiments, calcium chloride and sodium chloride described above may be sized and mixed to provide a second reagent stock solution.

In some alternative implementations of some embodiments, the calcium chloride and the sodium chloride described above may be sized and homogenized to provide a second reagent stock solution by:

and in the first step, the calcium chloride and the sodium chloride are subjected to constant volume treatment through purified water. Wherein the concentration of the calcium chloride after the constant volume treatment can be 0.2mol/L. The concentration of sodium chloride in the second reagent stock solution may be 0.9%. In practice, the calcium chloride and the sodium chloride can be placed in the same empty volumetric flask, and purified water is added to perform constant volume treatment.

And secondly, uniformly mixing the calcium chloride and the sodium chloride subjected to constant volume treatment to obtain a second reagent stock solution. In practice, the calcium chloride and the sodium chloride after the constant volume treatment can be mixed up and down reversely, so as to carry out uniform mixing treatment on the calcium chloride and the sodium chloride after the constant volume treatment.

Step 103, dissolving the tris and ethylenediamine tetraacetic acid to obtain a buffer solution.

In some embodiments, the buffer may be obtained by dissolving tris and ethylenediamine tetraacetic acid.

In some alternative implementations of some embodiments, the buffer may be obtained by dissolving tris and ethylenediamine tetraacetic acid by:

in the first step, purified water may be added to the tris (hydroxymethyl) aminomethane and the ethylenediamine tetraacetic acid to dissolve the tris (hydroxymethyl) aminomethane and the ethylenediamine tetraacetic acid so that the ph of the tris (hydroxymethyl) aminomethane and the ethylenediamine tetraacetic acid is in the range of 7.0 to 7.5. The concentration of the dissolved tris (hydroxymethyl) aminomethane may be 0.5%, and the concentration of the dissolved ethylenediamine tetraacetic acid may be 0.03%.

Alternatively, the buffer may be prepared from one or more of phosphoric acid, citric acid, acetic acid, ethylenediamine tetraacetic acid and tris.

And 104, adding bovine serum albumin, trehalose and sodium chloride into the buffer solution to obtain the freeze-drying protection solution.

In some embodiments, bovine serum albumin, trehalose, and sodium chloride may be added to the above buffer to provide a lyoprotectant. Wherein, the concentration of bovine serum albumin in the freeze-drying protection liquid can be 2.0%, the concentration of trehalose can be 1.5%, and the concentration of sodium chloride can be 0.6%.

Alternatively, the lyoprotectant may be prepared by adding one or more of sodium chloride, bovine serum albumin, arginine, glycine and trehalose to the buffer.

Step 105, the coagulation factor serine protease is added to the lyoprotectant to obtain a stock solution.

In some embodiments, a clotting factor serine protease can be added to the lyoprotectant described above to give a stock solution.

In some alternative implementations of some embodiments, the coagulation factor serine protease can be added to the lyoprotectant described above to give a stock solution by:

In the first step, the freeze-dried protective solution added with the coagulation factor serine protease is uniformly mixed. Wherein the concentration of the coagulation factor serine protease after the uniform mixing treatment can be 0.45u/L.

And secondly, carrying out constant volume treatment on the freeze-dried protection liquid after uniform mixing treatment to obtain stock solution. In practice, the freeze-dried protective liquid after the uniform mixing treatment can be added into purified water to carry out constant volume treatment.

And 106, sub-packaging the first reagent stock solution and the second reagent stock solution.

In some embodiments, the first reagent stock solution and the second reagent stock solution may be subjected to a dispensing process.

In some alternative implementations of some embodiments, the first reagent stock solution and the second reagent stock solution may be subjected to a split-charging process by:

and a first step of sub-packaging the first reagent stock solution according to a first preset sub-packaging volume. The first preset dispensing volume may be 20 μl of the first reagent stock solution dispensed in each empty reagent bottle. In practice, the first reagent stock solution may be dispensed in a volume of 20. Mu.L per reagent bottle.

And secondly, sub-packaging the second reagent stock solution according to a second preset sub-packaging volume. Wherein the second preset dispensing volume may be 1000 μl of the second preset dispensing volume dispensed in each empty reagent bottle. In practice, the second reagent stock solution may be dispensed in a volume of 1000. Mu.L per reagent bottle.

Step 107, sub-packaging and freeze-drying the stock solution to obtain the trypsin-like serine protease cup.

In some embodiments, the stock solution may be dispensed and lyophilized to provide a trypsin-like serine protease cup.

In some alternative implementations of some embodiments, the above stock solution may be dispensed and lyophilized to provide a trypsin-like serine protease cup by:

and a first step of sub-packaging the stock solution according to a third preset sub-packaging volume. Wherein the third predetermined dispensing volume may be 20 μl of the stock solution dispensed in each trypsin-like serine protease cup.

And secondly, freeze-drying the stock solution after the split charging treatment to obtain the trypsin-like serine protease cup.

And 108, forming an inhibitor detection kit by the first reagent stock solution after split charging, the second reagent stock solution after split charging, the common cup and the trypsin-like serine protease cup.

In some embodiments, the first reagent stock solution after the split-charging treatment, the second reagent stock solution after the split-charging treatment, the cuvette, and the trypsin-like serine protease cup described above may be combined into an inhibitor detection kit. Wherein, the ordinary cup can be an empty test cup.

The concentration of the blood coagulation factor serine protease is the active unit, the concentration of the calcium chloride is the mass concentration of the substance, the concentration of the other components is the mass volume concentration, and the concentration is converted into specific grams according to the volume of the prepared solution in practice. Adding purified water into the solution to enable the scale mark corresponding to the volume of the solution to reach a preset scale mark, wherein the preset scale mark can be the scale mark corresponding to the volume of the solution to be configured in practice. Ten human copies of the test reagent may be included in each inhibitor test kit.

In some alternative implementations of some embodiments, the following test may be performed during the preparation of the inhibitor detection kit by:

First, the concentration of the blood coagulation factor serine protease included in the inhibitor detection kit is tested to obtain the concentration of the corresponding blood coagulation factor serine protease.

And secondly, testing the components of the freeze-drying protection liquid contained in the inhibitor detection kit to obtain the freeze-drying protection liquid meeting the preset deviation condition.

Thirdly, performing validity test on the inhibitor detection kit to determine whether the inhibitor detection kit meets preset validity conditions.

Fourth, the inhibitor detection kit is subjected to in-batch precision test to determine whether the inhibitor detection kit meets the preset repeatability condition.

Fifthly, performing batch-to-batch precision test on the inhibitor detection kit to determine whether the inhibitor detection kit meets the preset range condition.

In some alternative implementations of some embodiments, the concentration of the coagulation factor serine protease included in the inhibitor detection kit described above can be tested to obtain the concentration of the corresponding coagulation factor serine protease by:

the concentration test steps of the blood coagulation factor serine protease included in the inhibitor detection kit are as follows:

And a first sub-step of placing the first blood sample into a common cup of the inhibitor detection kit to detect the blood coagulation time, thereby obtaining the blood coagulation time corresponding to the first blood sample.

And a second sub-step of placing the second blood sample into trypsin-like serine protease cups of the inhibitor detection kits to detect the blood clotting time, thereby obtaining the blood clotting time corresponding to the second blood sample. Wherein the concentration of the serine protease of the coagulation factor included in each of the inhibitor detection kits may vary. The concentration of each coagulation factor serine protease included in each inhibitor detection kit may correspond to each preset concentration in the set of preset concentrations. The concentration of the coagulation factor serine protease in the concentration of each coagulation factor serine protease may correspond to a preset concentration of the preset concentration set. The predetermined concentration set may be a set concentration value of the serine protease of the coagulation factor. For example, the set of preset concentrations may include 0.15u/L, 0.30u/L, 0.45u/L, 0.60u/L, 0.75u/L, and 0.90u/L.

And a third sub-step of determining an inhibitor detection kit corresponding to the blood coagulation time satisfying the preset blood coagulation time condition in each blood coagulation time as a target inhibitor detection kit. The preset blood coagulation time condition may be that a time difference between a blood coagulation time corresponding to the second blood sample and a blood coagulation time corresponding to the first blood sample is smaller than a preset difference. The target inhibitor detection kit may be a kit in which the detected blood coagulation time satisfies the preset blood coagulation time condition. For example, the preset difference may be 40.

A fourth sub-step of determining a target inhibitor detection kit having a minimum concentration of the coagulation factor serine protease from the respective target inhibitor detection kits in response to determining that the number of the target inhibitor detection kits is greater than a preset value. Wherein, the preset value may be 1. In practice, the target inhibitor detection kit with the smallest concentration corresponding to each target inhibitor detection kit can be determined.

And a fifth sub-step of determining the concentration of the coagulation factor serine protease corresponding to the target inhibitor detection kit, for which the determined concentration of the coagulation factor serine protease is the smallest, as the concentration of the corresponding coagulation factor serine protease. Thus, the concentration of serine protease corresponding to the coagulation factor can be obtained.

In some optional implementations of some embodiments, the components of the lyoprotectant included in the inhibitor detection kit may be tested to obtain the lyoprotectant that meets the preset deviation condition by:

the freeze-drying protective liquid contained in the inhibitor detection kit is tested by the following steps:

a first substep of determining a first set of predetermined concentration values corresponding to bovine serum albumin in the lyoprotectant. Wherein, the first preset concentration value set may include 1.0%, 1.5% and 2.0%. The first set of predetermined concentration values may characterize respective concentration values of predetermined bovine serum albumin. In practice, first, a predetermined concentration range corresponding to the above-mentioned bovine serum albumin may be determined. Wherein, the preset concentration range of the bovine serum albumin can be 0.5% -2.5%. Then, each concentration value is selected from the preset concentration range of the bovine serum albumin as a first preset concentration value set.

And a second substep of determining a second set of preset concentration values corresponding to trehalose in the lyoprotectant. Wherein the second set of preset concentration values may include 1.0%, 1.5%, and 2.0%. The second set of predetermined concentration values may be indicative of respective predetermined concentration values of trehalose. In practice, first, a predetermined concentration range corresponding to the above trehalose may be determined. Wherein, the preset concentration range of the trehalose can be 0.5% -2.5%. Then, each concentration value is selected from the preset concentration range of trehalose as a second preset concentration value set.

And a third sub-step of determining a third preset concentration value set corresponding to sodium chloride in the freeze-drying protection liquid. Wherein, the third preset concentration value set may include 0.3%, 0.6% and 0.9%. The third set of preset concentration values may characterize the respective concentration values of the preset sodium chloride. In practice, first, a preset concentration range corresponding to the above sodium chloride may be determined. Wherein, the preset concentration range of the sodium chloride can be 0.1% -1.0%. Then, each concentration value may be selected from the preset concentration ranges of sodium chloride as a third preset concentration value set.

And a fourth sub-step of combining each dose experiment group according to the first preset concentration value set, the second preset concentration value set and the third preset concentration value set. Wherein each of the above-described dose test groups may be a test group that characterizes each of the different dose components. Each of the dose experimental groups is composed of a preset number of first preset concentration values, second preset concentration values and third preset concentration values. For example, the preset number may be 1. In practice, first, each preset concentration value in the first preset concentration value set, the second preset concentration value set and the third preset concentration value set may be combined to obtain each combined dose experiment group. For example, each of the dose experimental groups described above may be each of the experimental groups in the following table:

wherein, EDTA can be ethylenediamine tetraacetic acid. Tris may be Tris.

And fifthly, sub-packaging and freeze-drying the freeze-drying protection liquid corresponding to each dose experiment group in the dose experiment groups according to preset doses to obtain the freeze-drying protection liquid of each trypsin-like serine protease cup. Wherein, the preset dose can be the dose divided by each cup. In practice, the freeze-drying protection liquid corresponding to each dose experiment group can be packaged and subjected to freeze-drying treatment according to preset doses. For example, the preset dose may be 20 μl/cup.

And a sixth sub-step, adding a calcium chloride solution into each trypsin-like serine protease cup lyoprotection solution in the trypsin-like serine protease cup lyoprotection solutions to obtain each target trypsin-like serine protease cup lyoprotection solution. Wherein, the concentration of the calcium chloride solution can be 0.2mol/L. The volume of the calcium chloride solution may be 20. Mu.l/cup.

And a seventh sub-step of detecting the blood coagulation time, the blood clot forming time, the blood coagulation rate and the blood clot strength of the freeze-dried protective liquid corresponding to each of the dose test groups before the freeze-drying treatment of the third blood sample according to the thromboelastography analyzer, and obtaining each average value corresponding to each dose test group. Wherein each of the above-mentioned average values includes a blood coagulation time average value, a blood clot formation time average value, a blood coagulation rate average value, and a blood clot intensity average value. The third blood sample may be a post-dose blood sample or a pre-dose blood sample. In practice, first, the executing body may detect the third blood sample by a preset number of detection times using the thromboelastography to obtain respective blood coagulation times, respective blood clot formation times, respective blood coagulation rates, and respective blood clot intensities corresponding to the third blood sample. Then, the average value corresponding to each of the blood coagulation time, each of the blood clot formation time, each of the blood coagulation rate, and each of the blood clot strength may be determined, respectively. Finally, the average value corresponding to each of the blood clotting time, each of the blood clot formation time, each of the blood clot rate and each of the blood clot intensity can be determined as each of the average values corresponding to each of the dose experimental groups. For example, the preset number of detections may be 3.

And an eighth substep, according to the thromboelastography apparatus, detecting the blood coagulation time, the blood clot forming time, the blood coagulation rate and the blood clot strength of each target trypsin-like serine protease cup freeze-drying protection liquid in the respective target trypsin-like serine protease cup freeze-drying protection liquids after the third blood sample is subjected to freeze-drying treatment, and obtaining respective target average values corresponding to each target trypsin-like serine protease cup freeze-drying protection liquid. Wherein each of the target averages may include a target clot time average, a target clot formation time average, a target clot rate average, and a target clot intensity average. The respective target average values may be average values corresponding to each target trypsin-like serine protease cup lyoprotectant. The target average value in each target average value of the above-mentioned target trypsin-like serine protease cup lyoprotectant corresponds to the average value in each average value of the corresponding dose experiment group. In practice, first, the executing body may detect each target trypsin-like serine protease cup lyoprotectant on which the third blood sample is placed by using the thromboelastography apparatus according to a preset detection frequency, so as to obtain a target blood coagulation time average value, a target blood clot forming time average value, a target blood coagulation rate average value and a target blood clot intensity average value of the third blood sample corresponding to each target trypsin-like serine protease cup lyoprotectant.

And a ninth substep of determining individual bias values for each dose experimental group based on the individual average values for each dose experimental group and the individual target average values for each target trypsin-like serine protease cup lyoprotectant. In practice, the absolute value of the difference of each target average value included for each target trypsin-like serine protease cup lyoprotectant from its corresponding average value may be determined. The ratio of the absolute value of the difference of the mean value corresponding thereto to the above-mentioned target mean value may then be determined as the deviation value of the dose experimental group. To obtain individual deviation values for each dose experimental group. For example, the respective bias values may be respective relative bias values in the following table:

wherein R may be the clotting time. K may be clot formation time. Angel may be the rate of blood clotting. MA may be clot strength.

And a tenth sub-step of selecting a dose experiment group satisfying the preset deviation condition from the dose experiment groups according to the deviation values. The preset deviation condition may be that a deviation value of the deviation values is the smallest and smaller than a preset deviation value. For example, the preset deviation value may be 10%.

In some alternative implementations of some embodiments, the inhibitor detection kit may be tested for effectiveness by:

the test procedure for the effectiveness of the inhibitor detection kit described above is as follows:

and a first sub-step of detecting the blood coagulation time of the blood sample without the coagulation factor serine protease inhibitor according to the common cup and the trypsin-like serine protease cup included in the inhibitor detection kit, so as to obtain a first detection result. The first detection result may be a detection result of a blood coagulation time of a blood sample corresponding to the blood sample without the coagulation factor serine protease inhibitor. In practice, the above-described thromboelastography may be used to detect the clotting time of blood samples without the factor serine protease inhibitor in each of the common cups and to detect the clotting time of blood samples without the factor serine protease inhibitor in each of the trypsin-like serine protease cups to obtain the first detection result. For example, the data for the detection of clotting time of a blood sample without a clotting factor serine protease inhibitor is shown in the following table:

Wherein the Xa cup can be a trypsin-like serine protease cup.

And a second sub-step of detecting the blood coagulation time of the blood sample containing the blood coagulation factor serine protease inhibitor according to the common cup and the trypsin-like serine protease cup included in the inhibitor detection kit, so as to obtain a second detection result. The second detection result may be a result of a blood coagulation time detection of the blood sample containing the inhibitor of the blood coagulation factor serine protease. In practice, the above-described thromboelastography may be used to detect the clotting time of the blood sample containing the above-described factor serine protease inhibitor in each of the common cups, and the clotting time of the blood sample containing the above-described factor serine protease inhibitor in each of the trypsin-like serine protease cups, to obtain the second detection result. For example, the data for the detection of clotting time of a blood sample without a clotting factor serine protease inhibitor is shown in the following table:

and a third sub-step of determining whether the inhibitor detection kit meets a first preset validity condition according to a first blood coagulation time set of each common cup and a second blood coagulation time set of each trypsin-like serine protease cup included in the first detection result. Wherein the first coagulation time in the first set of coagulation times corresponds to the second coagulation time in the second set of coagulation times. The first preset validity condition may be that each first blood coagulation time in the first blood coagulation time set is greater than or equal to a second blood coagulation time corresponding to the first blood coagulation time set. The first set of clotting times may be respective clotting times of a respective cuvette for detecting a blood sample comprising the factor serine protease inhibitor. The second set of clotting times may be respective clotting times of respective trypsin-like serine protease cups for detection of a blood sample containing the factor serine protease inhibitor.

And a fourth sub-step of determining whether the inhibitor detection kit meets a second preset validity condition according to a third blood coagulation time set of each common cup and a fourth blood coagulation time set of each trypsin-like serine protease cup included in the second detection result. Wherein the clotting time in the third clotting time set corresponds to a fourth clotting time in the fourth clotting time set. The second preset validity condition may be that a difference value between each third blood coagulation time in the third blood coagulation time set and a fourth blood coagulation time corresponding to the third blood coagulation time set is greater than or equal to a preset time difference value. For example, the preset time difference may be 60s. The third set of clotting times may be respective clotting times at which respective plain cups detect clotting times of a blood sample containing the factor serine protease inhibitor. The fourth set of clotting times may be respective clotting times at which respective trypsin-like serine protease cups detect clotting times of a blood sample containing the factor serine protease inhibitor.

And a fifth substep of determining that the inhibitor detection kit satisfies the preset validity condition in response to determining that the inhibitor detection kit satisfies the first preset validity condition and the second preset validity condition. The preset validity condition may be that the inhibitor detection kit satisfies the first preset validity condition and the second preset validity condition.

In some alternative implementations of some embodiments, the inhibitor detection kit may be tested for in-batch precision by:

the in-batch precision test procedure for the inhibitor detection kit described above was as follows:

and a first sub-step, detecting the blood coagulation time, the blood clot forming time, the blood coagulation rate and the blood clot strength of the quality control plasma without the blood coagulation factor serine protease inhibitor according to each target inhibitor detection kit and preset detection times, and obtaining a third detection result. Wherein, the batch numbers corresponding to the target inhibitor detection kits are consistent. For example, the preset number of detections may be 10. The third detection result may be a detection result of quality control plasma corresponding to the serpin-free serpin. In practice, first, the above-mentioned thromboelastography and the above-mentioned preset detection times may be used to detect the coagulation time, the clot formation time, the coagulation rate and the clot strength of the quality-controlled plasma without the coagulation factor serine protease inhibitor in each of the target inhibitor detection kits, thereby obtaining the coagulation time, the clot formation time, the coagulation rate and the clot strength corresponding to each of the target inhibitor detection kits. And combining the blood coagulation time, the blood clot forming time, the blood coagulation rate and the blood clot strength corresponding to each target inhibitor detection kit into a third detection result.

And a second sub-step of detecting the coagulation time, the clot formation time, the coagulation rate and the clot strength of the quality control plasma containing the coagulation factor serine protease inhibitor according to the target inhibitor detection kit and the preset detection times to obtain a fourth detection result. Wherein the final concentration of the coagulation factor serine protease inhibitor is 10 mug/ml. The fourth detection result may be a detection result of a quality control plasma corresponding to the blood coagulation factor serine protease inhibitor. In practice, first, the coagulation time, the clot formation time, the clot rate and the clot strength of the quality-controlled plasma containing the serpin in each of the target inhibitor detection kits may be detected using the thromboelastography machine and the preset number of times of detection, thereby obtaining the coagulation time, the clot formation time, the clot rate and the clot strength corresponding to each of the target inhibitor detection kits. And combining the respective clotting time, the respective clot formation time, the respective clotting rate and the respective clot strength in the respective target inhibitor detection kit as a fourth detection result. For example, the data for the detection of the quality control plasma without the factor serine protease inhibitor and the quality control plasma with the factor serine protease inhibitor according to each of the common cups included in each of the target inhibitor detection kits are shown in the following table:

Wherein R may be the clotting time. k may be the clot formation time. A may be the rate of blood clotting. MA may be clot strength.

For example, the data for the detection of the quality control plasma without the factor serine protease inhibitor and the quality control plasma with the factor serine protease inhibitor according to the respective trypsin-like serine protease cups included in the respective target inhibitor detection kits are shown in the following table:

And a third sub-step of determining whether each variation coefficient corresponding to each target inhibitor detection kit meets a preset variation coefficient condition according to the third detection result. The predetermined coefficient of variation condition may be that the coefficient of variation is smaller than a predetermined coefficient value. For example, the preset coefficient value may be 10%. In practice, first, the average value and standard deviation of the blood coagulation time, the blood clot formation time, the blood coagulation rate, and the blood clot strength corresponding to each of the respective target inhibitor detection kits may be determined. Then, the ratio of the average value and the standard deviation of the blood coagulation time, the blood clot formation time, the blood coagulation rate and the blood clot intensity may be determined as the coefficient of variation of the target inhibitor detection kit corresponding thereto. And finally, determining whether each variation coefficient is smaller than the preset coefficient value.

And a fourth sub-step of determining whether each target variation coefficient corresponding to each target inhibitor detection kit meets the preset variation coefficient condition according to the fourth detection result. In practice, first, the average value and standard deviation of the blood coagulation time, the blood clot formation time, the blood coagulation rate and the blood clot strength of the respective target inhibitor detection kits in the above fourth detection results may be determined. Then, the ratio of the average value and the standard deviation of the blood coagulation time, the blood clot formation time, the blood coagulation rate and the blood clot intensity may be determined as the coefficient of variation corresponding to its corresponding target inhibitor detection kit. And finally, determining whether each variation coefficient is smaller than the preset coefficient value.

And a fifth substep of determining that the inhibitor detection kit satisfies the preset repeatability condition in response to determining that the respective coefficient of variation and the respective target coefficient of variation satisfy the preset coefficient of variation condition. The predetermined repeatability condition may be that each of the coefficient of variation and each of the target coefficient of variation satisfy the predetermined coefficient of variation condition.

In some alternative implementations of some embodiments, the inhibitor detection kit may be tested for batch-to-batch precision by:

The batch-to-batch precision test procedure for the inhibitor detection kit described above was as follows:

and a first sub-step of detecting the clotting time, the clot formation time, the clotting rate and the clot strength of the quality control plasma without the clotting factor serine protease inhibitor and the quality control plasma containing the clotting factor serine protease inhibitor according to the common cups included in each inhibitor detection kit group to obtain a fifth detection result. Wherein, the lot numbers corresponding to the inhibitor detection kit groups can be different. The lot numbers corresponding to the respective inhibitor detection kits included in each of the above-described inhibitor detection kit groups may be the same. The number of inhibitor detection kits in each of the above-described inhibitor detection kit groups may be the same. The number of inhibitor detection kits in each of the above-described inhibitor detection kit groups may be all the target preset value. The concentration of the coagulation factor serine protease inhibitor may be 10. Mu.g/ml. The volume ratio of the quality control plasma containing the blood coagulation factor serine protease inhibitor to the blood coagulation factor serine protease inhibitor can be greater than or equal to 19:1. the final concentration of the factor serine protease inhibitor may vary within a range of + -10%. In practice, first, the quality control plasma without the coagulation factor serine protease inhibitor in the common cup and the quality control plasma containing the coagulation factor serine protease inhibitor in the common cup can be detected according to the thromboelastography to obtain detection results of the coagulation time, the clot formation time, the clot rate and the clot strength corresponding to each inhibitor detection kit group. Then, the average value of the blood coagulation time, the blood clot formation time, the blood coagulation rate and the blood clot strength corresponding to each inhibitor detection kit group can be obtained. Thereafter, the average value of the blood coagulation time, the blood clot formation time, the blood coagulation rate and the blood clot strength corresponding to each inhibitor detection kit group may be determined. Finally, the shortest and longest clotting times, the shortest and longest clot formation times, the smallest and largest clot rates, and the smallest and largest clot intensities in each inhibitor detection kit group can be determined. Differences corresponding to the shortest and longest clot times, the shortest and longest clot formation times, the minimum and maximum clot rates, and the minimum and maximum clot intensities may then be determined. Finally, the ratio of the difference between the shortest and longest blood coagulation times to the average value of the blood coagulation times, the ratio of the difference between the shortest and longest blood clot formation times to the average value of the blood clot formation times, the ratio of the minimum and maximum blood coagulation rates to the average value of the blood coagulation rates, and the ratio of the difference between the minimum and maximum blood clot intensities to the average value of the blood clot intensities are determined as the respective relative difference values for the respective inhibitor detection kit groups. And determining each relative difference value as a fifth detection result. For example, the data for the detection of the quality control plasma without the factor serine protease inhibitor and the quality control plasma containing the factor serine protease inhibitor according to the common cups included in each inhibitor detection kit set are shown in the following table:

And a second sub-step of detecting the clotting time, clot formation time, clotting rate and clot strength of the quality control plasma without the blood coagulation factor serine protease inhibitor and the quality control plasma containing the blood coagulation factor serine protease inhibitor according to trypsin-like serine protease cups included in each inhibitor detection kit group, thereby obtaining a sixth detection result. Note that, the manner of determining the sixth detection result is identical to the manner of determining the fifth detection result, and thus, a description thereof will not be repeated here. For example, the test data for the quality control plasma without the above-described factor serine protease inhibitors and the quality control plasma containing the above-described factor serine protease inhibitors according to the trypsin-like serine protease cups included in each inhibitor test kit set are shown in the following table:

And a third sub-step of determining whether each of the relative error values included in the fifth detection result and each of the relative error values included in the sixth detection result are smaller than a preset error value. For example, the preset tolerance value may be 15%.

And a fourth sub-step of determining that the inhibitor detection kit satisfies the preset limit condition in response to determining that each of the relative limit values included in the fifth detection result and each of the relative limit values included in the sixth detection result is smaller than the preset limit value. The preset margin condition may be that each of the margin values corresponding to the fifth detection result and the sixth detection result is smaller than the preset margin value.

The technical scheme is used as an invention point of the embodiment of the disclosure, and solves the second technical problem of the background art, namely when the detection kit is prepared, only the concentration of the liquid in the detection kit is tested repeatedly, so that the comprehensiveness of the test on the detection kit is lower, the detection accuracy of the prepared detection kit is lower, and the prepared detection kit is poor in practicability. The factors that lead to the lower accuracy of the detection kit and to the overdose or underdose are often as follows: only test is carried out on the detection reagent in the trypsin-like serine protease cup in the detection kit, so that the comprehensiveness of the test on the detection kit is low, and if the factors are solved, the effect of improving the detection accuracy of the detection kit can be achieved. To achieve this effect, the present disclosure provides for testing not only the concentration of the coagulation factor serine protease included in the inhibitor test kit and the composition of the lyoprotectant included in the inhibitor test kit, but also the effectiveness test, the in-batch precision test, and the in-batch precision test for the inhibitor test kit. Therefore, the comprehensiveness of the test on the detection kit is improved, the detection accuracy of the prepared detection kit is improved, and the condition of excessive or insufficient dosage is reduced.

Optionally, after step 108, the blood sample may also be tested according to the prepared inhibitor testing kit by:

first, according to a thromboelastography, the blood coagulation time of a first blood sample in a common cup is detected, and the blood coagulation time corresponding to the first blood sample is obtained. Wherein the first blood sample is a target blood sample which is not taken. The inhibitor detection kit comprises a first reagent stock solution, a second reagent stock solution, a common cup and a trypsin-like serine protease cup. The common cup may be an empty test cup. Stock solutions may be included in the trypsin-like serine protease cups described above. The stock solution may contain a lyoprotectant. The freeze-dried protective solution can comprise buffer solution and coagulation factor serine proteinase. The above-mentioned coagulation time may be a time required from the start of fibrin clot formation to the time when the thromboelastography amplitude reaches 2mm after the coagulation factor such as thrombin is sufficiently activated. In practice, the clotting time of the first blood sample in the common cup may be detected using a thromboelastography machine to obtain a clotting time corresponding to the first blood sample.

And secondly, detecting the blood coagulation time of the second blood sample in the trypsin-like serine protease cup according to the thromboelastography to obtain the target blood coagulation time corresponding to the second blood sample. The second blood sample may be a target blood sample after taking the drug. In practice, the thromboelastography apparatus may be used to detect the clotting time of a second blood sample in a trypsin-like serine protease cup to obtain a target clotting time corresponding to the second blood sample.

And thirdly, determining the detection result of the inhibitor drug residue corresponding to the target blood sample according to the difference value between the blood coagulation time and the target blood coagulation time. In practice, first, it may be determined whether the value of the above-described blood coagulation time minus the above-described target blood coagulation time is smaller than a preset blood coagulation time difference value. Then, in response to determining that the value of the blood clotting time minus the target blood clotting time is less than the preset blood clotting time difference, determining that no inhibitor drug remains or that the concentration of the remaining drug is in the normal range as the detection result corresponding to the target blood sample. And then, in response to determining that the value of the blood coagulation time minus the target blood coagulation time is greater than or equal to the preset blood coagulation time difference value, determining that more inhibitor medicine remains in the body as a detection result corresponding to the target blood sample. For example, the preset clotting time may be 1.0min.

The technical scheme is used as an invention point of the embodiment of the disclosure, solves the technical problem mentioned in the background art, namely, the detection of the inhibitor drug residue in the blood sample is realized by taking one person from a plurality of persons and one cup by using the sampler, the precision of the sampler is inconsistent, the detection error is larger, the repeated sampling operation steps are complicated, and the detection time is longer. The errors in detection are large, the dosage is too large or too small, and the factors with long detection time are often as follows: to detect inhibitor medicine residue, need use the sampler to get one person from many people a cup and detect, the precision of sampler is inconsistent, the operation procedure of repeated sampling is comparatively loaded down with trivial details if solved above-mentioned factor, just can reach the improvement and reduce the error that detects, reduce the condition of excessive or too little dosage, in order to reach this effect, this disclosure is when detecting inhibitor medicine residue, the detection mode of one person of a cup that adopts, the blood sample of one person is placed to each cup only, thereby reduced the detection error that is caused because of using the sampler to take a sample, and clinically need not sample by oneself, thereby improved the accuracy of detection, reduced the condition of excessive or too little dosage, shortened the time consuming of detecting.

The above embodiments of the present disclosure have the following advantageous effects: according to the preparation method of the inhibitor detection kit disclosed by the embodiment of the invention, the accuracy of detecting the inhibitor drug residues in the blood sample is improved, the condition of excessive or insufficient dosage is reduced, the detection time consumption is shortened, and the stability of detecting the inhibitor drug residues is improved. Specifically, the accuracy of the detection of the inhibitor drug residue is low, the stability of the detection of the inhibitor drug residue is poor, the dosage is too large or too small, and the time consumption of the detection of the inhibitor drug residue is long because: when the conventional blood coagulation experiment is used for detection, the type of the medicine, the type of the reagent and the experimental operation mode are easily affected, so that the accuracy of the obtained detection result is low, the accuracy of determining the taken medicine dosage is low, and the medicine dosage is excessive or too low; when the high performance liquid chromatography method is used for detection, the detection steps are more, and an additional detection machine is needed, so that the detection time is longer; when the antitrypsin-like serine protease activity is used for detection, the accuracy of the detection result is higher only when the proteins of the whole blood coagulation waterfall are complete, but the conditions of the whole blood coagulation waterfall are fewer when the proteins of the whole blood coagulation waterfall are complete, so that the accuracy of the detection result is lower, the detection stability is poor and the dosage is excessive or too little. According to the preparation method of the inhibitor detection kit, firstly, kaolin and sodium chloride are subjected to volume fixing and uniform mixing treatment to obtain a first reagent stock solution. Thus, a first reagent stock solution can be obtained, which can be used for preparing an inhibitor detection kit. Then, the calcium chloride and the sodium chloride are subjected to constant volume and uniform mixing treatment, and a second reagent stock solution is obtained. Thus, the method can be used for preparing an inhibitor detection kit. And then, carrying out dissolution treatment on the tris (hydroxymethyl) aminomethane and the ethylenediamine tetraacetic acid to obtain a buffer solution. Thus, can be used for preparing freeze-drying protective liquid. Then, bovine serum albumin, trehalose and sodium chloride were added to the above buffer to obtain a freeze-dried protective solution. Thus, it can be used for preparing stock solutions. Next, the coagulation factor serine protease was added to the above-mentioned lyoprotectant to obtain a stock solution. Thus, it can be used for preparing trypsin-like serine proteinase cups. Then, the first reagent stock solution and the second reagent stock solution are subjected to a split charging process. Thus, the method can be used for preparing an inhibitor detection kit. And secondly, sub-packaging and freeze-drying the stock solution to obtain the trypsin-like serine protease cup. Thus, the method can be used for further preparing an inhibitor detection kit. And finally, forming the inhibitor detection kit by the first reagent stock solution after the split charging treatment, the second reagent stock solution after the split charging treatment, the common cup and the trypsin-like serine protease cup. Thus, the prepared inhibitor detection kit can be obtained. Also, because the inhibitor detection kit only detects trypsin-like serine protease, and the conventional hemagglutination experiment is a universal clotting detection, the conventional hemagglutination experiment has different detection results for different drug types, so that the detection of the inhibitor detection kit is less influenced by the drug types, the accuracy of the detection result is improved, and the condition of excessive or insufficient dosage is reduced. And because the thromboelastography instrument is used for detection, an additional detection machine is not needed, and only the hemagglutination time is detected, so that the detection steps are reduced, the detection time consumption is shortened, and the detection resource consumption is reduced. In addition, the inhibitor detection kit of the scheme only needs to detect the protein in the first half section of the blood coagulation waterfall under the condition that the inhibitor drug residues are determined through the blood coagulation time, so that even if the protein integrity of the whole blood coagulation waterfall is lower, the influence on the accuracy of a detection result is smaller. Thereby improving the accuracy of the detection result and reducing the situation of excessive or insufficient dosage. Therefore, the accuracy of the detection result is improved, the time consumption of detection is shortened, and the situation of excessive or insufficient dosage is reduced.

The foregoing description is only of the preferred embodiments of the present disclosure and description of the principles of the technology being employed. It will be appreciated by those skilled in the art that the scope of the invention in the embodiments of the present disclosure is not limited to the specific combination of the above technical features, but encompasses other technical features formed by any combination of the above technical features or their equivalents without departing from the spirit of the invention. Such as the above-described features, are mutually substituted with (but not limited to) the features having similar functions disclosed in the embodiments of the present disclosure.

Claims

1. A method of preparing an inhibitor detection kit comprising:

carrying out constant volume and uniform mixing treatment on kaolin and sodium chloride to obtain a first reagent stock solution;

performing constant volume and uniform mixing treatment on the calcium chloride and the sodium chloride to obtain a second reagent stock solution;

dissolving tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid to obtain a buffer solution;

adding bovine serum albumin, trehalose and sodium chloride into the buffer solution to obtain a freeze-drying protection solution;

adding coagulation factor serine protease into the freeze-drying protection liquid to obtain a stock solution;

Subpackaging the first reagent stock solution and the second reagent stock solution;

subpackaging and freeze-drying the stock solution to obtain a trypsin-like serine protease cup;

and forming an inhibitor detection kit by the first reagent stock solution after split charging treatment, the second reagent stock solution after split charging treatment, the common cup and the trypsin-like serine protease cup.

2. The method of claim 1, wherein the sizing and blending of the kaolin clay and sodium chloride to obtain a first reagent stock solution comprises:

the kaolin and the sodium chloride are subjected to constant volume treatment through purified water, wherein the concentration of the kaolin after the constant volume treatment is 3.0mg/mL, and the concentration of the sodium chloride after the constant volume treatment is 0.9%;

and uniformly mixing the kaolin subjected to constant volume treatment and sodium chloride to obtain a first reagent stock solution.

3. The method of claim 1, wherein the sizing and mixing of the calcium chloride and the sodium chloride to obtain a second reagent stock solution comprises:

the calcium chloride and the sodium chloride are subjected to constant volume treatment through purified water, wherein the concentration of the calcium chloride after the constant volume treatment is 0.2mol/L, and the concentration of the sodium chloride after the constant volume treatment is 0.9%;

And uniformly mixing the calcium chloride and the sodium chloride subjected to the constant volume treatment to obtain a second reagent stock solution.

4. The method of claim 1, wherein the dissolving of the tris and ethylenediamine tetraacetic acid to obtain a buffer solution comprises:

purified water is added to the tris and ethylenediamine tetraacetic acid to carry out dissolution treatment on the tris and ethylenediamine tetraacetic acid so that the ph of the tris and ethylenediamine tetraacetic acid is in the range of 7.0 to 7.5, the concentration of tris after dissolution treatment is 0.5%, and the concentration of ethylenediamine tetraacetic acid after dissolution treatment is 0.03%.

5. The method of claim 1, wherein the concentration of bovine serum albumin in the lyoprotectant is 2.0%, the concentration of trehalose is 1.5%, and the concentration of sodium chloride is 0.6%.

6. The method of claim 1, wherein the adding a coagulation factor serine protease to the lyoprotectant to obtain a stock solution comprises:

uniformly mixing the freeze-dried protective solution added with the blood coagulation factor serine protease, wherein the concentration of the blood coagulation factor serine protease after uniformly mixing is 0.45u/L;

And (3) carrying out constant volume treatment on the freeze-dried protective liquid after uniform mixing treatment to obtain stock solution.

7. The method of claim 1, wherein the sub-packaging the first reagent stock solution and the second reagent stock solution comprises:

carrying out split charging treatment on the first reagent stock solution according to a first preset split charging volume;

and subpackaging the second reagent stock solution according to a second preset subpackaging volume.

8. The method of claim 1, wherein the dispensing and lyophilizing the stock solution to provide a trypsin-like serine protease cup comprises:

according to a third preset sub-packaging volume, sub-packaging treatment is carried out on the stock solution;

and carrying out freeze-drying treatment on the stock solution after the split charging treatment to obtain the trypsin-like serine protease cup.

9. The method of claim 1, wherein the inhibitor detection kit is prepared by the following test steps:

testing the concentration of the blood coagulation factor serine protease contained in the inhibitor detection kit to obtain the concentration of the corresponding blood coagulation factor serine protease;

testing the components of the freeze-drying protection liquid contained in the inhibitor detection kit to obtain the freeze-drying protection liquid meeting the preset deviation condition;

Performing a validity test on the inhibitor detection kit to determine whether the inhibitor detection kit meets a preset validity condition;

performing an in-batch precision test on the inhibitor detection kit to determine whether the inhibitor detection kit meets a preset repeatability condition;

and performing an inter-batch precision test on the inhibitor detection kit to determine whether the inhibitor detection kit meets a preset range condition.