CN117949661A

CN117949661A - Protein S activity determination kit

Info

Publication number: CN117949661A
Application number: CN202211325501.8A
Authority: CN
Inventors: 陈志宇
Original assignee: Shenzhen Dymind Biotechnology Co Ltd
Current assignee: Shenzhen Dymind Biotechnology Co Ltd
Priority date: 2022-10-27
Filing date: 2022-10-27
Publication date: 2024-04-30

Abstract

The invention discloses a protein S activity determination kit, which comprises an R1 reagent, an R2 reagent and an R3 reagent; the R1 reagent is freeze-dried powder, the components of the freeze-dried powder comprise human anticoagulated plasma of protein S, a first buffer solution and a first auxiliary material, and the first auxiliary material comprises sugar and inert protein; the R2 reagent is in a liquid state, the components of the reagent comprise activated protein C, a second buffer solution, calcium chloride and a second auxiliary material, and the second auxiliary material comprises a protein protecting agent; the R3 reagent is in a liquid state, the components of the reagent comprise protein C activator, phospholipid, a third buffer solution and a third auxiliary material, and the third auxiliary material comprises a protein protective agent; the kit provided by the invention has the advantages of simple preparation process, lower cost, convenience in operation and the like, and the R2 reagent and the R3 reagent are both in liquid state; in addition, the invention has good stability, high accuracy and good sensitivity, and the anti-interference capability is greatly improved.

Description

Protein S activity determination kit

Technical Field

The invention relates to the technical field of biology, in particular to a protein S activity determination kit.

Background

Protein S is a plasma protein involved in the regulation of complement activation process, protein S is a cofactor of protein C, protein C is vitamin K-dependent glycoprotein synthesized by liver, protein C has no anticoagulation activity, and active protein C is formed after activation, so that VIIIa and Va factors are inactivated; protein S is taken as a cofactor, can enhance the inactivation reaction of activated protein C, and human blood plasma regulates the formation of blood clots through the protein C and the protein S; when protein S is deficient, factors Va and VIIIa are reduced in inactivation and blood circulation fibrinolysis capacity is reduced, so that excessive fibrin formation is promoted to cause thrombosis, and measurement of protein S activity is important for prevention, monitoring and treatment of diseases.

The protein C activator used in the currently marketed reagent for determining protein S activity is mostly derived from purified fractions of Agkistrodon halys venom. In fact, the stability of the protein C activator extracted from the viper venom is slightly poor, so that in order to prolong the long-term stability and storage time of the reagent, the reagent in the protein S activity determination kit on the market at present is in a freeze-dried powder state, but the freeze-dried reagent has the following disadvantages: 1. the lyophilization process is too complex, resulting in increased costs; 2. the difference between the bottles of the reagent is large; 3, the freeze-dried reagent needs to be redissolved before use, and compared with a liquid reagent, the freeze-dried reagent is inconvenient to operate; 4. the freeze-dried reagent has poor stability after reconstitution, and needs to be used in a short time, otherwise, the reagent is wasted.

Therefore, a liquid type protein S activity determination kit which is simple to prepare, low in cost, good in stability, high in repeatability and accuracy and capable of meeting different requirements is urgently needed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the protein S activity determination kit which has the advantages of low price, high reagent sensitivity, high specificity, good stability, good repeatability and good accuracy.

The technical scheme adopted for solving the technical problems is as follows: a protein S activity assay kit, comprising an R1 reagent, an R2 reagent and an R3 reagent;

The R1 reagent is freeze-dried powder, the components of the R1 reagent comprise human anticoagulated plasma of protein S, a first buffer solution and a first auxiliary material, and the first auxiliary material comprises saccharides and inert proteins;

The R2 reagent is in a liquid state, and comprises activated protein C, a second buffer solution, calcium chloride and a second auxiliary material, wherein the second auxiliary material comprises a protein protecting agent;

The R3 reagent is in a liquid state, the components of the reagent comprise protein C activator, phospholipid, a third buffer solution and a third auxiliary material, and the third auxiliary material comprises a protein protective agent.

Further, in the R1 reagent, preferably, the activity of protein S in the human anticoagulated plasma of the protein S is not more than 1%, the human anticoagulated plasma of the protein S is prepared by an enzyme-linked immunosorbent assay method, the protein S in the human anticoagulated plasma is adsorbed by a polystyrene carrier, and the human anticoagulated plasma of the protein S does not contain C4 binding protein.

Further, in the R1 reagent, preferably, the concentration of the first buffer solution is 10 to 100mmol/L, the concentration of the saccharide is 10 to 50g/L, the concentration of the inert protein is 10 to 50g/L, and the mass percentage of the human anticoagulated plasma of the protein S is 80 to 98%.

Further, it is preferable that the saccharide is at least one of trehalose, sucrose and lactose; the inert protein is at least one of bovine serum albumin and calf serum; the first buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4.

Further, the concentration of the activated protein C is preferably 1-10 mg/mL, the concentration of the second buffer solution is preferably 10-100 mmol/L, the concentration of the calcium chloride is preferably 20-40 mmol/L, and the mass percentage of the protein protectant in the R2 reagent is preferably 1-5%.

Further, preferably, the activated protein C is at least one of human-derived activated protein C and bovine-derived activated protein C; the second buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH is stabilized at 6.0-7.4; the protein protectant is a composite protein protectant DSP50.

Further, in the R3 reagent, the concentration of the protein C activator is preferably 0.05 to 0.50IU/mL, the mass percentage of the phospholipid is preferably 0.05 to 0.5%, the mass percentage of the protein protectant is preferably 1 to 5%, and the concentration of the third buffer solution is preferably 10 to 100mmol/L.

Further, it is preferable that the protein C activator is a snake venom extract, or the protein C activator is a combination of thrombin and thrombomodulin; the phospholipid is at least one of phosphatidylserine, phosphatidylcholine and tissue factor esterified substance; the third buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4; the protein protectant is a composite protein protectant DSP50.

Further, preferably, the kit further comprises a diluting reagent, wherein the diluting reagent is in a liquid state, the components of the diluting reagent comprise inorganic salt and a preservative, the inorganic salt is sodium chloride, and the concentration of the sodium chloride is 50-200 mmol/L; the preservative is at least one of sodium azide and PC300, and the mass percentage of the preservative in the diluting reagent is 0.05-0.2%.

Further, it is preferable that the R2 reagent and the R3 reagent include a preservative, the preservative is at least one of sodium azide and PC300, and the mass percentages of the preservative in the R2 reagent and the R3 reagent are 0.05-0.2%, respectively.

The invention has the beneficial effects that: the protein S activity determination kit provided by the invention has the advantages that the R2 reagent and the R3 reagent are liquid, and are liquid reagents with few markets, so that compared with the traditional freeze-drying kit, freeze-drying is not needed, the kit has the advantages of simple preparation process, lower cost, convenience in operation, small inter-bottle difference, difficulty in wasting the reagents and the like, and is not needed to be re-dissolved, so that the operation is simple and convenient, the inter-bottle difference is small, and meanwhile, the corresponding protein protectant is added to facilitate the stability maintenance of the liquid reagents; in addition, compared with the protein S activity determination kit on the market, the stability, accuracy and sensitivity of the invention are obviously improved, and the anti-interference capability is also greatly improved. The protein S activity determination kit can meet the requirements of institutions such as clinical laboratory at all levels, biological research hospitals, pharmaceutical companies and the like.

Drawings

The invention will be further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a standard curve of protein S activity versus plasma clotting time for the present invention.

Detailed Description

Specific embodiments of the present invention will now be described in detail for a clearer understanding of the technical features, objects and effects of the present invention.

A protein S activity assay kit comprises an R1 reagent, an R2 reagent and an R3 reagent; the R1 reagent is freeze-dried powder, the components of the freeze-dried powder comprise human anticoagulated plasma of protein S, a first buffer solution and a first auxiliary material, and the first auxiliary material comprises sugar and inert protein; the R2 reagent is in a liquid state, the components of the reagent comprise activated protein C, a second buffer solution, calcium chloride and a second auxiliary material, and the second auxiliary material comprises a protein protecting agent; the R3 reagent is in a liquid state, the components of the reagent comprise protein C activator, phospholipid, a third buffer solution and a third auxiliary material, and the third auxiliary material comprises a protein protective agent.

The protein S activity determination kit provided by the invention has the advantages that the R2 reagent, the R3 reagent and the diluting reagent are all liquid, and are liquid reagents with few markets, so that compared with the traditional freeze-drying kit, freeze-drying is not needed, the kit has the advantages of simple preparation process, lower cost, convenience in operation, smaller inter-bottle difference, difficulty in reagent waste and the like, and the kit does not need to be redissolved, is simple and convenient to operate, has small inter-bottle difference, and is convenient for maintaining the stability of the liquid reagent by adding the corresponding protein protectant; in addition, compared with the protein S activity determination kit on the market, the stability, accuracy and sensitivity of the invention are obviously improved, and the anti-interference capability is also greatly improved. The protein S activity determination kit can meet the requirements of institutions such as clinical laboratory at all levels, biological research hospitals, pharmaceutical companies and the like.

The human anticoagulated plasma is human sodium citrate anticoagulated plasma, and the ratio of plasma to sodium citrate in the human anticoagulated plasma is 9:1; the addition of sodium citrate can play a role in anticoagulation, and can reduce the influence of endogenous interference on the measurement result so as to improve the accuracy of the detection result.

Further, in the R1 reagent, the activity of protein S in the human anticoagulated blood plasma of the protein S is preferably not more than 1%, the activity of protein S in the R1 reagent is too high, the measurement result of the activity of protein S in the blood plasma of a patient is influenced, the human anticoagulated blood plasma of the protein S is prepared by adopting an enzyme-linked immunosorbent assay, the protein S in the human anticoagulated blood plasma is adsorbed by adopting a polystyrene carrier, the polystyrene carrier has better adsorption performance, a low blank value and high hole bottom transparency, the adsorption rate of the enzyme-linked immunosorbent assay is high and is up to more than 99.5%, the protein S can be removed to the greatest extent, so that the interference on the measurement of the activity of the protein S in the blood plasma of the patient is eliminated, and the accuracy of the detection result is further improved.

Furthermore, preferably, the human anticoagulated plasma of the spent protein S does not contain C4 binding protein, otherwise, the C4 binding protein can form C4b-BP binding protein with free protein S to influence the test result, the concentration of the C4 protein in the human anticoagulated plasma of the spent protein S can be measured by using a C4 binding protein biological reagent ELISA detection reagent, and if the concentration is larger, the enzyme-linked immunosorbent assay is adopted to adsorb so as to eliminate interference of the C4 binding protein on the measurement of the protein S activity of the plasma of a patient, thereby improving the accuracy of the detection result.

Further, in the R1 reagent, the concentration of the first buffer solution is preferably 10-100 mmol/L, the concentration of the saccharide is preferably 10-50 g/L, the concentration of the inert protein is preferably 10-50 g/L, the mass percentage of human anticoagulated plasma of the protein S is preferably 80-98%, and the R1 reagent is favorable for long-term stability and higher detection accuracy under the above proportion.

Further, preferably, the saccharide is at least one of trehalose, sucrose and lactose, the saccharide can inhibit nonspecific adsorption in the system, slow down the aging effect of protein and increase hydrophilicity on the premise of not affecting the sensitivity of the reaction system, meanwhile, the saccharide can also play a certain role in protecting protease, can also effectively protect the human anticoagulated plasma of the protein S and improve the stability of the R1 reagent, and the trehalose, sucrose and lactose can also protect the human anticoagulated plasma of the protein S in the freeze-drying process, so that the loss of the human anticoagulated plasma of the protein S in the freeze-drying process is reduced, the due content and activity of the protein S are maintained, and the R1 reagent after freeze-drying is in an expected receiving range.

The inert protein is at least one of bovine serum albumin and calf serum, and the addition of the inert protein is beneficial to prolonging the stability of the R1 reagent in the kit and the long-term use and storage of the kit, and by increasing the concentration of the protein in the R1 reagent, the decomposition and nonspecific adsorption of enzymes are prevented, the denaturation of part of enzymes can be reduced, and the denaturation caused by adverse environmental factors such as heating, surface tension and chemical factors is reduced.

The first buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4. The buffer solution has strong buffering capacity and good solubility to protein, the R1 reagent can be effectively maintained within a preset pH range by adopting the buffer solution, namely the pH of the R1 reagent is stabilized between 6.0 and 7.4, and the activity of protein S in a sample to be detected is not adversely affected by adopting the first buffer solution.

Further, in the R2 reagent, the preferable concentration of activated protein C is 1-10 mg/mL, the concentration of the second buffer solution is 10-100 mmol/L, the concentration of calcium chloride is 20-40 mmol/L, and the mass percentage of the protein protectant in the R2 reagent is 1-5%, so that the R2 reagent is stable for a long time and the accuracy is higher during detection under the above proportion.

Further, it is preferable that the activated protein C is at least one of human-derived activated protein C and bovine-derived activated protein C.

The second buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH is stabilized at 6.0-7.4; the buffer solution has strong buffering capacity and good solubility to protein, the R2 reagent can be effectively maintained within a preset pH range by adopting the buffer solution, namely the pH of the R1 reagent is stabilized between 6.0 and 7.4, and the activity of protein S in a sample to be detected is not adversely affected by adopting the second buffer solution.

The protein protectant is a composite protein protectant DSP50, the composite protein protectant DSP50 and the addition of the composite protein protectant DSP50 can protect broad-spectrum enzymes, proteins and antibodies, the addition of the composite protein protectant can also increase the stability of an R2 reagent, and the addition of the component is helpful for maintaining the activity of activated protein C, so that the effect of improving the stability of a liquid reagent is achieved.

Further, in the R3 reagent, the concentration of the protein C activator in the R3 reagent is preferably 0.05-0.50 IU/mL, the mass percentage of the phospholipid is 0.05-0.5%, the mass percentage of the protein protectant is preferably 1-5%, and the concentration of the third buffer solution is preferably 10-100 mmol/L, so that the R3 reagent is stable for a long time and the accuracy in detecting the protein S is higher under the above proportion.

Further, it is preferred that the protein C activator is a snake venom extract, or that the protein C activator is a combination of thrombin and thrombomodulin; the combination of the snake venom extract, thrombin and thrombomodulin can specifically activate the anticoagulant function of protein C; preferably, the protein C activator is a snake venom extract, the snake venom extract can specifically activate protein C, and other substances in blood plasma can be eliminated to interfere with the protein C, preferably, the concentration of the protein C activator is 0.05-0.50 IU/mL, and at the concentration, the activation efficiency of the protein C activator on the protein C is higher, so that the protein C is activated into activated protein C rapidly.

The phospholipid is at least one of phosphatidylserine, phosphatidylcholine and tissue factor esterified substance. Preferably, the mass percentage of the phospholipid is 0.05-0.5%.

The third buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4; the buffer solution has strong buffering capacity and good solubility to protein, the R3 reagent can be effectively maintained within a preset pH range by adopting the buffer solution, namely the pH of the R1 reagent is stabilized between 6.0 and 7.4, and the protein S activity in the sample to be detected is not adversely affected by adopting the third buffer solution.

The protein protectant is a compound protein protectant DSP50, the mass percent of the protein protectant is 1-5%, the addition of the compound protein protectant DSP50 can protect broad-spectrum enzymes, proteins and antibodies, the addition of the compound protein protectant DSP can also increase the stability of an R3 reagent, and the addition of the compound protein protectant DSP50 is favorable for maintaining the activity of activated protein C, so that the effect of improving the stability of a liquid reagent is achieved.

Further, the kit also comprises a liquid diluting reagent, wherein the diluting reagent comprises inorganic salt and preservative, the inorganic salt is sodium chloride, and the concentration of the sodium chloride is 50-200 mmol/L. The addition of inorganic salt maintains the osmotic pressure of the whole reagent, thereby playing a role in protection. The preservative is one or more of sodium azide and PC300, the mass percentage of the preservative in the diluted reagent is 0.05-0.2%, and the preservative can achieve the preservative effect, prevent the failure of the kit caused by microbial contamination and be beneficial to prolonging the storage validity period of the kit.

Further, the R2 reagent and the R3 reagent preferably comprise preservative, and the mass percentages of the preservative in the R2 reagent and the R3 reagent are respectively 0.05-0.2%. The preservative is one or more of sodium azide and PC300, and the preservative can achieve the preservative effect, prevent the failure of the kit caused by microbial contamination and facilitate the prolongation of the storage validity period of the kit.

The dilution ratio of the plasma to be measured and the dilution reagent can be 5-20 times, for example, the dilution ratio can be 1:5, 1:10 or 1:20. The ratio of the amount of the R1 reagent to the amount of the R2 reagent is 2:1, the ratio of the amount of the R3 reagent to the amount of the R1 reagent is 3:1, and the ratio of the amount of the sample to be measured (plasma to be measured) to the amount of the R3 reagent is 1:2, for example: the plasma to be measured was 3. Mu.L, 30. Mu.L after 10-fold dilution, 20. Mu.L of R1 reagent, 10. Mu.L of R2 reagent and 60. Mu.L of R3 reagent.

(1) Determining a standard curve:

The protein S activity of the healthy human blood plasma is 100%, the blood plasma of the healthy human is diluted by using diluting agents according to different proportions, the diluted blood plasma is used as a calibrator for calibration operation, the abscissa is the protein S activity of the blood plasma, and the ordinate is the clotting time of the blood plasma.

TABLE 1 clotting time detection results for protein S Activity-Standard Curve determination

Dilution ratio	Protein S Activity (%)	Setting time(s)
			1：0.7	143％	210.5
1：0.8	125％	185.3
			1：1.0	100％	152.5
1：1.2	83％	126.4
			1：1.5	67％	99.6
1：2.0	50％	75.8
			1：2.5	40％	61.4

According to the data, a standard curve is drawn, and as shown in fig. 1, the clotting time of the plasma of a patient is detected by using the kit of the invention, and the activity of protein S corresponding to the clotting time is rapidly obtained according to the standard curve, so that the activity of protein S in the plasma of the patient is measured.

The invention is further illustrated by the following detailed description.

Example 1

A protein S activity assay kit comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the specific formula is as follows:

TABLE 2 formulation of the kit of example 1

Wherein Tris is Tris buffer solution, and the same is the same as the Tris; the phospholipid is phosphatidylserine, and the following is the same; the protein C activator is snake venom extract; the Tris buffer can be replaced by at least one of TBS buffer, PBS buffer and MES buffer; trehalose may be replaced with at least one of sucrose and lactose or a mixture with trehalose; bovine serum albumin may be replaced with calf serum or a mixture of both; the phosphatidylserine may be replaced with at least one of phosphatidylcholine, and tissue factor esters or a mixture with phosphatidylserine; protein C activator snake venom extract can be replaced with a combination of thrombin and thrombomodulin.

Example 2:

TABLE 3 formulation of the kit of example 2

Example 3:

TABLE 4 formulation of the kit of example 3

Example 4:

TABLE 5 formulation of the kit of example 4

Example 5:

TABLE 6 formulation of the kit of example 5

Comparative example 1

TABLE 7 formulation of the kit of comparative example 1

Acceleration stability experiment:

Examples 1 to 5 and comparative examples were subjected to an accelerated stability test (37 ℃ C.), as follows: sample testing was performed on days 0, 7 and 14 by placing examples 1-5 and comparative example 1 in a 37 ℃ oven, and testing the protein S activity of normal and abnormal plasma on SYSMEX CS5100 at test time points, respectively, and analysis of the relative deviation of the initial test values on days 7 and 14 from day 0 should be performed to meet the requirements: 7. the relative deviation of the test results from day 0 within 14 is within + -10%.

TABLE 8 acceleration stability test results for the kits of examples 1-5 and comparative example 1

As shown in Table 8, the deviations of the normal quality control and the abnormal quality control of examples 1 to 5 are all + -10%, and the acceleration stability meets the thermal stability requirements on days 7 and 14, thus demonstrating that the liquid type reagent of the present invention has good stability. Wherein, compared with the examples 1-5, the composite protein protectant DSP50 is reduced, the deviation reaches 10% boundary on the 7 th day, and the deviation exceeds 10% on the 14 th day, thus indicating that the composite protein protectant DSP50 has more obvious stability protection effect. Compared with example 1, example 4 increases the addition amount of the composite protein protectant DSP50, and the acceleration deviation of the composite protein protectant DSP50 on days 7 and 14 is obviously reduced, which proves that the increase of the addition amount is helpful for the stabilization of liquid reagents, and can effectively protect the APC and protein C activator with protein activity. As can be seen from examples 1 to 5, the deviations thereof were within.+ -. 10%, which means that the adjustment of the concentration ratio had less influence on the stability in the range of the claimed concentration of the present invention.

Accuracy test:

comparative experiments were performed with accuracy for examples 1 to 5 and comparative example 1, and the specific procedures are as follows: three levels of protein S calibrator were tested on SYSMEX CS5100 using the protein S activity kit of examples 1-5, with target values of 30%, 90% and 150%, respectively, three times, with relative deviations of the mean from the target value within 10% being a qualifying standard.

TABLE 9 accuracy test results for the kits of examples 1 to 5 and comparative example 1

As can be seen from Table 9, the test results of the kits of examples 1-5 of the present invention all meet the results, indicating good accuracy. In examples 1 to 5, compared with comparative example 1, the compound protein protectant was absent in comparative example 1, so that the deviation was more than 10% because of poor protection effect, poor precision and large fluctuation due to low activity.

As shown in the table above, the relative deviation of the accuracy of the kit of the embodiment of the invention at three different levels is low, which indicates that the linearity range of the kit of the invention is wide, and the linearity range of the invention can reach 30-150%.

Anti-interference experiment:

Representative examples 1 and 4 were selected for the tamper resistance test. And selecting normal protein s samples, adding interference substances of hemoglobin, bilirubin and triglyceride according to the concentration respectively, performing on-machine test, and comparing the interference sample test result with the sample test result without the interference substances. The result should meet the requirements: the relative deviation is within + -10%. The test results are shown in Table 10.

TABLE 10 accuracy test results for the kits of examples 1 and 4

From the data, it can be shown that the protein S active kit provided by the invention has good anti-interference capability on triglyceride, bilirubin and hemoglobin, and meets the requirements.

The data and the figures show that the related performance test is carried out by using the kit provided by the invention, and the obtained results all meet the acceptance standard. The feasibility and rationality of the invention is verified by the specific embodiments described above, which are only illustrative of alternative individual embodiments of the invention, but not limiting. It will be apparent to those skilled in the art that all changes, modifications and substitutions that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A protein S activity assay kit, which is characterized by comprising an R1 reagent, an R2 reagent and an R3 reagent;

2. The kit for determining protein S activity according to claim 1, wherein in the R1 reagent, the protein S activity in the human anticoagulated plasma of the protein S is not more than 1%, the human anticoagulated plasma of the protein S is prepared by an enzyme-linked immunosorbent assay method, the protein S in the human anticoagulated plasma is adsorbed by a polystyrene carrier, and the human anticoagulated plasma of the protein S does not contain C4 binding protein.

3. The kit for measuring protein S activity according to claim 1, wherein the concentration of the first buffer solution in the R1 reagent is 10-100 mmol/L, the concentration of the saccharide is 10-50 g/L, the concentration of the inert protein is 10-50 g/L, and the mass percentage of the human anticoagulated plasma of the protein S is 80-98%.

4. The kit for determining protein S activity according to claim 1, wherein the saccharide is at least one of trehalose, sucrose and lactose; the inert protein is at least one of bovine serum albumin and calf serum; the first buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4.

5. The kit for measuring protein S activity according to any one of claims 1 to 4, wherein the concentration of activated protein C is 1 to 10mg/mL, the concentration of the second buffer is 10 to 100mmol/L, the concentration of calcium chloride is 20 to 40mmol/L, and the mass percentage of the protein protectant in the R2 reagent is 1 to 5%.

6. The kit for determining protein S activity according to claim 5, wherein the activated protein C is at least one of human-derived activated protein C and bovine-derived activated protein C; the second buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4; the protein protectant is a composite protein protectant DSP50.

7. The kit for measuring protein S activity according to any one of claims 1 to 4, wherein the concentration of the protein C activator in the R3 reagent is 0.05 to 0.50IU/mL, the mass percentage of the phospholipid is 0.05 to 0.5%, the mass percentage of the protein protectant is 1 to 5%, and the concentration of the third buffer is 10 to 100mmol/L.

8. The kit for determining protein S activity according to claim 7, wherein the protein C activator is a snake venom extract or the protein C activator is a combination of thrombin and thrombomodulin; the phospholipid is at least one of phosphatidylserine, phosphatidylcholine and tissue factor esterified substance; the third buffer solution is at least one of TBS buffer solution, PBS buffer solution, MES buffer solution and Tris buffer solution, and the pH value is 6.0-7.4; the protein protectant is a composite protein protectant DSP50.

9. The kit for determining protein S activity according to any one of claims 1 to 4, wherein the kit further comprises a liquid dilution reagent comprising an inorganic salt and a preservative, wherein the inorganic salt is sodium chloride, and the concentration of the sodium chloride is 50-200 mmol/L; the preservative is at least one of sodium azide and PC300, and the mass percentage of the preservative in the diluting reagent is 0.05-0.2%.

10. The kit for determining protein S activity according to any one of claims 1 to 4, wherein the R2 reagent and the R3 reagent comprise a preservative, the preservative is at least one of sodium azide and PC300, and the mass percentage of the preservative in the R2 reagent and the R3 reagent is 0.05 to 0.2 percent respectively.