阅读说明：本技术 蛋白c活性测定试剂盒 (Protein C activity determination kit ) 是由 廖婉露 温昶钰 王高煊 包文海 于 2019-11-20 设计创作，主要内容包括：本发明涉及蛋白C活性测定试剂盒,包括：含有蛋白C激活剂的试剂R1和含有发色底物的试剂R2。本发明试剂盒中的试剂R1和试剂R2均为液体,制备工艺简单,成本低,具有显著提升的稳定性和抗血红蛋白干扰效果,可广泛应用于各级医院、卫生部门、医学生物科研单位等机构。(The invention relates to a protein C activity determination kit, which comprises: a reagent R1 containing a protein C activator and a reagent R2 containing a chromogenic substrate. The reagent R1 and the reagent R2 in the kit are both liquid, the preparation process is simple, the cost is low, the stability and the hemoglobin interference resistance effect are obviously improved, and the kit can be widely applied to institutions such as hospitals, health departments, medical and biological research institutions at all levels.)

1. The protein C activity determination kit is characterized by comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises a protein C activator, a buffer solution, polyethylene glycol, PC300 and an alcohol amine compound; the reagent R2 comprises a chromogenic substrate and a buffer.

2. The protein C activity assay kit according to claim 1, wherein the pH value of the reagent R1 is 7.2-8.5, preferably the pH value of the reagent R1 is 7.2-7.6.

3. The kit for measuring protein C activity according to claim 1, wherein the protein C activator is a protein C activator extracted from Agkistrodon halys venom in an amount of 0.05-0.50 IU/ml.

4. The kit for measuring protein C activity according to claim 1, wherein the polyethylene glycol is selected from one or a combination of two or more of PEG-20000, PEG-4000 and PEG-6000; preferably, the polyethylene glycol is PEG-20000; the dosage of the polyethylene glycol is 0.5-15.0 g/L.

5. The kit for measuring protein C activity according to claim 1, wherein the alcohol amine compound is one or a combination of two or more selected from tromethamine, 2-amino-2-methyl-1-propanol, monoethanolamine, diethanolamine, triethanolamine, 3-propanolamine, monoisopropanolamine, diisopropanolamine, triisopropanolamine, N-dimethylethanolamine and N, N-diethylethanolamine; preferably, the alcohol amine compound is selected from one or a combination of more than two of tromethamine, triethanolamine and 2-amino-2-methyl-1-propanol; preferably, the alcamines are triethanolamine; the dosage of the alcohol amine compound is 1-40 g/L.

6. The protein C activity assay kit according to claim 1, wherein the buffer solution in the reagent R1 and the reagent R2 is one or a combination of two or more selected from the group consisting of HEPES acid buffer solution, Tris buffer solution, imidazole buffer solution and barbiturate buffer solution.

7. The kit for measuring protein C activity according to claim 1, wherein the chromogenic substrate in the reagent R2 is one selected from the group consisting of PyroGlu-Pro-Arg-pNA-HCl, p-glu-Pro-Arg-MNA, and THC-Pro-Arg-pNA.

8. The protein C activity assay kit according to any one of claims 1 to 7, wherein the kit comprises a reagent R1 and a reagent R2;

the reagent R1 comprises:

HEPES buffer solution: 5.0-30.0 g/L;

protein C activator: 0.05-0.50 IU/ml;

bovine serum albumin: 5.0-30.0 g/L;

trehalose: 0.5-20.0 g/L;

PEG-20000：0.5～15.0g/L；

PC300：0.5～10.0mL/L；

triethanolamine: 10-30 mL/L;

the reagent R2 comprises:

HEPES buffer solution: 5.0-30.0 g/L;

chromogenic substrate: 0.1-0.8 mg/ml;

a stabilizer: 1.0-20.0 g/L;

inorganic salts: 1.0-30.0 g/L;

preservative: 0.1 to 10.0 g/L.

9. A preparation method of a protein C activity assay kit is characterized by comprising the following steps:

preparation of reagent R1: mixing a protein C activator, a buffer solution, a stabilizer, polyethylene glycol, PC300 and an alcamines compound, and adjusting the pH value to 7.2-8.5;

preparation of reagent R2: mixing the chromogenic substrate and the buffer solution, and adjusting the pH value to 6.0-7.2.

10. A preparation method of a protein C activity assay kit is characterized by comprising the following steps:

preparation of reagent R1: mixing a protein C activator, a buffer solution, bovine serum albumin, trehalose, PEG-20000, PC300 and triethanolamine, and adjusting the pH value to 7.2-8.5;

preparation of reagent R2: mixing a color development substrate, a buffer solution, a stabilizer, inorganic salt and a preservative, and adjusting the pH value to 6.0-7.2;

wherein the buffer solution in the reagent R1 and the reagent R2 is one or the combination of more than two of HEPES acid buffer solution, Tris buffer solution, imidazole buffer solution and barbital buffer solution.

Technical Field

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

Background

Protein C (PC for short) is a plasma serine protease zymogen dependent on vitamin K, is mainly synthesized in liver, can also be synthesized in kidney and testis, has a relative molecular mass of 62kD, and has a half-life period of 6 hours. It is converted into activated protein C (APC for short) by thrombin or thrombin-thrombomodulin complex. APC and its cofactor protein S (PS for short) form a complex, have activity of inactivating Va and VIIIa and increasing fibrinolysis, and thus have anticoagulation effect. When PC is deficient, inactivation of factors Va and VIIIa decreases and the fibrinolytic capacity of the blood circulation decreases, thus promoting excessive fibrin formation, leading to thrombosis.

PC plays an important role in aspects of anticoagulation, anti-inflammation, anti-apoptosis, protection of vascular endothelial cells and the like, and has a plurality of biological functions, so that the PC is closely related to a plurality of clinical diseases. Such as sepsis, septicemia, Disseminated Intravascular Coagulation (DIC), thrombotic diseases and liver diseases, such as cirrhosis and chronic hepatitis, a different degree of reduction in PC levels can be detected. Therefore, the determination of PC plays an important role in the prevention, monitoring and treatment of diseases. Therefore, the simple method for accurately detecting the activity of the PC in the blood plasma is established, and has important values for the prevention and diagnosis, the observation of the disease condition and the judgment of prognosis of various diseases.

Currently, the detection of protein C can be generally classified into 2 major categories: one is the measurement of its antigen content (PC: Ag) and the other is the measurement of its activity (PC: AC).

PC: ag can be used for quantitative analysis of PC antigen by radioimmunoassay, rocket electrophoresis, enzyme-linked immunosorbent assay (ELISA), etc. The rocket electrophoresis method has poor specificity and low sensitivity and is limited by the preparation scale of antiserum, so the method has great limitation; radioimmunoassay methods require special instruments and are difficult to perform in common laboratories; therefore, the content of the polypeptide is generally measured by adopting an ELISA method with higher specificity and simpler operation.

PC: the AC can be measured by activated partial thromboplastin reagent method (APTT) and chromogenic substrate method (CSA). The determination of the measurement result of the APTT method is often determined by the experience of inspectors, and the accuracy and the repeatability of the measurement result are directly influenced. The CAS method adopts a specific chromogenic substrate, so that the operation is simple and convenient, and the accuracy and the repeatability of the measurement result are better.

PC in patients with acquired PC deficiency: ag and PC: changes in AC are parallel, so PC: ag and PC: AC has the same clinical significance, and PC is generally measured by ELISA or Chromogenic Substrate Assay (CSA): ag or PC: and (6) AC.

The World Health Organization (WHO) recommends the PC detection method as a chromogenic substrate method (CSA). At present, the commercial protein C activity detection kit mainly adopts a chromogenic substrate method, the method can accurately and specifically reflect the PC activity, the result is real and stable, the operation is simple and easy, the method has the advantage of rapidness, and the detection can be completed in a few minutes.

The activator used in the current commercial protein C activity determination kit is mostly separated and purified from agkistrodon halys venom. However, the stability of the protein C activator extracted from the venom of Agkistrodon halys is poor, and in order to prolong the storage time of the reagent, in the currently marketed protein C activity determination kit, the reagent is all freeze-dried powder. However, lyophilized reagents have the following disadvantages: the preparation process is complicated and the cost is high; the difference between bottles of reagents is large; the freeze-dried reagent needs to be re-dissolved before actual use, and the operation is inconvenient; the re-dissolved reagent has poor stability and needs to be used in a short time, otherwise, the reagent is easy to waste.

Therefore, the liquid protein C activity assay kit which is simple to prepare, low in cost, good in stability, convenient to use and easy to popularize and use in hospitals and health departments at all levels is urgently needed in the field.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a liquid-type protein C activity assay kit having good stability.

In a first aspect, the present invention provides a protein C activity assay kit comprising reagent R1 and reagent R2. The reagent R1 contains a protein C activator, a buffer solution, polyethylene glycol, PC300 and an alcamines compound; the reagent R2 contains a chromogenic substrate and a buffer.

PC300, ProClin300, which comprises mainly 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one. An exemplary amount of PC300 is 0.5-10.0 mL/L, and for example, 0.5mL/L, 1mL/L, 2mL/L, 3mL/L, 4mL/L, 5mL/L, 6mL/L, 7mL/L, 8mL/L, 9mL/L, 10mL/L can be selected.

In some embodiments, the pH of the reagent R1 is 7.2-8.5, and can be, for example, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the reagent R2 is 6.0-7.2, and can be, for example, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, or 7.2.

In some embodiments, a protein C activator refers to a component extracted from Agkistrodon halys venom that specifically activates protein C. Exemplary amounts of protein C activator are 0.05-0.50 IU/ml, for example, 0.05IU/ml, 0.1IU/ml, 0.15IU/ml, 0.2IU/ml, 0.25IU/ml, 0.3IU/ml, 0.35IU/ml, 0.4IU/ml, 0.45IU/ml, or 0.5 IU/ml.

In some embodiments, the polyethylene glycol is selected from PEG-20000, PEG-4000, PEG-6000 in one or more combinations. In a further preferred embodiment, the polyethylene glycol is PEG-20000. Polyethylene glycol is illustratively used in an amount of 0.5 to 15.0g/L, for example, 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, or 15 g/L.

In some embodiments, the alkanolamine compound is selected from one or a combination of two or more of tromethamine, 2-amino-2-methyl-1-propanol, monoethanolamine, diethanolamine, triethanolamine, 3-propanolamine, monoisopropanolamine, diisopropanolamine, triisopropanolamine, N-dimethylethanolamine and N, N-diethylethanolamine. In a further preferred embodiment, the alkanolamine compound is one or a combination of two or more selected from the group consisting of tromethamine, triethanolamine and 2-amino-2-methyl-1-propanol. In a further preferred embodiment, the alkanolamine compound is triethanolamine. An exemplary amount of the alkanolamine compound is 1 to 40g/L, for example, may be 1g/L, 3g/L, 5g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 32g/L, 34g/L, 36g/L, 38g/L, 39g/L, 40 g/L.

In some embodiments, the buffer in reagents R1 and R2 is selected from one or a combination of two or more of HEPES acid buffer, Tris buffer, imidazole buffer, barbiturate buffer. An exemplary amount of buffer is 5.0-30.0 g/L

In some embodiments, the reagent R1 further comprises a stabilizer. The stabilizer can be one or more of protein, saccharide, amino acid, antioxidant, and suspending agent. In a more preferred embodiment, the stabilizer is selected from one or a combination of two or more of bovine serum albumin, trehalose, mannitol and sorbitol. In a further preferred embodiment, the stabilizer is a combination of bovine serum albumin and trehalose. The amount of stabilizer is conventional in the art, for example, bovine serum albumin is typically used in an amount of 5.0-30.0 g/L, and trehalose is typically used in an amount of 0.5-20.0 g/L.

In some embodiments, the chromogenic substrate in reagent R2 is selected from one of PyroGlu-Pro-Arg-pNA HCl, p-glu-Pro-Arg-MNA, THC-Pro-Arg-pNA, with an exemplary amount of chromogenic substrate being 0.1 to 0.8 mg/ml.

In some embodiments, the reagent R2 further comprises an inorganic salt. The inorganic salt may be selected from NaCl, KCl, or CSCl, and in certain embodiments, the inorganic salt is preferably CSCl. Exemplary amounts of inorganic salts are 1.0 to 30.0 g/L.

In some embodiments, a stabilizer is also included in reagent R2. The stabilizing agent in the reagent R2 is similar to the stabilizing agent in the reagent R1, and can be one or the combination of more than two of protein, sugar, amino acid, antioxidant and suspending agent, wherein, the protein can be bovine serum albumin; the saccharide can be trehalose, mannitol, sorbitol, etc., and the amino acid can be histidine, arginine, etc. In certain embodiments, the stabilizing agent in agent R2 is arginine. An exemplary amount of stabilizer is 1.0 to 20.0 g/L.

In some embodiments, a preservative is also included in the agent R2. The preservative may be selected from one or more of the sodium azide (Na3N), MIT (i.e., 2-methyl-4-isothiazolin-3-one), CMIT (i.e., 5-chloro-2-methyl-4-isothiazolin-3-one), phenol, ProClin series. In certain embodiments, the preservative in reagent R2 is sodium azide. An exemplary amount of preservative is 0.1 to 10.0 g/L.

In some embodiments, the protein C activity assay kit includes reagent R1 and reagent R2,

the reagent R1 includes:

HEPES buffer solution: 5.0-30.0 g/L;

protein C activator: 0.05-0.50 IU/ml;

bovine serum albumin: 5.0-30.0 g/L;

trehalose: 0.5-20.0 g/L;

PEG-20000：0.5～15.0g/L；

PC300：0.5～10.0mL/L；

triethanolamine: 10-30 mL/L;

the reagent R2 includes:

HEPES buffer solution: 5.0-30.0 g/L;

chromogenic substrate: 0.1-0.8 mg/ml;

a stabilizer: 1.0-20.0 g/L;

inorganic salts: 1.0-30.0 g/L;

preservative: 0.1 to 10.0 g/L.

In some embodiments, the protein C activity assay kit includes reagent R1 and reagent R2,

the reagent R1 includes:

HEPES buffer solution: 5.0-30.0 g/L;

protein C activator: 0.05-0.50 IU/ml;

bovine serum albumin: 5.0-30.0 g/L;

trehalose: 0.5-20.0 g/L;

PEG-20000：0.5～15.0g/L；

PC300：0.5～10.0mL/L；

triethanolamine: 10-30 mL/L;

the reagent R2 includes:

HEPES buffer solution: 5.0-30.0 g/L;

chromogenic substrate: 0.1-0.8 mg/ml;

arginine: 1.0-20.0 g/L;

cesium chloride: 1.0-30.0 g/L;

preservative: 0.1 to 10.0 g/L.

In another aspect, the present invention provides a method for preparing a protein C activity assay kit, comprising the following steps:

preparation of reagent R1: mixing a protein C activator, a buffer solution, polyethylene glycol, PC300 and an alcamines compound, and adjusting the pH value to 7.2-8.5;

preparation of reagent R2: mixing the chromogenic substrate and the buffer solution, and adjusting the pH value to 6.0-7.2.

In the preparation method of the protein C activity assay kit, PC300, namely ProClin300, is used in an exemplary amount of 0.5-10.0 mL/L, and for example, 0.5mL/L, 1mL/L, 2mL/L, 3mL/L, 4mL/L, 5mL/L, 6mL/L, 7mL/L, 8mL/L, 9mL/L, or 10mL/L can be selected.

In the above method for preparing the protein C activity assay kit, the protein C activator is a component extracted from Agkistrodon halys venom and capable of specifically activating protein C. Exemplary amounts of protein C activator are 0.05-0.50 IU/ml, for example, 0.05IU/ml, 0.1IU/ml, 0.15IU/ml, 0.2IU/ml, 0.25IU/ml, 0.3IU/ml, 0.35IU/ml, 0.4IU/ml, 0.45IU/ml, or 0.5 IU/ml.

In the preparation method of the protein C activity assay kit, the polyethylene glycol is selected from one or the combination of more than two of PEG-20000, PEG-4000 and PEG-6000. In a further preferred embodiment, the polyethylene glycol is PEG-20000. Polyethylene glycol is illustratively used in an amount of 0.5 to 15.0g/L, for example, 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, or 15 g/L.

In the method for preparing the protein C activity assay kit, the alcohol amine compound is one or a combination of two or more selected from tromethamine, 2-amino-2-methyl-1-propanol, monoethanolamine, diethanolamine, triethanolamine, 3-propanolamine, monoisopropanolamine, diisopropanolamine, triisopropanolamine, N-dimethylethanolamine and N, N-diethylethanolamine. In a further preferred embodiment, the alkanolamine compound is one or a combination of two or more selected from the group consisting of tromethamine, triethanolamine and 2-amino-2-methyl-1-propanol. In a further preferred embodiment, the alkanolamine compound is triethanolamine. An exemplary amount of the alkanolamine compound is 1 to 40g/L, for example, may be 1g/L, 3g/L, 5g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 32g/L, 34g/L, 36g/L, 38g/L, 39g/L, 40 g/L.

In the above method for preparing a protein C activity assay kit, the buffer solution used in the preparation of the reagent R1 and the reagent R2 is one or a combination of two or more selected from HEPES acid buffer, Tris buffer, imidazole buffer, and barbiturate buffer. An exemplary amount of buffer is 5.0-30.0 g/L.

In the above method for preparing the protein C activity assay kit, the stabilizing agent used in preparing the reagent R1 may be one or a combination of two or more selected from proteins, saccharides, amino acids, antioxidants, and suspending agents. In a more preferred embodiment, the stabilizer is selected from one or a combination of two or more of bovine serum albumin, trehalose, mannitol and sorbitol. In a further preferred embodiment, the stabilizer is a combination of bovine serum albumin and trehalose. The amount of stabilizer is conventional in the art, for example, bovine serum albumin is typically used in an amount of 5.0-30.0 g/L, and trehalose is typically used in an amount of 0.5-20.0 g/L.

In the above method for producing a protein C activity assay kit, the chromogenic substrate in the reagent R2 is selected from PyroGlu-Pro-Arg-pNA & HCl, p-glu-Pro-Arg-MNA, and THC-Pro-Arg-pNA, and an exemplary amount of the chromogenic substrate is 0.1 to 0.8 mg/ml.

In another aspect, the present invention provides a method for preparing a protein C activity assay kit, comprising the following steps:

preparation of reagent R1: mixing a protein C activator, a buffer solution, bovine serum albumin, trehalose, PEG-20000, PC300 and triethanolamine, and adjusting the pH value to 7.2-8.5;

preparation of reagent R2: mixing a color development substrate, a buffer solution, a stabilizer, inorganic salt and a preservative, and adjusting the pH value to 6.0-7.2;

wherein the buffer solution in the reagent R1 and the reagent R2 is one or the combination of more than two of HEPES acid buffer solution, Tris buffer solution, imidazole buffer solution and barbital buffer solution.

In certain embodiments, exemplary amounts of protein C activator in agent R1 are 0.05 to 0.50IU/ml, for example, 0.05IU/ml, 0.1IU/ml, 0.15IU/ml, 0.2IU/ml, 0.25IU/ml, 0.3IU/ml, 0.35IU/ml, 0.4IU/ml, 0.45IU/ml, or 0.5 IU/ml.

In some embodiments, the buffer in the reagent R1 is HEPES buffer in an amount of 5.0-30.0 g/L.

In some embodiments, the amount of bovine serum albumin in the reagent R1 is 5.0-30.0 g/L, and the amount of trehalose is 0.5-20.0 g/L.

In certain embodiments, PEG-20000 in the reagent R1 is illustratively used in an amount of 0.5-15.0 g/L, e.g., 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, or 15 g/L.

In certain embodiments, PC300 in reagent R1, ProClin300, is used in an exemplary amount of 0.5-10.0 mL/L, for example, 0.5mL/L, 1mL/L, 2mL/L, 3mL/L, 4mL/L, 5mL/L, 6mL/L, 7mL/L, 8mL/L, 9mL/L, 10mL/L can be selected.

In certain embodiments, the triethanolamine is illustratively used in an amount of 10 to 30mL/L in the reagent R1, for example, 10mL/L, 12mL/L, 14mL/L, 15mL/L, 17mL/L, 19mL/L, 20mL/L, 22mL/L, 24mL/L, 26mL/L, 28mL/L, 30 mL/L.

In certain embodiments, the chromogenic substrate in reagent R2 is selected from one of PyroGlu-Pro-Arg-pNA HCl, p-glu-Pro-Arg-MNA, THC-Pro-Arg-pNA, with an exemplary amount of chromogenic substrate being 0.1 to 0.8 mg/ml.

In certain embodiments, the buffer in reagent R2 is HEPES buffer. An exemplary amount of the buffer solution in the reagent R2 is 5.0-30.0 g/L.

In certain embodiments, the stabilizing agent in the reagent R2 can be selected from one or a combination of two or more of proteins, sugars, amino acids, antioxidants, and suspending agents, wherein the proteins can be bovine serum albumin; the saccharide can be trehalose, mannitol, sorbitol, etc., and the amino acid can be histidine, arginine, etc. In certain embodiments, the stabilizing agent in agent R2 is arginine. An exemplary amount of stabilizer is 1.0 to 20.0 g/L.

In certain embodiments, the preservative in agent R2 may be selected from one or more of the sodium azide (Na3N), MIT (i.e., 2-methyl-4-isothiazolin-3-one), CMIT (i.e., 5-chloro-2-methyl-4-isothiazolin-3-one), phenol, ProClin series. In certain embodiments, the preservative in reagent R2 is sodium azide. An exemplary amount of preservative is 0.1 to 10.0 g/L.

Advantageous effects

The protein C activity determination kit provided by the invention has the advantages of obviously improved stability and anti-hemoglobin interference effect. The reagent kit is a liquid reagent kit, and because the reagent R1 and the reagent R2 in the reagent kit are both liquid, freeze drying is not needed, the preparation process is simpler, and the production cost is reduced; before the actual use of the kit, the redissolution operation is not needed, the use is more convenient, and the difference between bottles is reduced.

The protein C activity determination kit provided by the invention is simple to prepare, low in cost, good in stability and convenient to use, and can be widely applied to institutions such as hospitals, health departments, medical and biological research institutions at all levels.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.