High-stability protein S activity determination kit and preparation method thereof

Abstract

The invention relates to the technical field of in-vitro diagnostic reagent preparation, in particular to a high-stability protein S activity determination kit and a preparation method thereof, and the high-stability protein S activity determination kit comprises an activator, a substrate, a stop buffer and a standard substance; the activating agent contains snake venom protease, trehalose and the like and can activate protein S; the substrate contains a chromogenic substrate S-2366 and the like for chromogenic reaction; the stop solution contains glacial acetic acid and the like and is used for stopping reaction and stably developing; the standard substance contains recombinant protein S and the like for calibration; special components are added into the components to improve the stability, for example, zinc and magnesium ions are added into an activator to enhance the enzyme activity, and arginine is used as a substrate to inhibit hydrolysis; the components of the kit are independently packaged, and the pH and performance stable period at 2-8 DEG C is as long as 6 months. Through innovative formula design, the reagent stability and the detection accuracy are remarkably improved; special components enhance enzyme and substrate stability and reduce background interference; a standard product is optimized to improve the calibration precision; the preparation process guarantees batch-to-batch consistency, and is simple and convenient to operate, high in interference resistance and suitable for various clinical scenes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnostic reagent preparation, in particular to a high-stability protein S activity assay kit and a preparation method thereof. BACKGROUND

[0002] Protein S is an important vitamin K-dependent anticoagulant protein in the human body, which plays a key role in the blood coagulation cascade. By synergistically activating protein C, it inhibits coagulation factors Va and VIIIa, thereby regulating thrombus formation. Clinically, abnormal protein S activity levels are closely related to various diseases. For example, congenital protein S deficiency can cause hereditary thrombophilia, while acquired protein S reduction is often seen in liver diseases, pregnancy complications, and autoimmune diseases. Therefore, accurate determination of protein S activity in plasma is of great significance for disease diagnosis, risk assessment, and treatment monitoring.

[0003] Currently, protein S activity assays mainly use coagulation and chromogenic substrate methods. Coagulation method is based on plasma clotting time determination, which requires specific protein S-deficient matrix plasma, and is complicated, time-consuming, and susceptible to interference from other coagulation factors, with low sensitivity and specificity. The chromogenic substrate method activates protein S with snake venom protease to hydrolyze the chromogenic substrate and release the chromogenic group (such as p-nitroaniline, pNA), and then measures the absorbance change to reflect the activity of protein S. This method has the advantages of simple operation and high automation, and has become the mainstream detection method in clinical practice.

[0004] However, existing chromogenic substrate method kits still have many technical bottlenecks. Firstly, the core reagent has poor stability: snake venom protease is easily affected by temperature, pH, and oxidation, leading to significant differences in activity between different batches of reagent kits; chromogenic substrate S-2366 is easily hydrolyzed in aqueous solution, producing background interference and reducing detection accuracy. Secondly, the detection system has insufficient anti-interference ability: other proteases, inhibitors, and endogenous chromogenic substances in plasma can compete with the substrate for binding or directly affect the color reaction, leading to false positive or false negative results. Thirdly, standard preparation is difficult: recombinant protein S is prone to aggregation and oxidation, and traditional protective agents (such as BSA) cannot effectively maintain its structural integrity, resulting in poor linearity of the standard curve and poor repeatability of the detection results.

[0005] Although attempts have been made to improve the performance of the kit, limitations remain. For example, the addition of sugars (such as sucrose) or amino acids to stabilize the enzyme protein has limited effectiveness and may affect substrate solubility. Surfactants are used to improve substrate stability, but this may alter the enzyme's conformation and activity. Furthermore, existing preparation processes lack coordinated optimization of the stability of each component, failing to meet clinical demands for highly accurate and reproducible test results. With the advancement of precision medicine, there is an urgent need to develop a protein S activity assay kit with high stability, strong anti-interference capabilities, and ease of use to improve the accuracy and efficiency of disease diagnosis. Summary of the Invention

[0006] (1) Technical problems solved In view of the shortcomings of the existing technology, the present invention provides a high-stability protein S activity assay kit and a preparation method thereof.

[0007] (2) Technical solution A kit for determining the activity of high-stability protein S, comprising the following components: Activator solution: containing snake venom protease EC 3.4.21.104, trehalose 20-50 mmol / L, β-cyclodextrin 10-30 mmol / L, and Triton X-100; wherein the content of snake venom protease is 1-5 U / mL, and the content of polyethylene glycol octyl phenyl ether is 0.05-0.2%; the structural formula of the polyethylene glycol octyl phenyl ether is:

[0008] Substrate solution: contains 0.5-2 mmol / L of chromogenic substrate S-2366, 50-100 mmol / L of 3-(N-morpholino)propanesulfonic acid buffer, and 0.1-0.5 mmol / L of disodium ethylenediaminetetraacetate; the structural formula of 3-(N-morpholino)propanesulfonic acid is:

[0009] Stop solution: contains 10-20% (v / v) glacial acetic acid, 1-5g / L polyvinyl pyrrolidone, and 0.01-0.05% thimerosal; the structural formula of the polyvinyl pyrrolidone is:

[0010] Standard: Contains recombinant protein S 0.5-2 IU / mL, bovine serum albumin 1-3 g / L, glycerol 5-10% (v / v), sodium azide 0.02-0.05%.

[0011] Preferably, the activator solution further comprises 0.01-0.1 mmol / L of zinc ions and 0.1-1 mmol / L of magnesium ions.

[0012] Preferably, 0.5-2 mmol / L of L-arginine and 0.05-0.2% of Tween 20 are added to the substrate solution.

[0013] Preferably, the standard contains 0.1-0.5 mmol / L reduced glutathione GSH and 0.01-0.05% ProClin 300.

[0014] Preferably, the stop solution further contains 0.5-2 g / L sodium dodecyl sulfate (SDS) and 5-15 mmol / L p-aminobenzenesulfonic acid.

[0015] Preferably, the components of the kit are packaged independently, and the pH values ​​of the activator solution and the substrate solution can be kept stable within ±0.1 unit for up to 6 months at 2-8°C.

[0016] Preferably, the preparation method of the highly stable protein S activity assay kit comprises the following steps: S1: Preparation of activator solution Dissolve snake venom protease in 3-(N-morpholino)propanesulfonic acid MOPS buffer containing 20-50 mmol / L trehalose and 10-30 mmol / L β-cyclodextrin, add Triton X-100, Zn²⁺, and Mg²⁺, filter sterilize with a 0.22 μm filter membrane, and dispense into brown glass bottles; S2: Prepare substrate solution S-2366 was dissolved in a buffer solution containing 50-100 mmol / L MOPS and 0.1-0.5 mmol / L EDTA-Na2, L-arginine and Tween 20 were added, the pH was adjusted to 7.4 ± 0.1, filtered through a 0.22 μm filter membrane, and then aliquoted. S3: Preparation of stop solution Dissolve glacial acetic acid, polyvinylpyrrolidone PVP K30, thimerosal, SDS and p-aminobenzenesulfonic acid in purified water, stir evenly, filter through a 0.45 μm filter membrane and dispense; S4: Preparation of Standards Recombinant protein S was dissolved in a buffer containing 1-3 g / L bovine serum albumin (BSA) and 5-10% glycerol, and GSH and ProClin 300 were added. The solution was dispensed into polypropylene tubes and stored frozen at -80°C. S5: Assembly kit The above components are packaged in aluminum foil bags according to the proportions, and desiccant and instructions are enclosed.

[0017] Preferably, in the preparation of the activator solution, the temperature during the dissolution of the snake venom protease is controlled at 4-8° C., the stirring speed is 100-200 rpm, and the dissolution time does not exceed 30 minutes.

[0018] Preferably, the substrate solution is nitrogen-filled after packaging, with the nitrogen filling pressure of each bottle being 0.05-0.1 MPa, and stored at 2-8° C. in the dark after packaging.

[0019] Preferably, the standard product is packaged and freeze-dried, with a pre-freezing temperature of -40°C for 2 hours, then heated to 25°C at a rate of 0.5°C / min, with the vacuum degree maintained at 0.1-0.5 mbar, and the freeze-drying time being 24-36 hours.

[0020] (3) Beneficial technical effects Compared with the existing technology, the beneficial effects of the present invention are: 1. Trehalose, β-cyclodextrin, and metal ions (Zn²⁺, Mg²⁺) are introduced into the activator solution to form a multi-layered protective mechanism: trehalose inhibits conformational changes in snake venom proteases through vitrification, β-cyclodextrin encapsulates the active center to prevent oxidation, and the metal ions enhance enzyme-substrate affinity. L-arginine and Tween 20 are added to the substrate solution. L-arginine inhibits substrate hydrolysis through hydrogen bonding, while Tween 20 enhances its dispersibility in the buffer, reducing the spontaneous hydrolysis rate and effectively eliminating background interference.

[0021] 2. The combination of reduced glutathione and ProClin 300 in the standard prevents protein S oxidative aggregation and inhibits microbial contamination, ensuring a linear correlation coefficient R² ≥ 0.995 for the standard curve over a three-month period. The SDS and p-aminobenzenesulfonic acid in the stop solution work synergistically to rapidly inactivate residual enzyme activity and stabilize the colorimetric product, significantly outperforming traditional kits.

[0022] 3. Through precise concentration ratios and pH control, the kit's components effectively shield against interference from plasma substances such as fibrinogen and plasmin. Clinical sample testing has shown that the kit has a detection error of less than 5% for hemolytic and lipemic samples, compared to 15-20% for conventional kits.

[0023] 4. Low-temperature dissolution, nitrogen-filled packaging, and lyophilization techniques are optimized for the characteristics of the enzyme, substrate, and standard. Low-temperature preparation of the activator solution minimizes enzyme activity loss, nitrogen-filled packaging of the substrate isolates oxygen, and lyophilization of the standard preserves protein structural integrity, achieving an intra-batch coefficient of variation (CV) of less than 3% and an inter-batch coefficient of variation (CV) of less than 5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of a method for preparing a high-stability protein S activity assay kit disclosed in the present invention; Figure 2 3. It is a broken line comparison diagram of enzyme activity retention rate and substrate spontaneous hydrolysis rate in Examples and Comparative Examples; Figure 33. This is a comparison chart of the anti-interference ability test of the embodiment and the comparative example; Figure 4 This is a radar comparison chart produced after unifying the dimensions of the stability test results of the embodiment and the comparative example. DETAILED DESCRIPTION

[0025] according to Figures 1 to 4 , the specific implementation of the present invention is as follows: 1. Materials and Instrument Preparation 1. Experimental Materials Activator solution ingredients: snake venom protease (EC 3.4.21.104, activity ≥10 U / mg), trehalose (purity ≥99%), β-cyclodextrin (pharmaceutical grade), Triton X-100 (analytical grade), ZnCl2 (analytical grade), MgCl2・6H2O (analytical grade), MOPS buffer (pH 7.4, 0.1 mol / L).

[0026] Substrate solution materials: chromogenic substrate S-2366 (purity ≥98%), MOPS buffer (pH 7.4, 0.1 mol / L), EDTA-Na2 (analytical grade), L-arginine (purity ≥99%), and Tween 20 (biological reagent grade).

[0027] Stop solution ingredients: glacial acetic acid (analytical grade), PVP K30 (pharmaceutical grade), thimerosal (analytical grade), SDS (electrophoresis grade), p-aminobenzenesulfonic acid (analytical grade).

[0028] Standard raw materials: recombinant protein S (activity ≥ 95%), BSA (purity ≥ 98%), glycerol (analytical grade), GSH (reduced form), ProClin 300 (preservative).

[0029] 2. Instruments and Equipment Low-temperature centrifuge (4°C, 10,000 rpm), pH meter (accuracy ±0.01), UV-visible spectrophotometer (wavelength range 200-800 nm), fully automatic biochemical analyzer (Hitachi 7180), freeze dryer (cold trap temperature ≤-50°C, vacuum degree ≤0.1 mbar).

[0030] 2. Example 1: Preparation of kit by standard process 1. Preparation of Activator Solution Dissolve 50 mg of snake venom protease in 100 mL of MOPS buffer containing 40 mmol / L trehalose and 20 mmol / L β-cyclodextrin. Stir at 150 rpm for 25 minutes at 4°C. Add 0.05 mmol / L ZnCl₂, 0.5 mmol / L MgCl₂·6H₂O, and 0.15% Triton X-100. Sterilize by filtration through a 0.22 μm filter and aliquot into brown glass bottles, 1 mL per bottle.

[0031] 2. Preparation of Substrate Solution Dissolve 1.5 mmol / L S-2366 in 100 mL of buffer containing 80 mmol / L MOPS and 0.3 mmol / L EDTA-Na2, add 1.2 mmol / L L-arginine and 0.12% Tween 20, adjust the pH to 7.40 ± 0.01 with 1 mol / L NaOH, filter through a 0.22 μm filter membrane, and dispense 2 mL into each bottle. Fill with nitrogen at a pressure of 0.08 MPa.

[0032] 3. Preparation of Stop Solution Dissolve 15% (v / v) glacial acetic acid, 3 g / L PVP K30, 0.03% thimerosal, 1.2 g / L SDS, and 10 mmol / L p-aminobenzenesulfonic acid in 100 mL of purified water, stir evenly, filter through a 0.45 μm filter membrane, and dispense into 3 mL bottles.

[0033] 4. Preparation of Standards Recombinant protein S was dissolved in a buffer containing 2 g / L BSA and 8% glycerol to a concentration of 1.2 IU / mL. 0.3 mmol / L GSH and 0.03% ProClin 300 were added and aliquoted into polypropylene tubes, 0.5 mL per tube. Freeze at -40°C for 2 hours and then lyophilize at a heating rate of 0.5°C / min, a vacuum of 0.3 mbar, and a lyophilization time of 30 hours.

[0034] 5. Kit Assembly The above components are packaged in aluminum foil bags according to the proportion, with silica gel desiccant inside. Each box contains 10 bottles of activator, 20 bottles of substrate, 15 bottles of stop solution, 5 bottles of standard and instructions.

[0035] Example 2: Optimizing stability process 1. Preparation of Activator Solution Dissolve 50 mg of snake venom protease in 100 mL of MOPS buffer containing 50 mmol / L trehalose and 15 mmol / L β-cyclodextrin. Stir at 150 rpm for 25 minutes at 4°C. Then, add 0.05 mmol / L ZnCl₂, 0.5 mmol / L MgCl₂·6H₂O, and 0.15% Triton X-100. Mix thoroughly, filter sterilize through a 0.22 μm filter, and aliquot into brown glass bottles (1 mL per bottle).

[0036] 2. Preparation of Substrate Solution Dissolve 1.5 mmol / L of the chromogenic substrate S-2366 in 100 mL of buffer containing 80 mmol / L MOPS and 0.3 mmol / L EDTA-Na2. Add 1.8 mmol / L L-arginine and 0.08% Tween 20. Adjust the pH to 7.40 ± 0.01 with 1 mol / L NaOH. Filter through a 0.22 μm filter and aliquot into 2 mL bottles. Fill with nitrogen to a pressure of 0.08 MPa to exclude oxygen.

[0037] 3. Preparation of Stop Solution Dissolve 15% (v / v) glacial acetic acid, 3 g / L PVP K30, 0.03% thimerosal, 1.2 g / L SDS, and 10 mmol / L p-aminobenzenesulfonic acid in 100 mL of purified water. Stir well, filter through a 0.45 μm filter membrane, and dispense into transparent glass bottles, 3 mL per bottle.

[0038] 4. Preparation of Standards Recombinant protein S was dissolved in a buffer containing 2 g / L BSA and 8% glycerol to a concentration of 1.2 IU / mL. 0.5 mmol / L reduced glutathione (GSH) and 0.03% ProClin 300 were added. Aliquots were placed into polypropylene tubes, 0.5 mL per tube, and pre-frozen at -40°C for 2 hours before lyophilization. The temperature was maintained at a rate of 0.5°C / min and a vacuum of 0.3 mbar. The lyophilization time was extended to 36 hours.

[0039] 5. Kit Assembly Package the activator, substrate, stop solution, and standard in aluminum foil bags according to the appropriate ratio, with silica gel desiccant inside. Each box contains 10 bottles of activator, 20 bottles of substrate, 15 bottles of stop solution, 5 bottles of standard, and instructions.

[0040] Example 3: Scale-up production verification 1. Activator Solution Preparation (Single Batch) Dissolve 2.5 g of snake venom protease in 5 L of MOPS buffer containing 40 mmol / L trehalose and 20 mmol / L β-cyclodextrin. Stir at 150 rpm for 25 minutes at 4°C. Add 2.5 mmol / L ZnCl₂, 25 mmol / L MgCl₂·6H₂O, and 7.5% Triton X-100. Mix thoroughly, filter sterilize through a 0.22 μm filter, and aliquot into brown glass bottles (1 mL per bottle, single batch yield: 5000 bottles).

[0041] 2. Substrate Solution Preparation (Single Batch) Dissolve 75 mmol / L S-2366 in 5 L of buffer containing 80 mmol / L MOPS and 0.3 mmol / L EDTA-Na2. Add 60 mmol / L L-arginine and 6% Tween 20. Adjust the pH to 7.40 ± 0.01 with 1 mol / L NaOH. Filter through a 0.22 μm filter and aliquot into 2 mL bottles (2500 bottles per batch). Maintain a nitrogen pressure of 0.08 MPa.

[0042] 3. Stop Solution Preparation (Single Batch) Dissolve 15% (v / v) glacial acetic acid, 150 g PVP K30, 1.5 g thimerosal, 60 g SDS, and 500 mmol / L p-aminobenzenesulfonic acid in 5 L of purified water. Stir well, filter through a 0.45 μm filter membrane, and dispense into transparent glass bottles, 3 mL per bottle (single batch yield is approximately 1,666 bottles).

[0043] 4. Standard Preparation (Single Batch) Recombinant protein S was dissolved in 5 L of buffer containing 100 g BSA and 8% glycerol to a concentration of 1.2 IU / mL. 15 mmol / L GSH and 1.5% ProClin 300 were added. Aliquots were made into polypropylene tubes, 0.5 mL per tube (10,000 tubes per batch), pre-frozen at -40°C for 2 hours, and then lyophilized at a heating rate of 0.5°C / min, a vacuum of 0.3 mbar, and a lyophilization time of 30 hours.

[0044] 5. Continuous Production and Kit Assembly Ten batches were produced continuously according to the above process, and the cumulative amount obtained was: 50,000 bottles of activator solution; 25,000 bottles of substrate solution; 16,660 bottles of stop solution; and 100,000 tubes of standard product. The components were assembled into a test kit according to the ratio of Example 1 (each box contained 10 bottles of activator, 20 bottles of substrate, 15 bottles of stop solution, and 5 bottles of standard). A total of 1000 boxes were produced (10 batches × 100 boxes / batch). Each box contained a silica gel desiccant and instructions.

[0045] Comparative Example: Preparation of Kit Using Traditional Process Steps: Activator solution: Snake venom protease (1 U / mL) was dissolved in 10 mmol / L HEPES buffer (pH 7.4) containing 0.1% BSA; Substrate solution: S-2366 (1 mmol / L) was dissolved in 50 mmol / L Tris-HCl buffer (pH 8.0); Termination solution: 50% acetic acid solution; Standard: Recombinant protein S (1 IU / mL) was dissolved in 1% BSA in PBS buffer; After aliquot, it was stored normally, without nitrogen charging and without lyophilization.

[0046] The stability test of the examples and the comparative examples is compared as follows: Table 1

[0047] After the kit of the examples 1-3 of the present application was stored at 2-8℃ for 6 months, the enzyme activity retention rate was more than 95%, the substrate hydrolysis rate was less than 5%, and the standard linear degree R² was ≥0.996; while the enzyme activity of the comparative example was only retained 72.1%, and the substrate hydrolysis rate was as high as 21.5%, proving that the formula of the present application significantly improves the stability of the reagent. The anti-interference ability test of the examples and the comparative examples is compared as follows: Table 2

[0048] In the face of hemolysis (hemoglobin), lipemia (triglyceride), jaundice (bilirubin) samples, the detection error of the example 1 of the present application was less than 3.1%, while the error of the comparative example was more than 15.6%, indicating that the reagent kit of the present application has stronger anti-interference ability and more reliable results.

[0049] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A kit for determining the activity of high-stability protein S, characterized in that: Includes the following components: Activator solution: contains snake venom protease EC 3.4.21.104, trehalose 20-50 mmol / L, β-cyclodextrin 10-30 mmol / L, and Triton X-100; the content of snake venom protease is 1-5 U / mL, and the content of Triton X-100 is 0.05-0.2%; Substrate solution: Contains chromogenic substrate S-2366 at a content of 0.5-2 mmol / L, 3-(N-morpholino)propanesulfonic acid buffer at a content of 50-100 mmol / L, and disodium ethylenediaminetetraacetate at a content of 0.1-0.5 mmol / L; Stop solution: contains 10-20% (v / v) glacial acetic acid, 1-5g / L polyvinylpyrrolidone, and 0.01-0.05% thimerosal; Standard: Contains recombinant protein S 0.5-2 IU / mL, bovine serum albumin 1-3 g / L, glycerol 5-10% (v / v), sodium azide 0.02-0.05%.

2. The highly stable protein S activity assay kit according to claim 1, characterized in that The activator solution further comprises 0.01-0.1 mmol / L of zinc ions and 0.1-1 mmol / L of magnesium ions.

3. The highly stable protein S activity assay kit according to claim 2, characterized in that The substrate solution is added with 0.5-2 mmol / L L-arginine and 0.05-0.2% Tween 20.

4. The highly stable protein S activity assay kit according to claim 3, characterized in that The standard contains 0.1-0.5 mmol / L reduced glutathione GSH and 0.01-0.05% ProClin 300.

5. The highly stable protein S activity assay kit according to claim 4, characterized in that The stop solution also contains 0.5-2 g / L sodium dodecyl sulfate (SDS) and 5-15 mmol / L p-aminobenzenesulfonic acid.

6. The highly stable protein S activity assay kit according to any one of claims 1 to 5, characterized in that: Each component of the kit is packaged independently, and the pH values ​​of the activator solution and the substrate solution can be kept stable within ±0.1 unit for up to 6 months at 2-8°C.

7. A method for preparing a kit for determining the activity of highly stable protein S according to claim 5, characterized in that: The following steps are involved: S1: Preparation of activator solution Dissolve snake venom protease in 3-(N-morpholino)propanesulfonic acid MOPS buffer containing 20-50 mmol / L trehalose and 10-30 mmol / L β-cyclodextrin, add Triton X-100, Zn²⁺, and Mg²⁺, filter sterilize with a 0.22 μm filter membrane, and dispense into brown glass bottles; S2: Prepare substrate solution S-2366 was dissolved in a buffer solution containing 50-100 mmol / L MOPS and 0.1-0.5 mmol / L EDTA-Na2, L-arginine and Tween 20 were added, the pH was adjusted to 7.4 ± 0.1, filtered through a 0.22 μm filter membrane, and then aliquoted. S3: Preparation of stop solution Dissolve glacial acetic acid, polyvinylpyrrolidone PVP K30, thimerosal, SDS and p-aminobenzenesulfonic acid in purified water, stir evenly, filter through a 0.45 μm filter membrane and dispense; S4: Preparation of Standards The recombinant protein S was dissolved in a buffer containing 1-3 g / L bovine serum albumin (BSA) and 5-10% glycerol, GSH and ProClin300 were added, and the solution was dispensed into polypropylene tubes and stored frozen at -80°C. S5: Assembly kit The above components were packed in aluminum foil bags according to the proportions.

8. The method for preparing the highly stable protein S activity assay kit according to claim 7, wherein: In the preparation of the activator solution, the temperature is controlled at 4-8° C. when the snake venom protease is dissolved, the stirring speed is 100-200 rpm, and the dissolution time does not exceed 30 minutes.

9. The method for preparing the highly stable protein S activity assay kit according to claim 7, wherein: The substrate solution is filled with nitrogen for protection after packaging, with the nitrogen pressure of each bottle being 0.05-0.1 MPa. After packaging, it is stored at 2-8° C. in the dark.

10. The method for preparing the highly stable protein S activity assay kit according to claim 7, wherein: The standard product was packaged and freeze-dried using a pre-freezing temperature of -40°C for 2 hours, then heated to 25°C at a rate of 0.5°C / min, with the vacuum maintained at 0.1-0.5 mbar, and the freeze-drying time was 24-36 hours.

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