Protein C detection reagent

Abstract

The present application relates to protein C detection technical field, especially to protein C detection reagent, the kit can measure wide linear range, linear correlation coefficient is excellent, and also has higher sensitivity, stability and anti-interference ability, market competition advantage is big, can realize the substitution of import product, reduces the cost of clinical examination, satisfies the demand of clinical examination.

Description

Technical Field

[0001] This invention relates to the field of protein C detection technology, and more particularly to protein C detection reagents. Background Technology

[0002] Protein C (PC) is a vitamin K-dependent double-chain glycoprotein synthesized by the liver. It has a molecular weight of 62 kDa, contains 461 amino acids, and consists of a light chain and a heavy chain linked by a single disulfide bond. In plasma, PC exists as an inactive proenzyme and has no biological function. It only exerts its anticoagulant activity when activated into activated protein C (APC) by the thrombin-thrombomodulin complex. Once activated, APC primarily inactivates factors V and VIII, reduces plasminogen activator inhibitors, and inhibits factor X binding to platelet membrane phospholipids. Therefore, APC possesses both anticoagulant and fibrinolytic effects.

[0003] Monitoring protein C activity is of great clinical significance. The level of protein C activity in healthy individuals is approximately 70-140%. Decreased protein C activity is commonly seen in congenital PC defects, but can also be associated with acquired PC defects manifesting as disseminated intravascular coagulation (DIC), liver diseases (such as acute hepatitis, chronic active hepatitis, and cirrhosis), malignant tumors, vitamin K deficiency, and acute respiratory distress syndrome. Thrombotic diseases pose a serious threat to human health due to their high incidence and mortality. Protein C deficiency is associated with recurrent venous thrombosis, and patients with protein C deficiency have an increased tendency for thrombosis; therefore, protein C can also serve as an indicator for diagnosing thrombotic diseases.

[0004] Based on the quantitative or qualitative abnormalities of PC, PC deficiency can be divided into Type I PC deficiency and Type II PC deficiency. Type I deficiency is a quantitative deficiency, characterized by varying degrees of decrease in both PC antigen content and anticoagulant activity, with an incidence of approximately 75%–80%. Patients with Type II PC deficiency have PC antigen content within the normal range, but their activity level is decreased. Based on the detection principle, current methods for quantitative detection of protein C mainly include the assessment of PC anticoagulant activity and the detection of PC antigen content.

[0005] Methods for detecting PC antigen content mainly include antigen content detection and anticoagulant activity detection. While PC antigen assays can detect the amount of PC in plasma, they cannot accurately assess its function and therefore cannot detect type II PC defects. Combining PC anticoagulant activity detection with antigen content measurement can differentiate between type I and type II PC defects.

[0006] Methods for detecting the anticoagulant activity of PC include the coagulation method and the chromogenic substrate method. Both methods are based on detecting the anticoagulant activity of PC after it has been activated into APC. Specifically:

[0007] The anticoagulant activity of cysteine ​​(PC) can be detected using coagulation methods based on activated partial thromboplastin time (APTT) and Russell's snake venom coagulation time. The APTT method is the most frequently used and the most commonly employed method for detecting PC activity using coagulation methods. Coagulation methods can detect all functional domains of PC, but the results are easily affected by various interfering factors.

[0008] Chromogenic substrate assays for detecting the anticoagulant activity of protein C (PC) offer advantages such as good stability, resistance to interference, and high reproducibility, making them the mainstream method for assessing the anticoagulant activity of PC. However, a search of currently available chromogenic substrate assay kits reveals that most are imported products; furthermore, due to the instability of protein C activators, they are all lyophilized reagents, resulting in a limited number of tests, high prices, and high detection costs, thus restricting the implementation of protein C assays in hospitals across China. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a protein C detection reagent in order to improve the stability of the reagent.

[0010] The protein C assay kit provided by this invention includes reagent R1 and reagent R2.

[0011] The R1 reagent contains a protein C activator, a 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer, a buffer, a stabilizer, and a preservative.

[0012] The R2 reagent comprises a chromogenic substrate, PPACK dihydrochloride, 2-methacryloyloxyethylphosphorylcholine (MPC) copolymer, stabilizer, preservative and buffer.

[0013] The detection principle of this kit is as follows: Protein C in plasma samples is activated into activated protein C by a protein C activator; activated protein C can act on a specific chromogenic substrate, cleaving the chromogenic group p-nitroaniline (pNA), and the intensity of the color is directly proportional to the activity of protein C. When using the kit of this invention for sample detection, in terms of reaction time, after mixing the sample with the diluent in the kit of this invention and incubating for 0.5 to 1 minute, adding reagent R1 in the kit of this invention and incubating for 2 to 4 minutes, followed by adding reagent R2 in the kit of this invention, no further incubation is required. The entire reaction time is shorter than that of existing commercially available products, the reaction is faster, the time required for a single test is shorter, and the sample waiting time is reduced.

[0014] The R1 and R2 reagents described in the above kit can be liquid or lyophilized. The concentrations of the reagent components described below are the working concentrations, i.e., the concentrations used. During reagent storage or transportation, the concentration can be the working concentration or 1.5 to 5 times the working concentration. For example, the concentration factor of reagent R1 is 1.5 to 5, and the concentration factor of reagent R2 is 1.5 to 10. As a feasible example, in the preparation of the lyophilized reagents, each component is prepared at twice the working concentration and then lyophilized.

[0015] In this invention, the detection reagents for protein C include reagent R1 and reagent R2;

[0016] The composition of the R1 reagent is as follows: protein C activator 0.2-0.5 U / mL, Tris 40-120 mM, BSA 0.3%-1%, mannitol 2%-10%, Biolipidure-103 0.5%-1.5%, ciprofloxacin 2-5 mg / mL, and the balance being water;

[0017] The composition of reagent R2 is as follows: S-2366 1.0-2.0 mg / mL, Tris 40-120 mM, mannitol 2%-10%, Biolipidure-406 1%-3%, PPACK dihydrochloride 0.05-2 mg / mL, cesium chloride or magnesium chloride 0.1-0.5 M, ciprofloxacin 2-5 mg / mL, and the balance being water.

[0018] In some embodiments, the R1 reagent consists of the following components at the following concentrations: protein C activator 0.2 U / mL, Tris 40 mM, BSA 0.3%, mannitol 2%, Biolipidure-103 0.5%, ciprofloxacin 2 mg / mL, with the balance being water;

[0019] In other embodiments, the components consist of the following concentrations: protein C activator 0.5 U / mL, Tris 120 mM, BSA 1.0%, mannitol 10%, Biolipidure-103 1.5%, ciprofloxacin 5 mg / mL, with the balance being water.

[0020] In other embodiments, the components consist of the following concentrations: protein C activator 0.35 U / mL, Tris 80 mM, BSA 0.65%, mannitol 6%, Biolipidure-103 1.0%, ciprofloxacin 3.5 mg / mL, with the balance being water.

[0021] In reagent R1 of this invention, the protein C activator is a snake venom extract. Preferably, its working concentration is 0.2-0.5 U / ml.

[0022] The stabilizers used in reagents R1 and R2 of this invention are one or more of proteins, amino acids, sugars, and surfactants. As feasible examples, proteins that can be used as stabilizers include bovine serum albumin (BSA) and Prionex; amino acids that can be used as stabilizers include histidine and arginine; sugars that can be used as stabilizers include sucrose and trehalose; alcohols that can be used as stabilizers include mannitol and sorbitol; and surfactants can be PEG, PVP, and Tween. Based on the linearity, sensitivity, and stability of the reagents, the combination of BSA and mannitol in R1, and mannitol in R2, allows for better compatibility with other components, thus ensuring better stability, sensitivity, and linearity. Preferably, reagent R1 contains 0.3–1.0 wt% BSA and 2–10 wt% mannitol; reagent R2 contains 2–10 wt% mannitol.

[0023] In this invention, reagents R1, R2, and the diluent further include a 2-methacryloyloxyethylphosphorylcholine (MPC) copolymer, which is an ionic surfactant. Compared to other ionic surfactants, MPC copolymer has good dispersing and solubilizing effects, and can better cooperate with other components in the reagents (for example, experiments show that Biopidure-103 and BSA can have a synergistic effect, improving the stability of reagent R1), inhibiting non-specific adsorption in the system, and improving the sensitivity and stability of the reaction system. Preferably, reagent R1 and the diluent contain Biopidure-103 at a concentration of 0.5–1.5 wt%; reagent R2 contains Biopidure-406 at a concentration of 1.0–3.0 wt%. The diluent contains Biopidure-103 at a concentration of 0.5–1.5 wt%.

[0024] In this invention, the R2 reagent further includes PPACK dihydrochloride, a protease inhibitor. When used in conjunction with Biolipidure-406, it effectively inhibits non-specific reactions between other proteases in plasma samples and the substrate, improving the reaction's resistance to interference. Compared to other enzyme inhibitors, PPACK exhibits better compatibility with other components in the system. The concentration of PPACK dihydrochloride added is 0.05–2 mg / mL.

[0025] In this invention, the R2 reagent further includes either cesium chloride or magnesium chloride at a concentration of 0.1–0.5 mol / L. Compared to other ionic strength modifiers, cesium chloride or magnesium chloride can better coordinate with other components. Besides ensuring the ionic strength in the R2 reagent, their presence can also accelerate the reaction rate, working synergistically with Biolipidure-103 to improve the sensitivity of the kit.

[0026] Reagents R1 and R2, as well as the diluent, all include preservatives. The preservatives are conventional preservatives in the art. As a feasible example, the preservative is any one of Procllin 300, ciprofloxacin, gentamicin sulfate, or sodium azide, which can effectively kill bacteria, has no side effects, and effectively extends the shelf life of the kit. In a specific embodiment, the preservative is 2–5 mg / mL of ciprofloxacin.

[0027] In some embodiments, the R2 reagent consists of the following components at the following concentrations: S-2366 1.0 mg / mL, Tris 40 mM, mannitol 2%, Biolipidure-40 61%, PPACK dihydrochloride 0.05 mg / mL, cesium chloride 0.1 M, ciprofloxacin 2 mg / mL, with the balance being water;

[0028] In other embodiments, the components consist of the following concentrations: S-2366 2.0 mg / mL, Tris 120 mM, mannitol 10%, Biolipidure-40 63%, PPACK dihydrochloride 2 mg / mL, cesium chloride 0.5 M, ciprofloxacin 5 mg / mL, with the balance being water;

[0029] In other embodiments, the components consist of the following concentrations: S-2366 1.5 mg / mL, Tris 80 mM, mannitol 6%, Biolipidure-40 62%, PPACK dihydrochloride 1.0 mg / mL, magnesium chloride 0.3 M, ciprofloxacin 3.5 mg / mL, with the balance being water;

[0030] In other embodiments, the components consist of the following concentrations: S-2366 1.5 mg / mL, Tris 80 mM, mannitol 6%, Biolipidure-40 62%, PPACK dihydrochloride 1.0 mg / mL, cesium chloride 0.3 M, ciprofloxacin 3.5 mg / mL, with the balance being water.

[0031] The chromogenic substrate in reagent R2 of the kit of the present invention is any one of pGlu-Pro-Arg-pNA·HCl(S-2366), pGlu-Pro-Arg-MNA(S-2366), or THC-Pro-Arg-pNA. Experiments have shown that the chromogenic substrate pGlu-Pro-Arg-pNA·HCl(S-2366) is more conducive to improving stability and sensitivity. As a preferred option, its working concentration is 1.0-2.0 mg / ml.

[0032] The detection reagent for protein C in this invention also includes a diluent containing sodium chloride, 2-methacryloyloxyethylphosphorylcholine (MPC), a preservative, and a buffer.

[0033] The diluent for the kit of this invention is a buffer solution containing 0.6 wt% to 1.2 wt% sodium chloride. Experiments have shown that this diluent can ensure good compatibility with the sample when mixed with it, and ensure that the activity of protein C in the sample is not easily destroyed.

[0034] The diluent consists of 0.6%–1.2% sodium chloride, 10–60 mM Tris, 0.5%–1.5% Biolipidure-103, 2–5 mg / mL ciprofloxacin, and the remainder is water.

[0035] In some embodiments, the diluent consists of the following components at the following concentrations: sodium chloride 0.6%, Tris 10mM, Biolipidure-103 0.5%, ciprofloxacin 2mg / mL, with the remainder being water;

[0036] In other embodiments, the components consist of the following concentrations: sodium chloride 0.9%, Tris 35mM, Biolipidure-103 1.0%, ciprofloxacin 3.5mg / mL, with the balance being water;

[0037] In other embodiments, the components consist of the following concentrations: 1.2% sodium chloride, 60 mM Tris, 1.5% Biolipidure-103, 5 mg / mL ciprofloxacin, with the balance being water.

[0038] In this invention, the buffers for reagents R1 and R2 and the diluent are all HEPES buffer, Tris buffer, or Tris-HEPES buffer. Based on sensitivity, stability, and linear range, Tris-HCl buffer is preferred as the buffer for reagents R1 and R2 and the diluent.

[0039] In some embodiments, the buffer solution in reagent R1 has a pH range of 7.5-8.5 and a final Tris concentration of 40-120 mM; the buffer solution in reagent R2 has a pH range of 6.0-7.0 and a final Tris concentration of 40-120 mM; and the diluent has a pH range of 7.0-7.5 and a final Tris concentration of 10-60 mM.

[0040] Furthermore, the present invention also provides a method for preparing the aforementioned detection reagent, comprising:

[0041] The protein C activator, Tris, BSA, mannitol, Biolipidure-103 and ciprofloxacin were dissolved in water to prepare reagent R1.

[0042] The R2 reagent was prepared by dissolving S-2366, Tris, mannitol, Biolipidure-406, PPACK dihydrochloride, cesium chloride, and ciprofloxacin in water.

[0043] Sodium chloride, Tris, Biolipidure-103, and ciprofloxacin were dissolved in water to prepare a diluted solution.

[0044] As a feasibility example, the preparation method of the reagent described in this invention further includes the step of freeze-drying reagent R1 and / or reagent R2;

[0045] The lyophilization parameters for reagents R1 and R2 are the same, as shown in the table below:

[0046]

[0047] The present invention also provides the application of the detection reagents as described above or the detection reagents prepared by the preparation methods as described above in the preparation of diagnostic reagents for thrombotic diseases.

[0048] The kit of this invention has a wide linear range and excellent linear correlation coefficient; it also has high sensitivity, stability and anti-interference ability; it has a strong competitive advantage in the market, can replace imported products, reduce the cost of clinical testing, and meet the needs of clinical testing. Attached Figure Description

[0049] Figure 1 Correlation analysis of the detection results of experimental group 1 in Example 1 of the reagent kit of the present invention and the IL reference kit;

[0050] Figure 2 Correlation analysis of the detection results of experimental group 2 in Example 1 of the present invention and the IL reference kit;

[0051] Figure 3 Correlation analysis of the detection results of experimental group 3 in Example 1 of the present invention and the IL reference kit;

[0052] Figure 4 Correlation analysis of the detection results of experimental group 4 in Example 1 of the present invention and the IL reference kit;

[0053] Figure 5 Correlation analysis of the detection results of experimental group 5 in Example 1 of the present invention and the IL reference kit;

[0054] Figure 6 This study analyzes the correlation between the detection results of experimental group 6 in Example 1 of the present invention and the IL reference kit. Detailed Implementation

[0055] This invention provides a protein C detection reagent. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0056] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0057] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0058] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0059] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0060] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0061] The embodiments and comparative examples of this invention describe some examples. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples. In fact, good sensitivity, linearity, anti-interference ability, and stability can be achieved at any concentration of the components between the two endpoint values ​​shown in the embodiments.

[0062] The nine control groups only listed some cases where the results were not good in the experiment. In addition, many other attempts were made during the research and development process, but the results of these attempts were not as good as those of the experimental groups 1 to 5, which will not be elaborated here.

[0063] The test materials used in this invention are all common commercially available products, and can be purchased on the market. Among them:

[0064] Protein C activator is from DSM Nutritional Products Ltd Branch Pentapharm, and its brand name is [Brand Name].

[0065] The S-2366 activated protein C chromogenic substrate was obtained from Shanghai Chuanfu Biotechnology Co., Ltd. or Shanghai Taiyang Biotechnology Co., Ltd.

[0066] Biolipidure-406 or Biolipidure-103 are from Nagase (China) Co., Ltd.;

[0067] PPACK dihydrochloride is from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0068] Hirudin comes from Xi'an Ruilin Biotechnology Co., Ltd.

[0069] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0070] Example 1: Preparation of the reagent kit and control experimental group of the present invention

[0071] (1) The reagent kit of the present invention

[0072] Table 1. Formulation of the reagent kit of the present invention (liquid reagent) (Experimental Group 1)

[0073]

[0074] Table 2. Formulation of the reagent kit of the present invention (liquid reagent) (Experimental group 2 and experimental group 3)

[0075]

[0076] Note: The R2 reagent in experimental group 2 was prepared using cesium chloride; the R2 reagent in experimental group 3 was prepared using magnesium chloride.

[0077] Table 3. Formulation of the reagent kit of the present invention (liquid reagent) (Experimental group 4)

[0078]

[0079] Table 4. Reagent formulations of the present invention (lyophilized reagents) (Experimental Groups 5 and 6)

[0080]

[0081] Note: ① All lyophilized reagents were dispensed in 1ml vials and lyophilized. Before use, the lyophilized products were reconstituted with 2ml of distilled water. The concentrations of each component after reconstitution (i.e., working concentrations) are shown in Table 4. ② The R2 reagent in experimental group 5 was prepared using cesium chloride; the R2 reagent in experimental group 6 was prepared using magnesium chloride.

[0082] (2) Preparation of the control experimental group

[0083] Table 5. Formulas for the control group

[0084]

[0085] Example 2: Calibration results of the standard curve of the reagent kit of the present invention

[0086] Table 4. Determination results of the standard curve of the experimental group of the reagent kit of the present invention.

[0087]

[0088] Table 5. Results of the determination of the standard curve of the control group of the reagent kit of the present invention.

[0089]

[0090] The results in Tables 4 and 5 show that: First, the correlation coefficients of the calibration curves in experimental groups 1 to 6 are superior, all greater than 0.995. While the correlation coefficients of the calibration curves in control groups 1, 4, 6, 11, and 12 are lower than 0.995, they are still greater than 0.985. However, the correlation coefficient between control group 6 and control group 7 is lower than 0.985, which does not meet the requirement of this kit (not lower than 0.985). Second, compared with the control group, the absorbance at the zero concentration point in the experimental groups is lower than that in the control group, while the results at all other points are higher than those in the control group, and the test results at the highest calibration point are significantly higher than those in the control group. This indicates that Biopidure-406 and Biopidure-103 can effectively inhibit non-specific adsorption and enhance the signal intensity of the reaction. Furthermore, the synergistic effect of Biopidure-406 with cesium chloride or magnesium chloride further accelerates the reaction rate and improves the sensitivity of the kit.

[0091] Example 3: Analytical performance evaluation of the kit of the present invention

[0092] (1) Sensitivity

[0093] The blank samples were tested using reagents from experimental groups 1-6 and control groups 1, 4, 6-8, 11, and 12, respectively. The tests were repeated 20 times, and the OD values ​​of each sample were calculated. 405nm The mean (X) and standard deviation (SD) were substituted into the corresponding calibration curves of each kit in Example 2 (subtracting twice the standard deviation from the blank mean) to calculate the limit of detection (LOD). The results are shown in Tables 6 and 7. The results show that the LOD obtained by the experimental group is much lower than that of the control group, further verifying the conclusion of Example 2. The LOD of the kit of this invention for detecting protein C can reach 3%, which is lower than the LOD (5%) stated in the instructions for use in commercial products.

[0094] Table 6. Analysis results of the lowest detection limit in the experimental group of the reagent kit of the present invention.

[0095]

[0096]

[0097] Table 7. Analysis results of the lowest detection limit in the control group of the kit of the present invention.

[0098]

[0099] (2) Linear

[0100] Seven samples of different concentrations were formed by mixing high-value samples (near the upper limit of the linear range) with low-value samples (near the lower limit of the linear range). Each concentration sample was tested using reagents from experimental groups 1-6 and control groups 1, 4, 6-8, 11, and 12, with the results repeated three times. The mean values ​​were calculated for each sample. Using the theoretical concentration as X and the measured mean as Y, the correlation coefficient of the linear regression equation was calculated. The results are shown in Tables 8-9. The results showed that, except for the control group 8 whose linearity did not meet the correlation coefficient requirement (not less than 0.985), the correlation coefficients of the linearity measurement results of the other groups were all higher than 0.985; however, the linear range of the control group did not meet the requirement of 10% to 150%, which adversely affected the reliability of the clinical sample test results; the linear range obtained by the experimental group exceeded the requirement of 10% to 150%, and the correlation coefficients were all greater than 0.995, further verifying the conclusion of Example 2, that is, the sensitivity of the kit of the present invention is improved and the measurable linear range is wider when Biolipidure-406, Biolipidure-103, cesium chloride or magnesium chloride are used together; while replacing cesium chloride or magnesium chloride with calcium chloride does not improve the sensitivity of the kit, but instead has an inhibitory effect.

[0101] Table 8. Linearity determination results of the experimental group of the reagent kit of the present invention.

[0102]

[0103] Table 9. Linearity assay results of the control group of the reagent kit of the present invention.

[0104]

[0105] (3) Anti-interference capability

[0106] According to the instructions for coagulation factor VIII, coagulation factor IX, thrombin, low molecular weight heparin, and unfractionated heparin, the above substances were dissolved to prepare stock solutions of a certain concentration. Then, the stock solutions were diluted to the required concentrations using normal plasma samples containing PC (theoretical value 117%) and low-PC plasma samples (theoretical value 61%), respectively, to obtain test samples containing different concentrations of coagulation factor VIII, coagulation factor IX, thrombin, low molecular weight heparin, and unfractionated heparin. The test samples were tested using reagents from experimental groups 1-6, control groups 6, and control groups 9-11, respectively. The measurements were repeated 5 times, and the mean values ​​of the protein C activity assay results for each group were calculated. The results are shown in Tables 10-12. The results showed that the combined action of Biolipidure-406 and PPACK dihydrochloride effectively improved the anti-interference ability of the reagent of the present invention. When the activity of coagulation factor VIII in the sample plasma was below 200 U / mL, the activity of coagulation factor IX was below 150 U / mL, the thrombin activity was below 20 U / mL, and the heparin content was below 2.4 U / mL, the kit of the present invention did not affect the detection of protein C activity in the sample. However, when PPACK dihydrochloride was replaced with hirudin, the anti-interference ability of hirudin combined with Biolipidure-406 was not as good as that of PPACK dihydrochloride and Biolipidure-406 alone, especially against thrombin interference.

[0107] Table 10. Detection results of the anti-interference ability of the experimental group of the reagent kit of the present invention (normal plasma samples)

[0108]

[0109] Table 11. Detection results of the anti-interference ability of the experimental group of the reagent kit of the present invention (low-value plasma samples)

[0110]

[0111] Table 12. Detection results of the anti-interference ability of the control group of the reagent kit of the present invention (normal plasma samples).

[0112]

[0113] (3) Stability

[0114] (I) Stability of R1 reagent

[0115] The R1 reagents of experimental groups 1–6 and control groups 1–5 were placed in a 37℃ electric thermostatic incubator and tested on days 0, 7, and 14. At each testing interval, the reagents of experimental groups 1–6 and control groups 1–5 were used to test two quality control samples: a normal control sample and an abnormal control sample. The tests were repeated three times. The relative deviations between the mean results on day 7 or 14 and day 0 were calculated based on the mean results, and the results are shown in Table 13. The results show that BSA alone has limited effect on the stability of the R1 reagent. The addition of Biolipidure-103 has a positive synergistic effect with BSA, further improving the stability of the R1 reagent without the need for additional stabilizers. While replacing BSA with Prinex, although the stability of Prinex combined with Biolipidure-103 also met the requirements, the stability effect was not as good as that of BSA and Biolipidure-406 alone.

[0116] Table 13 shows the stability results of reagent R1.

[0117]

[0118] (II) Stability of R2 reagent

[0119] The R2 reagents for experimental groups 1-6, control group 6, and control group 11 were placed in a 37℃ thermostatic incubator and tested on days 0, 7, and 14. At each testing interval, the reagents for experimental groups 1-6, control group 6, and control group 11 were used to test two quality control samples: a normal control sample and an abnormal control sample. The tests were repeated three times. The relative deviations between the mean results on day 7 or 14 and the mean results on day 0 were calculated based on the mean results. The results are shown in Table 14. The results show that Biolipidure-406 can effectively improve the stability of the R2 reagent without the need for additional stabilizers.

[0120] Table 14 Stability results of R2 reagent

[0121]

[0122] (III) Stability testing of the kit of the present invention

[0123] The reagents from experimental groups 1 to 6 were placed in a 37°C thermostatic incubator and tested on days 0, 7, 14, 21, 28, and 35. At each testing time interval, two quality control samples (one normal and one abnormal) were tested using the reagents from experimental groups 1 to 6, with the tests repeated three times. The results are shown in Table 15. The results show that the reagent kit of this invention exhibits almost no change in the test results of the normal and abnormal quality control samples within 35 days at 37°C, indicating that the reagent kit of this invention is effectively stable at 37°C for 35 days.

[0124] Table 15. Stability of the reagent kit of the present invention at 37°C.

[0125]

[0126] (III) Determination of the reconstitution stability of the lyophilized product of the kit of the present invention

[0127] After the reagents in experimental groups 5 and 6 met the requirements for pre-lyophilization test results, they were lyophilized. After lyophilization, the lyophilized products were reconstituted with distilled water to the indicated volume and then tested again after lyophilization. The test result after lyophilization was taken as the test result on day 0. Simultaneously, the reconstituted reagents were placed at room temperature (18-25℃) and tested on days 7, 15, 30, and 40. For each test, two quality control samples (one normal and one abnormal) were tested, and the tests were repeated three times. The mean of the test results was calculated, and the results are shown in Table 16. Table 16 shows that within 45 days of storage at room temperature after reconstitution of the lyophilized reagent kit of this invention, the test results of the normal and abnormal quality control samples remained almost unchanged. This indicates that the lyophilized reagent kit of this invention is stable at room temperature for 45 days after reconstitution, exceeding the shelf life (15 days) stated in the instructions of commercially available protein C detection kits (chromogenic substrate method).

[0128] Table 16 shows the stability of the lyophilized product of the reagent kit of the present invention at room temperature after reconstitution.

[0129]

[0130] Example 4 Comparison with Marketed Products

[0131] The kits obtained in Example 1 of this invention (experimental groups 1 to 6) and the reference kit (Instrumentation Laboratory Co. Protein C Assay Kit (chromogenic substrate method), catalog number: 0020300500) were used to test a set of samples covering the linear range, respectively. The correlation coefficient between the two kits was calculated and linear regression was performed. Figures 1-6The results showed that the slope of the linear regression equation for the comparison of the samples of the embodiments of the present invention with the reference kit was between 0.95 and 1.05, and the correlation coefficient r was greater than 0.99.

[0132] According to the requirements of the Clinical Laboratory Standards Institute (CLSI) document (r > 0.975), the test data of the kit of this invention and the imported kit from Instrumentation Laboratory Co. show good consistency.

[0133] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A protein C detection reagent comprising an Rl reagent, an R2 reagent, and a diluent; the Rl reagent consists of a protein C activator 0.2-0.5 U / mL, Tris 40-120 mM, BSA 0.3 wt%-1 wt%, mannitol 2 wt%-10 wt%, Biolipidure-103 0.5 wt%-1.5 wt%, ciprofloxacin 2-5 mg / mL, and the balance being water, the protein C activator being a snake venom extract; the R2 reagent consists of S-2366 1.0-2.0 mg / mL, Tris 40-120 mM, mannitol 2 wt%-10 wt%, Biolipidure-406 1 wt%-3 wt%, PPACK dihydrochloride 0.05-2 mg / mL, cesium chloride or magnesium chloride 0.1-0.5 M, ciprofloxacin 2-5 mg / mL, and the balance being water; the S-2366 being pGlu-Pro-Arg-pNA-HCl; the diluent consists of sodium chloride 0.6 wt%-1.2 wt%, Tris 10-60 mM, Biolipidure-103 0.5 wt%-1.5 wt%, ciprofloxacin 2-5 mg / mL, and the balance being water; the pH of the Rl reagent is 7.5-8.5, the pH of the R2 reagent is 6.0-7.0, and the pH of the diluent is 7.0-7.

5.

2. The detection reagent according to claim 1, wherein the Rl reagent consists of a protein C activator 0.2 U / mL, Tris 40 mM, BSA 0.3 wt%, mannitol 2 wt%, Biolipidure-103 0.5 wt%, ciprofloxacin 2 mg / mL, and the balance being water; or consists of a protein C activator 0.5 U / mL, Tris 120 mM, BSA 1.0 wt%, mannitol 10 wt%, Biolipidure-103 1.5 wt%, ciprofloxacin 5 mg / mL, and the balance being water; or consists of a protein C activator 0.35 U / mL, Tris 80 mM, BSA 0.65 wt%, mannitol 6 wt%, Biolipidure-103 1.0 wt%, ciprofloxacin 3.5 mg / mL, and the balance being water.

3. The detection reagent according to claim 1, wherein the R2 reagent consists of S-2366 1.0 mg / mL, Tris 40 mM, mannitol 2 wt%, Biolipidure-406 1 wt%, PPACK dihydrochloride 0.05 mg / mL, cesium chloride 0.1 M, ciprofloxacin 2 mg / mL, and the balance being water; or consists of the following components at the following concentrations: S-2366 2.0 mg / mL, Tris 120 mM, mannitol 10 wt%, Biolipidure-406 3 wt%, PPACK dihydrochloride 2 mg / mL, cesium chloride 0.5 M, ciprofloxacin 5 mg / mL, the remainder being water; or consists of the following components at the following concentrations: S-2366 1.5 mg / mL, Tris 80 mM, mannitol 6 wt%, Biolipidure-406 2 wt%, PPACK dihydrochloride 1.0 mg / mL, magnesium chloride 0.3 M, ciprofloxacin 3.5 mg / mL, the remainder being water; or consists of the following components at the following concentrations: S-2366 1.5 mg / mL, Tris 80 mM, mannitol 6 wt%, Biolipidure-406 2 wt%, PPACK dihydrochloride 1.0 mg / mL, cesium chloride 0.3 M, ciprofloxacin 3.5 mg / mL, the remainder being water.

4. The test reagent according to claim 1, characterized in that, the diluent consists of the following components at the following concentrations: sodium chloride 0.6 wt%, Tris 10 mM, Biolipidure-103 0.5 wt%, ciprofloxacin 2 mg / mL, the remainder being water; or consists of the following components at the following concentrations: sodium chloride 0.9 wt%, Tris 35 mM, Biolipidure-103 1.0 wt%, ciprofloxacin 3.5 mg / mL, the remainder being water; or consists of the following components at the following concentrations: sodium chloride 1.2 wt%, Tris 60 mM, Biolipidure-103 1.5 wt%, ciprofloxacin 5 mg / mL, the remainder being water.

5. A method for the preparation of a test reagent according to any one of claims 1 to 4, characterised in that, comprises: dissolving a protein C activator, Tris, BSA, mannitol, Biolipidure-103 and ciprofloxacin in water to obtain a R1 reagent; dissolving S-2366, Tris, mannitol, Biolipidure-406, PPACK dihydrochloride, cesium chloride and ciprofloxacin in water to obtain a R2 reagent; dissolving sodium chloride, Tris, Biolipidure-103 and ciprofloxacin in water to obtain a diluent.

6. The production method according to claim 5, characterized by, comprises a step of freeze-drying the R1 reagent and / or the R2 reagent; the freeze-drying parameters of the R1 reagent and the R2 reagent are the same and comprise: a pre-freezing phase at -50°C for 240 min; a sublimation phase at -30°C for 270 min at a controlled vacuum of 8.0 Pa, then at -20°C for 315 min; a desorption phase at -10°C for 315 min, then at 0°C for 150 min at a controlled vacuum of 12.0 Pa, then at 22°C for 480 min without vacuum control.

7. Use of the detection reagent according to any one of claims 1 to 4 or the detection reagent prepared by the preparation method according to any one of claims 5 to 6 in the preparation of a diagnostic reagent for thrombotic diseases.

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