Compositions, detection reagents, detection kits, and uses thereof

Abstract

The present application relates to the field of biological detection, and particularly relates to a composition, a detection reagent, a detection kit and application thereof.The present application provides a composition comprising: a protein C activator 0.2-0.5 U / mL, polyvinylpyrrolidone 0.1-1.0 w / v% and L-lysine hydrochloride 0.1-0.5 w / v%. The present application provides a protein C activity determination kit and a preparation method thereof, through optimization of a reaction system and screening of specific additives, a protein C activity determination kit based on interaction between activated protein C and a chromogenic substrate is provided, the kit has many advantages such as good stability, high sensitivity, strong anti-interference, high correlation and convenient use, and of course, the kit also improves the disadvantages of slow speed and high price of the determination method mentioned in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to compositions, detection reagents, detection kits, and their applications. Background Technology

[0002] As a major component of the protein C system and one of the most important anticoagulants in the human body, protein C (hereinafter referred to as PC) is a vitamin-dependent glycoprotein synthesized by the liver and circulates in the blood in its inactive proenzyme form. Activated by the thrombin-thrombomodulin complex, protein C is rapidly converted into active protein C under the mediation of protein S, inactivating coagulation factors Va and VIIIa, thereby controlling the processes of blood coagulation and fibrinolysis and maintaining the body's coagulation balance. Therefore, the protein C system plays a crucial regulatory role in coagulation and fibrinolysis.

[0003] Studies have shown that low levels or activity of protein C can be both hereditary and acquired. Congenital protein C deficiency is divided into type I and type II. Type I patients have reduced protein C levels and activity; type II patients have normal protein C levels, but some PC activity is lost, resulting in reduced activity. Acquired PC deficiency has various causes, such as reduced PC synthesis due to liver disease; PC loss due to kidney disease; PC loss due to oral anticoagulants like warfarin, post-surgery, and vitamin K deficiency; and PC consumption due to disseminated intravascular coagulation. Both hereditary and acquired PC deficiency can lead to thrombus formation, therefore, clinical measurement of plasma PC or dynamic monitoring of its changes is essential and has certain reference value for clinical diagnosis, prognosis estimation, and efficacy monitoring.

[0004] Currently, there are two methods for detecting protein C: one is to quantitatively detect the content of PC antigen, such as immunoassay. This method is relatively specific and sensitive, but it is cumbersome, time-consuming, and cannot detect type II PC deficiency; the other is to quantitatively detect PC activity, such as the APTT coagulation method and the chromogenic substrate method. PC activity assays must use the chromogenic substrate method because the APTT coagulation method has many influencing factors on PC activity in the sample, resulting in poor specificity and accuracy, and is also cumbersome. Currently, commercially available kits of this type all use the chromogenic substrate method to determine plasma PC activity. This method has the advantages of high sensitivity, good accuracy, and short detection time, and is compatible with various automated analytical instruments. It is the preferred screening test for detecting protein C activity recognized by the WHO. However, these kits are all imported products and are expensive, making widespread clinical application difficult.

[0005] The principle of the chromogenic substrate method: Protein C exists in plasma in an inactive proenzyme form. Upon activation by a protein C activator, plasma protein C is converted into activated protein C (APC). APC acts on a specific chromogenic substrate, cleaving the substrate to produce a chromogenic group of 4-nitroaniline (pNA). The amount of 4-nitroaniline is positively correlated with the change in absorbance at 405 nm, therefore, the amount of 4-nitroaniline produced is positively correlated with protein C activity. Thus, by measuring the change in absorbance at 405 nm, the activity level of protein C in plasma can be calculated.

[0006] Currently, patents both domestically and internationally have disclosed reagent kits for detecting protein C activity. One patent develops a novel protein C activator extracted from the venom of the Qinling viper in China; however, this type of activator is not yet mass-produced or widely used. Another patent discloses a method for detecting protein C activity, mainly describing the merging of the two separate processes of "activation" and "color development" into a single "activation and color development" step, shortening the experimental time. Furthermore, another patent discloses a method for determining protein C activity, the reagents used for determination, and the preparation method. This patent is based on the chromogenic substrate method and optimizes the concentration of the protein C activator, buffer solution, stabilizer, and inorganic salts.

[0007] Based on the chromogenic substrate method, the reaction time of the protein C activity assay is relatively long, which limits the detection speed. In addition, the amount of assay reagents required is large and the price is expensive, which is not conducive to widespread clinical application.

[0008] Currently, the chromogenic substrate method for detecting protein C activity still needs improvement. Summary of the Invention

[0009] In view of this, the present invention provides compositions, detection reagents, detection kits, and their applications. The present invention provides a protein C activity assay kit and its preparation method. Through optimization of the reaction system and screening of specific additives, a protein C activity assay kit based on the interaction between activated protein C and a chromogenic matrix is ​​provided. This kit has many advantages such as good stability, high sensitivity, strong anti-interference ability, high correlation, and ease of use. It also overcomes the drawbacks of slow speed and high cost of the assay methods mentioned in existing patent literature.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a composition comprising:

[0012] Protein C activator 0.2–0.5 U / mL Polyvinylpyrrolidone 0.1–1.0 w / v%

[0013] L-Lysine hydrochloride 0.1–0.5 w / v%.

[0014] In some embodiments of the present invention, the protein C activator in the above composition includes: protein C activator Protac.

[0015] In some embodiments of the present invention, the above composition further includes:

[0016] Sorbitol 1.0–5.0 w / v%

[0017] Trehalose 5.0–10.0 w / v% Buffer solution 30–100 mmol / L.

[0018] In some embodiments of the present invention, the above composition further includes:

[0019]

[0020] In some embodiments of the present invention, the buffer solution in the above composition comprises: a Tris-HCl buffer solution with a pH of 7.2 to 8.4.

[0021] In some embodiments of the present invention, the above composition includes:

[0022]

[0023] The present invention also provides a detection reagent, comprising: the above-described composition, a chromogenic substrate reagent, and a diluent.

[0024] In some embodiments of the present invention, the chromogenic substrate reagent in the above-mentioned detection reagent comprises: 1.0 to 4.0 mg / L chromogenic substrate.

[0025] In some embodiments of the present invention, the chromogenic substrate reagent in the above-mentioned detection reagent includes: 1.0 mg / L, 2.0 mg / L, 3.0 mg / L or 4.0 mg / L chromogenic substrate.

[0026] In some embodiments of the present invention, the chromogenic substrate in the above-mentioned detection reagent includes: chromogenic substrate S2366.

[0027] In some embodiments of the present invention, the chromogenic substrate reagent in the above-mentioned detection reagent further includes:

[0028] Sorbitol 0.5–5.0 w / v% Trehalose 5.0–10.0 w / v% Buffer solution 30–100 mmol / L.

[0029] In some embodiments of the present invention, the chromogenic substrate reagent in the above-mentioned detection reagent further includes:

[0030]

[0031] In some embodiments of the present invention, the buffer solution in the above-mentioned detection reagent comprises: Tris-HCl buffer solution with pH 7.2 to 8.4.

[0032] In some embodiments of the present invention, the diluent in the above-mentioned detection reagent comprises: 0.05–0.50 w / v%.

[0033] The present invention also provides the use of the above-described composition and / or the above-described detection reagent in the preparation of a kit for detecting protein C activity.

[0034] In some embodiments of the present invention, the detection in the above application includes the following steps: taking the sample to be tested and mixing it with the diluent, then mixing it with the composition and the chromogenic substrate reagent, and after the colorimetric reaction, obtaining the activity of the protein C based on the OD value.

[0035] In some embodiments of the present invention, in the above applications, the volume ratio of the sample to be tested to the diluent is 1:4.

[0036] In some embodiments of the present invention, in the above applications, the volume ratio of the composition to the chromogenic substrate reagent is 1:1.

[0037] This invention provides the use of the above-described composition and / or the above-described detection reagents in the preparation of a kit for detecting protein C.

[0038] The present invention also provides a detection kit comprising: the above-described composition and / or the above-described detection reagent, and acceptable adjuvants or carriers.

[0039] In some embodiments of the present invention, the auxiliary agent in the above-mentioned detection kit includes:

[0040] Sorbitol 1.0–5.0 w / v% Trehalose 5.0–10.0 w / v% Buffer solution 30–100 mmol / L.

[0041] In some embodiments of the present invention, the buffer solution in the above-mentioned detection reagent comprises: Tris-HCl buffer solution with pH 7.2 to 8.4.

[0042] The present invention also provides a method for preparing the above-mentioned detection kit, wherein the composition, the chromogenic substrate reagent and the diluent are obtained respectively, and the composition and the chromogenic substrate reagent are freeze-dried to obtain the detection kit.

[0043] This invention provides a composition comprising:

[0044] Protein C activator 0.2–0.5 U / mL Polyvinylpyrrolidone 0.1–1.0 w / v%

[0045] L-Lysine hydrochloride 0.1–0.5 w / v%.

[0046] The kit provided by this invention includes a diluent, an activation reagent, and a chromogenic substrate reagent. The diluent is physiological saline containing a surfactant. The activation reagent includes protein C activator Protac, polyvinylpyrrolidone, L-lysine hydrochloride, sorbitol, trehalose, and a buffer solution. The chromogenic substrate reagent includes a chromogenic substrate, sorbitol, trehalose, and a buffer solution. In this invention, the diluent contains a surfactant, which neutralizes hemoglobin in plasma, preventing hemoglobin from interfering with the reaction rate of the activated protein C cleavage substrate and improving the accuracy of protein C activity detection. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0048] Figure 1 This demonstrates the effect of different concentrations of surfactants and activators on the calibration curves;

[0049] Figure 2 The calibration curves of Examples 6 and 7 and commercially available similar reagent kits are shown;

[0050] Figure 3 The correlation and relative deviation between Example 6 and commercially available similar reagent kits are shown; the upper figure shows the correlation, and the lower figure shows the relative deviation.

[0051] Figure 4 The linearity of the kits in Examples 1 and 6 is shown; the upper figure shows the linearity of the kit in Example 1, and the lower figure shows the linearity of the kit in Example 6.

[0052] Figure 5 The figure shows the differences between the kit of Example 6 on the ACL TOP CTS, ZK-600 and CS 5100 instruments. The upper figure shows the differences between the kit of Example 6 on the ACL TOP CTS and CS 5100 instruments; the lower figure shows the differences between the kit of Example 6 on the ZK-600 and CS 5100 instruments. Detailed Implementation

[0053] This invention discloses compositions, detection reagents, detection kits, and their applications.

[0054] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0055] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0056] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0057] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0058] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0059] This invention provides a protein C activity assay kit, comprising a diluent, an activating reagent, and a chromogenic substrate reagent.

[0060] The diluent is physiological saline containing surfactant;

[0061] The activating agent comprises protein C activator (DMS), sorbitol, polyvinylpyrrolidone, L-lysine hydrochloride, trehalose, and buffer solution;

[0062] The chromogenic substrate reagent comprises a chromogenic substrate, sorbitol, trehalose, and a buffer solution;

[0063] The coagulation process in plasma samples produces thrombin, which binds to its receptor, thrombomodulin, thereby activating protein C and converting it into activated protein C. Activated protein C, with the assistance of protein S, can inactivate coagulation factors Va and VIIIa, thus inhibiting excessive coagulation and maintaining the balance between coagulation and anticoagulation. Based on the chromogenic substrate method, protein C activators can rapidly and efficiently activate protein C in plasma samples, converting it into active protein C. Activated protein C then acts on a specific substrate, dissociating to release a chromogenic group. Compared to the thrombin-thrombomodulin complex, protein C activators can more rapidly and fully activate protein C without affecting the coagulation and anticoagulation systems, shortening reaction time and thus improving sample detection rate and the accuracy of protein C activity detection.

[0064] To avoid interference from hemoglobin and bilirubin in plasma samples with protein C activation, the diluent of this invention dilutes the sample, reducing interference from other coagulation factors (such as Va and VIIIa) that can affect the detection of activated protein C activity, thus reducing the consumption of activated protein C and decreasing the amount of protein C activator and substrate used in the kit, saving production costs. Furthermore, the diluent contains a surfactant. and L23 (preferably one or more) can neutralize hemoglobin and bilirubin, causing a conformational change and eliminating the influence of interference with protein C activity detection.

[0065] Preferably, in the diluent The concentration ranges from 0.05 w / v% to 0.50 w / v%.

[0066] Preferably, the concentrations of each component in the activating reagent are as follows: Protein C activator Protac 0.2 U / mL to 0.5 U / mL, polyvinylpyrrolidone 0.1 w / v% to 1.0 w / v%, L-lysine hydrochloride 0.1% to 0.5% (w / v), sorbitol 1.0 w / v% to 5.0 w / v%, trehalose 5 w / v% to 10.0 w / v%, and the balance of buffer solution.

[0067] As a preferred choice, the protein C activator Protac is manufactured by DSM.

[0068] Preferably, the concentrations of each component of the chromogenic substrate reagent are as follows: chromogenic substrate 1.0 mg / L to 4.0 mg / mL, sorbitol 0.5 w / v% to 5.0 w / v%, trehalose 5.0 w / v% to 10.0 w / v%, and buffer solution balance.

[0069] Preferably, the buffer solution is a 30 mmol / L to 100 mmol / L, pH 7.2-8.4 tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution.

[0070] The present invention also provides a method for preparing the kit, comprising preparing a diluent, an activating reagent and a chromogenic substrate reagent, and freeze-drying the activating reagent and the chromogenic substrate reagent.

[0071] This invention provides a protein C activity assay kit and its preparation method. The kit's preparation materials include a diluent, an activating reagent, and a chromogenic substrate reagent; the diluent contains... physiological saline;

[0072] The activating reagents include protein C activator Protac, polyvinylpyrrolidone, L-lysine hydrochloride, sorbitol, trehalose, and a buffer solution; the chromogenic substrate reagents include a chromogenic substrate, sorbitol, trehalose, and a buffer solution. The advantages of this invention are as follows:

[0073] In this invention, diluent is added. It can neutralize interfering substances bilirubin and hemoglobin in plasma, preventing these interfering substances from interfering with the activation reaction of protein C and improving the accuracy of detecting protein C activity;

[0074] In this invention, the additives polyvinylpyrrolidone, L-lysine hydrochloride and sorbitol can be further optimized in the activating reagent, which can not only improve the stability of the protein C activator Protac aqueous solution, but also enhance the precision for detecting protein C activity.

[0075] To address the issue that activator Protac and chromogenic substrates are generally unstable in aqueous solutions and cannot be stored for long periods, specific additives can be used to effectively improve the stability of activator Protac and chromogenic substrates in aqueous solutions. Furthermore, their dry powder form further enhances their stability.

[0076] Experiments show that the protein C activity assay kit of the present invention has excellent performance, is stable for 24 months at 2-8℃ with a relative deviation of no more than ±10%; precision CV value: no more than 8%; inter-vial difference CV value: no more than 5%; linear range: [5, 160]%, with a goodness of fit R2>0.99; after reconstitution, the kit can be stably maintained at 37℃ for 3 days and at 2-8℃ for at least 60 days; it has high stability.

[0077] In Examples 1 to 12 of this invention, all raw materials and reagents used can be purchased from the market.

[0078] The present invention will be further illustrated below with reference to the embodiments:

[0079] Example 1

[0080] Preparation of diluent: Weigh 0.05g Dissolve in 100 mL of physiological saline.

[0081] Preparation of activating reagents: The proportions of each component are as follows: 0.1 U / mL protein C activator Protac, 0.5 w / v% polyvinylpyrrolidone, 0.1 w / v% L-lysine hydrochloride, 0.5 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH 8.1 Tris-HCl buffer.

[0082] Preparation of substrate reagents: The composition ratio of each group is as follows: 2 mg / L chromogenic substrate S2366, 2 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH7.2 Tris-HCl buffer.

[0083] After the above-mentioned activating reagent and chromogenic substrate reagent were prepared, they were dispensed, freeze-dried, and made into lyophilized powder. Example 1 serves as a comparative example.

[0084] Example 2

[0085] Preparation of diluent: Weigh 0.05g Dissolve in 100 mL of physiological saline.

[0086] Preparation of activating reagents: The proportions of each group were 0.2 U / mL protein C activator Protac, 0.2 w / v% polyvinylpyrrolidone, 0.3 w / v% L-lysine hydrochloride, 2.0 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH 8.1 Tris-HCl buffer.

[0087] Preparation of substrate reagents: The composition ratio of each group is 2 mg / L chromogenic substrate S2366, 2 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH7.2 Tris-HCl buffer.

[0088] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0089] Example 3

[0090] Preparation of diluent: Weigh 0.20g Dissolve in 100 mL of physiological saline.

[0091] Preparation of activating reagents: The proportions of each group were 0.2 U / mL protein C activator Protac, 0.5 w / v% polyvinylpyrrolidone, 0.2 w / v% L-lysine hydrochloride, 3.0 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH 7.4 Tris-HCl buffer.

[0092] Preparation of substrate reagents: The composition ratio of each group is 1.0 mg / L chromogenic substrate S2366, 2 w / v % sorbitol, 7 w / v % trehalose, and 30 mmol / L pH 7.2 Tris-HCl buffer.

[0093] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0094] Example 4

[0095] Preparation of diluent: Weigh 0.30g Dissolve in 100 mL of physiological saline.

[0096] Preparation of activating reagents: The proportions of each group were as follows: 0.3 U / mL protein C activator Protac, 0.1 w / v% polyvinylpyrrolidone, 0.1 w / v% L-lysine hydrochloride, 1.0 w / v% sorbitol, 5 w / v% trehalose, and 60 mmol / L pH 8.4 Tris-HCl buffer.

[0097] Preparation of substrate reagents: The composition ratio of each group is 3 mg / L chromogenic substrate S2366, 0.5 w / v % sorbitol, 5 w / v % trehalose, and 30 mmol / L pH 7.2 Tris-HCl buffer.

[0098] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0099] Example 5

[0100] Preparation of diluent: Weigh 0.50g Dissolve in 100 mL of physiological saline.

[0101] Preparation of activating reagents: The proportions of each component are as follows: 0.5 U / mL protein C activator Protac, 1.0 w / v% polyvinylpyrrolidone, 0.5 w / v% L-lysine hydrochloride, 5.0 w / v% sorbitol, 10 w / v% trehalose, and 100 mmol / L pH 8.4 Tris-HCl buffer.

[0102] Preparation of substrate reagents: The composition ratio of each group is 4 mg / L chromogenic substrate S2366, 5 w / v % sorbitol, 10 w / v % trehalose, and 30 mmol / L pH7.2 Tris-HCl buffer.

[0103] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0104] Example 6

[0105] Preparation of diluent: Weigh 0.25g Dissolve in 100 mL of physiological saline.

[0106] Preparation of activating reagents: The proportions of each component are as follows: 0.3 U / mL protein C activator Protac, 0.3 w / v% polyvinylpyrrolidone, 0.1 w / v% L-lysine hydrochloride, 1.0 w / v% sorbitol, 5 w / v% trehalose, and 60 mmol / L pH 8.2 Tris-HCl buffer.

[0107] Preparation of substrate reagents: The composition ratio of each group is 1 mg / L chromogenic substrate S2366, 1.0 w / v % sorbitol, 5 w / v % trehalose, and 30 mmol / L pH 7.2 Tris-HCl buffer.

[0108] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0109] Example 7

[0110] Preparation of diluent: Weigh 0.25g Dissolve in 100 mL of physiological saline.

[0111] Preparation of activating reagents: The proportions of each group were 0.6 U / mL protein C activator Protac, 1.0 w / v% polyvinylpyrrolidone, 1.0 w / v% L-lysine hydrochloride, 5.0 w / v% sorbitol, 12 w / v% trehalose, and 60 mmol / L pH 8.4 Tris-HCl buffer.

[0112] Preparation of substrate reagents: The composition ratio of each group is 4 mg / L chromogenic substrate S2366, 4.0 w / v % sorbitol, 9 w / v % trehalose, and 30 mmol / L pH 7.2 Tris-HCl buffer.

[0113] After the above-mentioned activating reagent and chromogenic substrate reagent were prepared, they were dispensed, freeze-dried, and made into lyophilized powder. Example 7 serves as a comparative example.

[0114] Example 8

[0115] Preparation of diluent: Weigh 0.20g Triton X-100 and dissolve it in 100mL of physiological saline.

[0116] Preparation of activating reagents: The proportions of each group were 0.2 U / mL protein C activator Protac, 0.5 w / v% polyvinylpyrrolidone, 0.2 w / v% L-lysine hydrochloride, 3.0 w / v% sorbitol, 7 w / v% trehalose, and 30 mmol / L pH 7.4 Tris-HCl buffer.

[0117] Preparation of substrate reagents: The composition ratio of each group is 1.0 mg / L chromogenic substrate S2366, 2 w / v % sorbitol, 7 w / v % trehalose, and 30 mmol / L pH 7.2 Tris-HCl buffer.

[0118] After the above-mentioned activating reagent and chromogenic substrate reagent are prepared, they are packaged and freeze-dried to produce lyophilized powder.

[0119] Example 9: Procedure for Protein C Activity Assay

[0120] The protein C activity assay kit of this invention, after reconstitution and standing for 10 min, is used with a Sysmex 5100 fully automated coagulation analyzer. The parameters are set according to the manufacturer's instructions: the sample (calibrator, quality control sample, or plasma) is mixed with the diluent at a 1:4 ratio, and 50 μL of the mixture is taken out. An equal volume of activating reagent is added, mixed, and incubated for 4 min. Then, an equal volume of chromogenic substrate reagent is added and mixed. The mixture is reacted under 405 nm wavelength irradiation for 90 s, and the signal intensity (absorbance OD value) of the sample is read from 15 s to 75 s. Finally, the activity of protein C in plasma is obtained by calculating the signal intensity and comparing it with a standard curve of known concentration standards.

[0121] Figure 1 and Figure 2 This is a standard curve performance evaluation of the protein C activity assay kit described in this invention.

[0122] Example 10 Effects of the interfering agents bilirubin and hemoglobin

[0123] The Siemens universal calibrator with a known protein C activity of 98% was reconstituted and calibrated according to the automatic calibration procedure in the instrument's operation manual. A multi-point calibration method was used, setting a series of concentrations (1 / 1, 1 / 2, 1 / 4, 1 / 8, 0 / 1) for calibration. A series of surfactants or activators were prepared, such as the kits from Examples 1, 2, 3, 4, 5, and 8, and a commercially available similar kit (Siemens Medical Diagnostics GmbH, Protein C Activity Assay Kit (Chromogenic Substrate Method), batch number: 01619). The absorbance (OD) values ​​corresponding to the protein C activity of the universal calibrator were measured. Each sample was tested twice, and the average value was calculated. Calibration curves for each example were plotted. The results are shown in the appendix. Figure 1 .

[0124] Depend on Figure 1 As shown, the linearity R-values ​​of the calibration curves in Examples 1, 2, 3, 4, and 5 are... 2 All values ​​are greater than 0.9985, which is better than the linearity R-value of the calibration curve in Example 8. 2 And with surfactants The addition of an activator increases the absorbance OD value of the standard protein C activity detection, and the absorbance OD values ​​of Examples 2, 3, 4 and 5 are highly consistent and greater than those of Example 1, thus improving the activation efficiency of protein C.

[0125] Fresh mixed plasma samples (with PC activity approximately 110% mixed samples) were collected and mixed with bilirubin solution (800 mg / mL) and hemoglobin solution (2000 mg / mL), respectively, to obtain four gradients (10, 20, 40, 80 mg / dL) of bilirubin mixed plasma and four gradients (25, 50, 100, 200 mg / dL) of hemoglobin mixed plasma, as well as deionized water blank control plasma. The protein C activity of the above plasma samples was detected using kits from Examples 1, 2, 3, 4, 5, and 8, as well as commercially available similar kits. Each sample was tested twice, and the average value was calculated. The relative deviation between the protein C activity detection results with added interfering substances and those without interfering substances was calculated. The results are shown in Tables 1 to 5.

[0126] Table 1. Resistance of the kits used in Examples 1, 2, and 3 to bilirubin interference.

[0127]

[0128] Table 2 shows the resistance of the kits used in Examples 4, 5, and 8 to bilirubin interference.

[0129]

[0130] Table 3. Resistance of the kits used in Examples 1, 2, and 3 to hemoglobin interference.

[0131]

[0132]

[0133] Table 4. Resistance of the kits used in Examples 4, 5, and 8 to hemoglobin interference.

[0134]

[0135] Table 5. Resistance to interference from bilirubin and hemoglobin in commercially available similar reagent kits.

[0136]

[0137] As shown in Tables 1-5, the relative deviations between the protein C activity results of plasma with added interfering substances (80 mg / dL bilirubin, 200 mg / dL hemoglobin) and those of plasma without added interfering substances in Examples 2, 3, 4, and 5 are all within ±8.5%, and are significantly better than those in Examples 1 and 8 and commercially available similar kits, i.e., surfactants. The resistance to interference from bilirubin and hemoglobin is significantly better than that of the surfactant Triton X-100 and commercially available kits of the same type. This indicates that the surfactant enables the kit of this invention to exhibit strong resistance to interference from bilirubin and hemoglobin when detecting plasma protein C activity.

[0138] Example 11 Effect of stabilizer composition on the stability of protein C activity assay kit

[0139] Reconstitute the reagent kits from Examples 1, 2, 3, 4, 5, 6, and 7 with commercially available similar kits, and perform calibration according to the automatic calibration procedure in the instrument's operation manual. Figure 1 and Figure 2 The absorbance (OD) values ​​of Examples 2, 3, 4, 5, 6, and 7 are basically the same and significantly greater than those of Example 1 and commercially available similar kits.

[0140] Examples 1, 2, 3, 4, 5, 6, and 7, along with commercially available similar kits, were reconstituted and divided into three groups. These groups were then placed at 2℃–8℃, 25℃, and 37℃, respectively, at intervals. Following the standard procedure for protein C activity assays, the assays were performed by the same operator or a group of operators on the same instrument. Normal and abnormal quality control plasmas were tested, and changes in protein C activity were recorded to determine whether the activation effect of the protein C activator in the kit was reduced. The results are shown in Tables 6–8.

[0141] Table 6 shows the stability of the protein C activity assay kit at 2℃–8℃.

[0142]

[0143]

[0144]

[0145] Table 7. Stability of the Protein C Activity Assay Kit at 25°C

[0146]

[0147]

[0148] Table 8. Stability of the Protein C Activity Assay Kit at 37°C

[0149]

[0150]

[0151]

[0152] Tables 6-8 show that the relative deviations of the protein C activity detection values ​​after reconstitution of the reagents in Examples 2, 3, 4, 5, 6, and 7 at 2℃-8℃, 25℃, and 37℃ compared to day 0 are all within ±3.6%, which is significantly better than that of Example 1 and commercially available similar reagents. Therefore, the protein C activity detection reagents after reconstitution of the reagents in Examples 2, 3, 4, 5, 6, and 7 can be stable for at least 60 days at 2℃-8℃; can be stably stored for 30 days at 25℃; and can maintain activity for 3 days at 37℃, with consistent stability. This indicates that the composition (L-lysine hydrochloride, polyvinylpyrrolidone, and sorbitol) provided by this invention can maintain the stability of the protein C activator for a relatively long time, thereby maintaining the stability of the protein C activity detection reagent.

[0153] Example 12 Analytical performance evaluation of the kit of the present invention

[0154] 1. Plotting the calibration standard curves of Examples 6 and 7 and commercially available similar reagents.

[0155] The Siemens universal calibrator with a known protein C activity of 98% was reconstituted and operated according to the automatic calibration procedure in the instrument's operation manual. A multi-point calibration method was used, setting a series of concentrations (1 / 1, 1 / 2, 1 / 4, 1 / 8, 0 / 1) to calibrate both the kit of this invention and commercially available similar kits. The OD values ​​of the calibrators were measured on a Sysmex 5100 coagulation analyzer. A standard curve was plotted with OD values ​​on the ordinate and the corresponding protein C activity values ​​on the abscissa. Figure 2 As shown.

[0156] Depend on Figure 2 As shown, based on the chromogenic substrate method, the linear relationship function between the absorbance OD value of the calibrator detected by the kit of this invention and commercially available similar kits and the corresponding protein C activity is defined as Y = 0.0011X + 0.0003, R... 2 =0.9994, Y=0.0011X-0.0001, R 2 =0.9995 and Y = 0.0009X - 0.0003, R 2=0.9990. This demonstrates that the linear relationships in Examples 6 and 7 are highly consistent, exhibiting good linearity and high sensitivity compared to similar commercially available kits. Furthermore, the high content of components in the reagent of Example 7 resulted in a long lyophilization process and high raw material costs.

[0157] 2. Precision detection of Examples 1, 6 and 7

[0158] Following the standard procedure for protein C activity assay, the assay was performed by the same operator or a group of operators on the same instrument. Examples 1, 6, and 7 involved repeated measurements of normal and abnormal quality control plasma 10 times each. The mean, standard deviation, and coefficient of variation (CV) of the measured values ​​were calculated as follows:

[0159] Table 9 Precision of the Protein C Activity Assay Kit

[0160]

[0161] The coefficient of variation (CV) is commonly used to measure the precision of an assay method; a smaller CV value indicates better precision. Table 9 shows that the CV values ​​for detecting protein C activity in normal quality control plasma using reagents from Examples 6 and 7 were 1.02% and 1.00%, respectively; while the CV values ​​for detecting protein C activity in abnormal quality control plasma were 2.81% and 2.00%, respectively, significantly better than those of Example 1. This indicates that the precision performance of the reagents from Examples 6 and 7 is superior to that of Example 1.

[0162] 3. Accuracy testing in Example 6

[0163] Following the standard procedure for protein C activity assay, the assay was performed by the same operator or a group of operators on the same instrument. The international standard NIBSC 02 / 342 was measured three times repeatedly. The average value was calculated and compared with the target value of this international standard to calculate the relative deviation, as follows:

[0164] Table 10 Accuracy of the Protein C Activity Assay Kit

[0165]

[0166] As shown in Table 10, the kit of the present invention detected an average value of 85.7% for the activity of protein C of the international standard NIBSC 02 / 342, with a relative deviation of 0.82%, which is within ±10%. Therefore, the kit of the present invention has high accuracy.

[0167] 4. Example 6: Detection of inter-batch differences

[0168] Take out three batches of the reagent kit of this invention, reconstitute them, and perform the protein C activity assay by the same operator or a group of operators on the same instrument. Perform 10 tests on both normal and abnormal quality control plasma, and calculate the mean, standard deviation, and coefficient of variation (CV) of the measured values ​​as follows:

[0169] Table 11. Inter-batch variation of the protein C activity assay kit in detecting normal quality control plasma.

[0170]

[0171] Table 12. Inter-batch variation in detecting abnormal control plasma using the Protein C Activity Assay Kit

[0172]

[0173] The coefficient of variation (CV) is commonly used to measure the precision of an assay method; a smaller CV value indicates better precision. Tables 11 and 12 show that the precision (CV) values ​​of the kit in this invention for detecting protein C activity (%) in normal and abnormal quality control plasma were 0.97% and 2.26%, respectively, indicating that the kit has excellent inter-batch variation.

[0174] 5. Example 6: Detection of intra-batch differences

[0175] Using 10 different kits from the same batch of this invention, the plasma was reconstituted and, following the procedure for protein C activity assay, performed by the same operator or a group of operators on the same instrument. Normal control plasma and abnormal control plasma were tested 10 times per vial, and 10 vials were tested once per vial, respectively. The mean, standard deviation, and coefficient of variation (CV) of the protein C activity assay values ​​were calculated as follows:

[0176] Table 13 Intra-batch variation of the Protein C Activity Assay Kit in detecting normal quality control plasma

[0177]

[0178] Table 14. Intra-batch variation of protein C activity assay kit in detecting abnormal control plasma.

[0179]

[0180] As shown in Tables 13 and 14, the precision (CV) values ​​of the kit of the present invention for detecting normal quality control plasma and abnormal quality control plasma were 0.79% and 2.98%, respectively, indicating that the kit of the present invention has excellent intra-batch variation.

[0181] 6. Correlation detection between Example 6 and commercially available similar reagent kits

[0182] The kit of this invention and commercially available kits were used on a Sysmex 5100 fully automated coagulation analyzer to detect protein C activity in 32 fresh plasma samples (including normal and abnormal samples), and correlation analysis was performed on the measured values ​​(see results). Figure 3 The X-axis represents the measured value of commercially available reagent kits, and the Y-axis represents the measured value of the reagent kit of this invention or the relative deviation from the measured value of commercially available similar reagent kits.

[0183] Figure 3 The linear equation showing the correlation between the reagent kit of this invention and commercially available similar reagent kits is: y = 0.9913x + 1.1608, with a correlation coefficient of: R0. 2 =0.9954, with relative deviations all within ±10%, indicating that the detection results are basically consistent. This demonstrates that the kit of this invention has good correlation with commercially available similar kits. The linear equation is: [The results show that the kit of this invention has good correlation with commercially available similar kits.]

[0184] 7. Detection of linear range in Examples 1 and 6

[0185] Plasma samples with approximately 170% protein C activity were diluted to a gradient of 1, 0.8, 0.6, 0.5, 0.4, 0.2, 0.1, and 0.05. In Examples 1 and 6, each plasma sample was tested twice, and the mean value of the results for each sample was calculated. Using the dilution factor as the independent variable and the mean value of the results as the dependent variable, a linear regression equation and correlation coefficient R were derived. Figure 4 Substituting the dilution concentration into the linear regression equation, the estimated value and the relative or absolute deviation between the mean and the estimated value are calculated, as shown in the table below:

[0186] Table 15 Linearity of the Protein C Activity Assay Kit in Example 1

[0187]

[0188] Table 16 Linearity of the Protein C Activity Assay Kit in Example 6

[0189]

[0190] Depend on Figure 4 As shown in Tables 15 and 16, the linear regression equation for Example 1 is y = 153.89x + 6.2729, R0 2 =0.9828, with a relative deviation between -33.77% and 7.25%; the linear regression equation for Example 6 is y = 170.57x + 0.9975, R0 2 =0.9996, with a relative deviation between -7.10% and 2.57%. Example 6 exhibits better linearity than Example 1; therefore, the linear range of the kit of this invention is 9% to 170%.

[0191] 8. Example 6: Detection of Model Differences

[0192] The kit from Example 6 was calibrated on a Sysmex 5100, ACL TOP CTS, and ZK-6000 fully automated coagulation analyzer. Based on the calibration curves on each instrument, the protein C activity of 40 plasma samples was detected. The protein C activity of these plasma samples was within the range of [10, 160]%, and the results are shown in Table 17. To analyze the performance difference of the kit on these three instruments, a performance difference curve was plotted with the results of the Sysmex 5100 instrument as the x-axis and the results of the ACL TOP CTS and ZK-6000 instruments as the y-axis. Figure 5 .

[0193] Table 17 Differences in the Model of Protein C Activity Assay Kit

[0194]

[0195]

[0196] Table 17 shows that the relative deviations of the plasma protein C activity detection results using the kit of the present invention on Sysmex 5100, ACL TOP CTS, and ZK-6000 fully automated blood coagulation analyzers were all within ±10%. Figure 5 This reflects the correlation R between the reagent kit of the present invention and the detection of plasma sample protein C activity on these three instruments. 2 All values ​​are greater than 0.99, therefore the model difference of the kit of the present invention is small.

[0197] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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 composition, characterized in that, include: Protein C activator 0.2–0.5 U / mL Polyvinylpyrrolidone 0.1–1.0 w / v% L-Lysine hydrochloride 0.1–0.5 w / v%.

2. A detection reagent, characterized in that, include: The composition, chromogenic substrate reagent, and diluent as described in claim 1.

3. The detection reagent as described in claim 2, characterized in that, The chromogenic substrate reagent includes: 1.0 to 4.0 mg / L chromogenic substrate.

4. The detection reagent as described in claim 2 or 3, characterized in that, The diluent comprises: 0.05–0.50 w / v%.

5. The use of the composition of claim 1 and / or the detection reagent of any one of claims 2 to 4 in the preparation of a kit for detecting protein C activity.

6. The application as described in claim 5, characterized in that, The detection includes the following steps: the sample to be tested is mixed with the diluent, then mixed with the composition and the chromogenic substrate reagent. After the color reaction, the activity of the protein C is obtained according to the OD value.

7. The use of the composition of claim 1 and / or the detection reagent of any one of claims 2 to 4 in the preparation of a kit for detecting protein C system.

8. A test kit, characterized in that, include: The composition as described in claim 1 and / or the detection reagent as described in any one of claims 2 to 4, and acceptable adjuvants or carriers.

9. The detection kit as described in claim 8, characterized in that, The adjuvants include: Sorbitol 1.0–5.0 w / v% Trehalose 5.0–10.0 w / v% Buffer solution 30–100 mmol / L.

10. The method for preparing the detection kit as described in claim 8 or 9, characterized in that, The composition, the chromogenic substrate reagent, and the diluent were obtained separately, and the composition and the chromogenic substrate reagent were freeze-dried to obtain the detection kit.

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