Compositions, detection reagents, detection kits, and uses in making detection of coagulation factor x activity

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 in preparation of detection of coagulation factor X activity. The present application provides a composition, which comprises: plasma without coagulation factor X and a buffer system. The present application provides a kit for detecting factor X activity by coagulation method, which has the characteristics of simple operation, high sensitivity, good repeatability, good stability and strong market competition advantage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a composition, a detection reagent, a detection kit and the use in the preparation of detecting the activity of coagulation factor X. BACKGROUND

[0002] Coagulation factor X (FX) is synthesized in the liver and is a vitamin K-dependent coagulation factor. Its active site and amino-terminal region are very similar to other vitamin K-dependent coagulation factors, especially FIX. FX is in the common pathway of the intrinsic and extrinsic coagulation pathways. After activation by the intrinsic (FIXa-FVIIIa) and extrinsic (FVIIa-TF) coagulation pathways, FX is converted to activated FXa. FXa, in the presence of Ca 2+ In the presence of phospholipid membranes and FVa, FXa forms a prothrombinase complex that activates prothrombin to thrombin, which has enzymatic activity. In addition to its proteolytic activity, FXa also has esterase activity. In plasma, FXa can be inactivated by binding to antithrombin or TFPI.

[0003] Patients with reduced FX activity in plasma have severe bleeding, second only to hemophilia A and hemophilia B. Patients who are homozygous or compound heterozygous have moderate to severe bleeding tendencies, including deep hematoma, joint cavity hemorrhage, menorrhagia and postoperative bleeding. Intracranial hemorrhage in infants and newborns, even fatal gastrointestinal bleeding or umbilical cord stump bleeding. These diseases are caused by coagulation factor X deficiency or defect, so the detection of coagulation factor X activity has important clinical significance for the diagnosis and treatment of the disease.

[0004] The main method for determining coagulation factor X is the coagulation method, which is based on detecting the correction ability of the test sample on the prolongation of prothrombin time (PT) caused by factor X-deficient plasma. After mixing the diluted test plasma with factor X-deficient plasma, PT is detected, and the test results are negatively correlated with the activity of coagulation factor X in the test plasma. By using plasma with known coagulation factor X activity for serial dilution and mixing with factor X-deficient plasma, and then performing PT detection to establish a calibration curve, the activity of coagulation factor X in the test plasma can be obtained, which has the characteristics of high sensitivity and simple operation.

[0005] There are already several kits on the domestic market for the determination of factor X activity, but they are all produced abroad, with high prices, high detection costs, long delivery times, limited clinical applications, and cannot meet market demand. SUMMARY

[0006] Therefore, the application provides a composition, a detection reagent, a detection kit and an application in preparation of detection of coagulation factor X activity.

[0007] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides a composition, which comprises: plasma without coagulation factor X and a buffer system.

[0009] In some embodiments of the application, the plasma without coagulation factor X in the above-mentioned composition is provided by the German Biomex GmbH company.

[0010] In some embodiments of the application, the buffer system in the above-mentioned composition comprises: one or more of potassium caprylate, mannitol, glycylglycine, a non-ionic surfactant, a buffer and / or a preservative.

[0011] In some embodiments of the application, the pH value of the composition in the above-mentioned composition is 7.6-8.4.

[0012] In some embodiments of the application, the pH value of the composition in the above-mentioned composition is 8.0.

[0013] In some embodiments of the application, the concentration of the potassium caprylate in the above-mentioned composition is 0.1-1 mM; the concentration of the mannitol is 1-5% (w / v); and the concentration of the glycylglycine is 0.5-1.5% (w / v).

[0014] In some embodiments of the application, the concentration of the potassium caprylate in the above-mentioned composition is 0.5 mM; the concentration of the mannitol is 3% (w / v); and the concentration of the glycylglycine is 1% (w / v).

[0015] In some embodiments of the application, the concentration of the non-ionic surfactant in the above-mentioned composition is 0.01-0.1% (w / v); and the non-ionic surfactant comprises: polyoxyethylene (23) lauryl ether.

[0016] In some embodiments of the application, the concentration of the non-ionic surfactant in the above-mentioned composition is 0.05% (w / v).

[0017] In some embodiments of the application, the concentration of the buffer in the above-mentioned composition is 30-70 mM; and the buffer comprises: one or more of HEPES buffer, Tris buffer and / or phosphate buffer.

[0018] In some embodiments of the present invention, the buffer in the above composition comprises 30-70 mM HEPES buffer.

[0019] In some embodiments of the present invention, the buffer in the above composition comprises 50 mM HEPES buffer.

[0020] In some embodiments of the present invention, the concentration of the preservative in the above composition is 0.01 to 0.1% (w / v); the preservative includes one or more of sodium azide, ciprofloxacin, thimerosal, gentamicin and / or Proclin-300.

[0021] In some embodiments of the present invention, the preservative in the above composition comprises 0.01 to 0.1% (w / v) sodium azide.

[0022] In some embodiments of the present invention, the preservative in the above composition comprises 0.05% (w / v) sodium azide.

[0023] The present invention also provides a detection reagent comprising the above-described composition and acceptable adjuvants.

[0024] The present invention also provides a detection kit comprising the above-described composition and / or the detection reagents described above, as well as an acceptable carrier or device.

[0025] The present invention also provides the use of the above-described composition, the above-described detection reagent and / or the above-described detection kit in the preparation of products for detecting coagulation factor X activity.

[0026] In some embodiments of the present invention, the detection in the above applications includes the following steps:

[0027] S1: Take the pretreated test sample and mix it with the composition, the detection reagent and / or the detection kit, incubate, and obtain mixed plasma;

[0028] S2: Mix the aforementioned mixed plasma with prothrombin time assay reagent, incubate, and obtain absorbance values;

[0029] S3: Obtain the coagulation factor X activity of the sample to be tested based on the absorbance value and the standard curve.

[0030] In some embodiments of the present invention, in the above application, the pretreatment in the detection S1 includes a dilution step using a diluent; the volume ratio of the diluent to the sample to be tested is 19:1.

[0031] In some embodiments of the present invention, in the above application, the incubation temperature in the detection S1 is 37°C and the time is 2 min.

[0032] In some embodiments of the present invention, in the above applications, the volume ratio of the test sample to the composition, the detection reagent and / or the detection kit in the detection S1 is 1:1.

[0033] In some embodiments of the present invention, in the above application, the volume ratio of the mixed plasma to the prothrombin time assay reagent in the detection S2 is 1:1.

[0034] In some embodiments of the present invention, in the above application, the incubation temperature in the detection S2 is 37°C and the time is 3 min.

[0035] The present invention provides a composition comprising: plasma free of coagulation factor X and a buffer system.

[0036] The beneficial effects of this invention include:

[0037] (1) The presence of potassium octanoate and polyoxyethylene (23) lauryl ether in the plasma of factor X deficiency described in this invention can effectively improve the sensitivity of the kit of this invention.

[0038] (2) The factor X-deficient plasma of the present invention contains both mannitol and glycine, which can effectively reduce the impact of the freeze-drying process on factor X-deficient plasma.

[0039] (3) The factor X deficiency plasma of the present invention contains both glycyl glycine and polyoxyethylene (23) lauryl ether, which can effectively improve plasma stability. Attached Figure Description

[0040] 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.

[0041] Figure 1 This paper presents a correlation analysis between the reagent kit of the present invention and similar reagent kits already on the market. Detailed Implementation

[0042] This invention discloses compositions, detection reagents, detection kits, and their use in the preparation of methods for detecting coagulation factor X activity.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] This invention provides a coagulation factor X assay kit, the kit components of which are coagulation factor X deficient plasma. The coagulation factor X deficient plasma in this kit includes potassium caprylate, mannitol, glycyl glycine, polyoxyethylene (23) lauryl ether, preservatives, and buffers. The concentration of potassium caprylate is 0.1–1 mM, the concentration of mannitol is 1–5%, the concentration of glycyl glycine is 0.5–1.5%, the concentration of polyoxyethylene (23) lauryl ether is 0.01–0.1%, and the pH range is 7.6–8.4.

[0049] The preservative in the coagulation factor X deficient plasma of the present invention is one of sodium azide, ciprofloxacin, thimerosal, gentamicin or Proclin-300. In some embodiments, the preservative is sodium azide at a concentration of 0.01% to 0.1%.

[0050] The buffer for coagulation factor X-deficient plasma described in this invention is selected from one or more of HEPES, Tris, and phosphate buffer. In some embodiments, the buffer is HEPES buffer, and the concentration of HEPES in the buffer is 30–70 mM.

[0051] In some embodiments, the concentration of potassium caprylate in coagulation factor X-deficient plasma is 0.5 mM, the concentration of mannitol is 3%, the concentration of glycyl glycine is 0.5-1.5%, preferably 1%, the concentration of polyoxyethylene (23) lauryl ether is 0.01-0.1%, preferably 0.05%, the concentration of sodium azide is 0.05%, the concentration of HEPES is 50 mM, and the pH is 8.0.

[0052] The present invention also provides specific steps for detecting the activity of coagulation factor X in a sample using the above-mentioned kit:

[0053] (1) Dilute the plasma sample with coagulation analysis diluent at a ratio of 1:20, pipette it into a test tube, and preheat it at 37°C.

[0054] (2) Coagulation factor X deficient plasma and diluted plasma sample were mixed at a volume ratio of 1:1, mixed at 37°C and incubated for 2 minutes;

[0055] (3) Add the prothrombin time (PT) assay reagent and mix it with the mixed plasma obtained in step (2) at a volume ratio of 1:1, react at 37°C for 3 minutes, and measure the coagulation time.

[0056] (5) The activity of factor X in the plasma sample was calculated based on the known standard curve.

[0057] In Examples 1 to 7 of this invention, all raw materials and reagents used can be purchased from the market.

[0058] The present invention will be further illustrated below with reference to the embodiments:

[0059] Example 1: Preparation of the reagent kit and control experimental group of the present invention

[0060] (1) The reagent kit of the present invention

[0061] The plasma used in this invention, which is free of coagulation factor X, is human plasma supplied by Biomex GmbH, Germany. The buffer system is added to the plasma free of coagulation factor X and mixed thoroughly to a pH of 8.0. The kit formulation of this invention is shown in Table 1.

[0062] Table 1. Formulation of plasma buffer system for factor X deficiency (experimental group)

[0063] Component Amount HEPES 50 mM Potassium octanoate 0.5 mM Polyoxyethylene (23) lauryl ether 0.05％ Glycylglycine 1％ Mannitol 3％ Sodium azide 0.05％

[0064] (2) Control group

[0065] Table 2. Plasma formulation for factor X deficiency (control group)

[0066]

[0067]

[0068] Example 2 Repeatability Test

[0069] Factor X-deficient plasma from the kit of this invention (experimental group) and commercially available kits (main ingredient: 20 g / L mannitol) were used to detect normal value control plasma and abnormal value control plasma, respectively. The tests were repeated 10 times, and the coefficient of variation (CV) was calculated.

[0070] Table 3 shows that the coefficient of variation (CV) of the experimental group was significantly lower than that of the commercially available kits, indicating that the reproducibility of the coagulation factor X assay kit (coagulation method) of this invention is good and significantly better than that of commercially available kits.

[0071] Table 3 Repeatability Test Results

[0072]

[0073]

[0074] Example 3 Detection Limit Test

[0075] The samples were diluted (L) with diluent to obtain 11 low-concentration samples (0.1L–0.9L). The low-concentration samples were tested for three consecutive days using the kit of this invention (experimental group) and control groups 1–4 (plasma lacking coagulation factor X). Each sample was tested 10 times daily, and the number of positive results (i.e., the number of results with non-zero activity) was counted. The positive rate was calculated, and the results are shown in Table 4. The activity measurement results of the low-concentration samples were logarithmically transformed using log10 as the x-axis and the positive rate as the y-axis for fitting. The measured activity corresponding to a positive rate of 95% was calculated based on the fitted equation; this value is the limit of detection for that plasma. The calculated limits of detection for the experimental group and control groups 1–4 were 1.0%, 4.5%, 6.0%, 4.5%, and 4.0%, respectively.

[0076] In addition, five low-value samples with concentrations of approximately 1.0% (samples 1-5), 4.0% (samples 6-10), 4.5% (samples 11-15), and 6.0% (samples 16-20) were prepared for testing. Each sample was tested five times, and the results are shown in Table 5.

[0077] Tables 4 and 5 illustrate that the presence of both potassium octanoate and polyoxyethylene (23) lauryl ether in coagulation factor X-deficient plasma can lower the detection limit of the kit of this invention, effectively improving its detection sensitivity. The detection limit of the kit of this invention is 1.0%, lower than the 6.2% detection limit stated in the instructions of commercially available kits. When other nonionic surfactants are used to replace polyoxyethylene (23) lauryl ether, their sensitivity is not as good as that of the kit of this invention.

[0078] Table 4 Detection Limit Results

[0079]

[0080]

[0081] Table 5 Detection Limit Verification Results

[0082]

[0083]

[0084]

[0085] Example 4: Study on the protective effect of freeze-drying

[0086] The coagulation factor X-deficient plasma from the kit of this invention (experimental group) and control groups 5-6 were lyophilized. Normal and abnormal values ​​of the quality control plasma were measured before and after lyophilization. Simultaneously, the activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen (FIB) levels of the plasma were measured. The measurements were repeated three times, and the mean and difference of the results before and after lyophilization were calculated. Table 6 shows that the difference in the measured values ​​of the quality control samples before and after lyophilization in the experimental group was significantly lower than that in the control group 5-6. Furthermore, the differences in APTT, PT, and FIB levels in the experimental group plasma before and after lyophilization were smaller, and the lyophilization attenuation was significantly lower than that in the control group 5-6. This indicates that the presence of glycylglycine and mannitol in the coagulation factor X-deficient plasma can effectively reduce the impact of the lyophilization process on the coagulation factor X-deficient plasma and protect the activity of various coagulation factors and fibrinogen in the plasma.

[0087] Table 6. Test results before and after freeze-drying

[0088]

[0089]

[0090] Example 5: Stabilizer Concentration Screening

[0091] Plasma samples lacking coagulation factor X from the kit of this invention (experimental group) and control group 7-10 were freeze-dried. The freeze-dried samples were reconstituted with 1 mL of distilled water and stored at +18 to +25°C. Tests were performed at 0 h, 5 h, 15 h, and 20 h after reconstitution. Each test involved testing with normal and abnormal control plasma samples, repeated three times. The mean of the test results was calculated, and the relative deviation from 0 h was also calculated for each time point. Table 7 shows that when the glycylglycine concentration was 0.5-1.5% and the polyoxyethylene (23) lauryl ether concentration was 0.01-0.1%, the relative deviation from 0 h was less than 10% at each time point. Specifically, the relative deviation was minimized when the glycylglycine concentration was preferably 1.0% and the polyoxyethylene (23) lauryl ether concentration was 0.05%.

[0092] Table 7. Detection results of stabilizers at different concentrations

[0093]

[0094]

[0095] Example 6 Reconstitution Stability Test

[0096] Plasma samples lacking coagulation factor X from the kit of this invention (experimental group), control group 2, and control group 5 were freeze-dried. The freeze-dried products were reconstituted with 1 mL of distilled water and stored at +18 to +25°C. Measurements were performed every 2 hours after reconstitution. Each measurement involved testing with both normal and abnormal control plasma samples, repeated three times, and the mean value was calculated. Linear regression was performed on the results, and a two-tailed t-test was conducted to assess the significance of the slope b versus 0. From the table, α = 0.05, ν = 11, and tα(ν) = 2.201. The p-values ​​for the activity assay results of the kit of this invention were all greater than 0.05, which fully demonstrates that the activity of the freeze-dried products from the kit of this invention remained essentially unchanged after 24 hours at +18 to +25°C, with a stability time longer than the 8-hour stability time stated in the instructions of commercially available kits, indicating good usability. The results of the test kit of the present invention, compared with those of control groups 2 and 5, fully demonstrate that the presence of glycyl glycine and polyoxyethylene (23) lauryl ether in factor X-deficient plasma can effectively improve the stability of enzyme activity during the reconstitution of factor X-deficient plasma.

[0097] Table 8 Results of Reconstitution Stability Test

[0098]

[0099]

[0100] Example 7: Comparison of the reagent kit of the present invention with similar commercially available reagent kits.

[0101] One hundred clinical samples were randomly selected from inpatients and outpatients at Shanghai Yangsi Hospital. Blood was mixed with 0.11 mol / L sodium citrate anticoagulant at a ratio of 9:1, centrifuged at 1500g for 15 minutes, and plasma was obtained. The clinical samples were measured on a CS5100 using the kit obtained in Example 1 (experimental group) and the Siemens Factor X Activity Assay Kit (coagulation method), respectively. The correlation coefficient between the two kits was calculated, and linear regression was performed. The results showed that the correlation coefficient r = 0.99522 for the sample measurements on the CS5100, and the linear regression equation was y = 1.01463x - 0.39963. Figure 1 As shown.

[0102] 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 Siemens, Germany, show good consistency.

[0103] 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. Composition, characterized in that, Compositions comprising: plasma free of coagulation factor X and a buffer system; said buffer system comprising: 0.5 mM potassium octanoate, 3% (w / v) mannitol, 1% (w / v) glycylglycine, 0.05% (w / v) polyoxyethylene (23) lauryl ether, 50 mM HEPES and 0.05% (w / v) sodium azide.

2. A test reagent, characterized by Compositions as claimed in claim 1, together with acceptable adjuvants.

3. A test kit characterized in that, Compositions as claimed in claim 1 and / or detection reagents as claimed in claim 2, together with acceptable carriers or devices.

4. Use of compositions as claimed in claim 1, detection reagents as claimed in claim 2 and / or kits as claimed in claim 3 for the preparation of a product for detecting the activity of coagulation factor X.

5. The use according to claim 4, wherein the compound is ###0002### Said detection comprising the following steps: S1 : mixing the pretreated sample to be tested with the composition, the detection reagent and / or the kit, incubating, obtaining a mixed plasma; S2: mixing the mixed plasma with a prothrombin time assay reagent, incubating, obtaining an absorbance value; S3: obtaining the activity of coagulation factor X of the sample to be tested from the absorbance value and a standard curve.

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