Antithrombin III determination kit and application thereof

Abstract

The invention discloses an antithrombin III determination kit and an application thereof. The kit is composed of the following components: a reagent R1 is a buffer solution component and comprises a buffer solution, sodium chloride, propyl alcohol, BSA and a preservative; the reagent R2 is a chromogenic substrate component and comprises a heparin-AT compound, thrombin, a specific chromogenic substrate S-2238, a novel stable peptide A, a stabilizer and a standard substance. The stable peptide A is innovatively adopted to improve the stability of the reagent, and the AT-III activity in the plasma is indirectly quantified by measuring the hydrolysis reaction absorbance change of the specific chromophoric substrate S-2238 at the wavelength of 405 nm. The kit has the remarkable technical effects that the reagent stability is excellent, the detection specificity is high, the anti-interference capability is strong, the operation is convenient, the linear range is wide (0.5-12 IU / ml), the sensitivity is improved by 30% compared with the prior art, and the clinical conventional detection requirements can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of medical diagnosis, and particularly relates to a kit for antithrombin III activity and its application. Background Art

[0002] Antithrombin III (AT-III) is the most important anticoagulant protein in the human body and is also a key factor involved in regulating the blood coagulation system. AT-III is mainly synthesized by the liver, is a single-chain glycoprotein with a molecular weight of about 58,000 daltons, and exists in plasma. Its main function is to bind to coagulation factors such as thrombin and activated coagulation factor X, thereby inhibiting the coagulation process. Low AT-III levels may lead to a tendency to form thrombi and are associated with a variety of clinical diseases, including disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), pulmonary embolism (PE), pregnancy complications, and liver diseases, etc.

[0003] Currently, the commonly used methods for AT-III determination in clinical practice mainly include the coagulation method and the chromogenic substrate method. The principle of the coagulation method is to use heparin as a catalyst to accelerate the binding of AT-III to thrombin, thereby prolonging the coagulation time. However, this method is greatly affected by other factors in the blood sample and has low precision. The chromogenic substrate method reacts a specific chromogenic substrate with thrombin and indirectly determines the activity of AT-III based on the color change of the hydrolysis product of the substrate, and has the advantages of high specificity and good sensitivity.

[0004] However, the existing AT-III determination kits have the following problems: First, the reagent stability is poor, and the activity loss is serious during transportation and storage; second, the determination steps are complex and the operation is cumbersome; third, the determination results are interfered by various factors, resulting in unsatisfactory accuracy and repeatability; fourth, the components of the kit are not optimized enough, resulting in unstable reaction conditions. Therefore, it is of important clinical application value to develop an AT-III determination kit with good stability, simple operation, and accurate and reliable determination results. Summary of the Invention

[0005] The object of the present invention is to provide an antithrombin III determination kit with good stability, high specificity, strong sensitivity, and simple operation, so as to solve the above problems existing in the prior art.

[0006] The present invention provides an antithrombin III assay kit, and the innovative points are as follows: 1) The reagent formulation is optimized, especially by adding a special new stabilizing peptide A, which significantly improves the stability of the reagent; 2) The ratio of heparin-AT complex to thrombin is improved, making the reaction faster and more specific; 3) By using the improved chromogenic substrate S-2238 concentration and reaction conditions, the detection sensitivity is increased by about 30% compared with the traditional method, and the lower detection limit can reach 0.5 IU / ml; 4) The volume ratio of reagent R1 to R2 is innovatively optimized to 5:1, which not only improves the reaction efficiency but also reduces the sample consumption during the determination; 5) The upper limit of the linear range of high-concentration samples is increased to 12 IU / ml, making the detection results more accurate and reliable.

[0007] To achieve the above object, the present invention provides the following technical solutions: An antithrombin III assay kit, comprising: Reagent R1: A buffer solution containing 50 mM Tris-HCl buffer (pH 7.4 ± 0.2), sodium chloride, propanol, BSA and a preservative; Reagent R2: A chromogenic substrate containing a heparin-AT complex, thrombin, and a specific chromogenic substrate S-2238, stabilizing peptide A and a stabilizer; Among them, the volume ratio of the reagent R1 to the reagent R2 is 5:1; Standard solution: Containing antithrombin III with known activity, and the activity range is 2 - 10 IU / ml.

[0008] Preferably, the concentration of Tris-HCl buffer in the reagent R1 is 50 mM, the pH value is 7.4 ± 0.2; the concentration of sodium chloride is 7.3 g / L; the concentration of propanol is 2%; the concentration of BSA is 0.5%, and the preservative is sodium azide with a concentration of 0.02%.

[0009] Wherein the percentages are mass percentages.

[0010] Preferably, in the reagent R2, the stabilizing peptide A has a mass concentration of 10%, and the sequence is: LFEGLRPLFEKKSLEDKTERELLRELRED; The stabilizer is a mixture containing polyethylene glycol 6000, glycerol, dithiothreitol and sodium EDTA, with a total concentration of 0.2%; the concentration of heparin-AT complex is 0.3 U / ml; the mass concentration of thrombin is 0.2 U / ml.

[0011] Preferably, the chemical name of the specific chromogenic substrate S-2238 in the reagent R2 is D-phenylalanine-piperidine-arginine-p-nitroaniline, and its mass concentration is 5%; this chromogenic substrate has a high reaction sensitivity with the AT-III-heparin-thrombin complex, can detect the AT-III activity as low as 0.5 IU / ml, and the sensitivity is increased by about 30% compared with the prior art. Preferably, the AT-III activity range of the standard solution is 2-10 IU / ml, which is suitable for the determination of normal plasma samples. For patients with coagulation disorders, it can be diluted to the range of 1-10 IU / ml for determination.

[0012] The present invention also provides a preparation method of the above-mentioned antithrombin III assay kit, which includes the following steps: (1) Preparation of reagent R1: Dissolve Tris-HCl in deionized water, adjust the pH value to 7.4±0.2, add sodium chloride, propanol, BSA and preservatives, make up the volume with deionized water, mix well, filter and sterilize, and dispense; (2) Preparation of reagent R2: Dissolve the heparin-AT complex in an appropriate amount of deionized water, add thrombin and chromogenic substrate S-2238, add stable peptide A and stabilizer, make up the volume with deionized water, mix well, filter and sterilize, and dispense; (3) Preparation of standard solution: Dissolve the freeze-dried powder of human plasma AT-III with known activity in deionized water to the required concentration, and dispense.

[0013] The present invention also provides the application of the above-mentioned antithrombin III assay kit in the determination of antithrombin III activity in human plasma, including the following determination steps: (1) Sample collection and treatment: Collect venous blood from the subject, anticoagulate it with 3.8% sodium citrate (1:9), centrifuge at 3000 rpm for 15 min, and take the upper plasma as the test sample; (2) Preparation before determination: Warm the reagent R1, reagent R2 and the sample to room temperature (15-25°C); (3) Determination operation: a) Add an appropriate amount of reagent R1, sample and standard to the determination wells, mix well, and incubate at 37°C for 3 minutes; b) Add an appropriate amount of reagent R2, mix well, and incubate at 37°C for 3 minutes; c) Measure the absorbance at a wavelength of 405 nm; (4) Result calculation: Draw a standard curve according to the known activity and absorbance value of the standard, and find the corresponding AT-III activity from the absorbance value of the sample on the standard curve.

[0014] The beneficial effects of the present invention: 1) The kit provided by the present invention has good stability. The R1 and R2 reagents can be stably stored at 2 - 8 °C for at least 12 months, and the lyophilized standard can be stably stored at 2 - 8 °C for at least 24 months. Compared with traditional kits, by adding a special stabilizer formulation, the reagent stability of the present invention is increased by more than 40%; 2) The present invention uses a specific chromogenic substrate S - 2238, and can directly measure the activity of AT - III by the change in absorbance at a wavelength of 405 nm, avoiding the interference factors of traditional coagulation methods, with high specificity and good sensitivity; 3) The operation steps of the present invention are simple and the degree of automation is high, and it can be applied to a variety of fully automatic coagulation analyzers, such as Sysmex CA series, STAGO series, ACL series, etc.; 4) By optimizing the proportion of reagent components, the present invention reduces the possible interference factors in plasma, such as hemoglobin (≤7 g / L), bilirubin (≤200 mg / L), and lipemia (≤6.5 g / L), etc., on the measurement results, with good accuracy and repeatability; 5) The method provided by the present invention has a wide linear range (0.5 - 12 IU / ml), can meet the needs of measuring the activity of AT - III in normal populations and patients in pathological states, its sensitivity is increased by about 30% compared with similar products on the market, and the upper limit of high - concentration sample measurement is increased by 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the binding of the novel stable peptide A to the heavy chain of thrombin; Figure 2 It is the conformational change caused by the binding of the novel stable peptide A to the heavy chain of thrombin; Figure 3 It is a comparison chart of the kit of the present invention and imported kits. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0017] Example 1 Preparation of Antithrombin III Assay Kit This example provides an antithrombin III assay kit, including: Reagent R1: 50 mM Tris - HCl buffer (pH 7.4), 7.3 g / L sodium chloride, 2% propanol, 0.5% BSA, 0.02% sodium azide (preservative); Reagent R2: Heparin-AT complex (0.3 U / ml), thrombin (0.2 U / ml), 5% specific chromogenic substrate S-2238, 0.2% stabilizer mixture (containing polyethylene glycol, glycerol, dithiothreitol and sodium EDTA), 10% stable peptide A sequence (LFEGLRPLFEKKSLEDKTERELLRELRED); Figure 1 Shows the binding mode of the novel stable peptide A (represented by the protein backbone) to the heavy chain of thrombin (represented by gray simulation). The red area marks the binding region between the two.

[0018] Figure 2 Shows the conformational changes of the novel stable peptide A before and after binding to the heavy chain of thrombin. Gray represents the novel stable peptide A and the heavy chain of thrombin in the unbound state. Red and yellow respectively represent the conformational changes that occur after the novel stable peptide A binds to the heavy chain of thrombin.

[0019] Standard solution: Lyophilized human plasma standard with an AT-III activity of 5.0 IU / ml. In addition, calibrators of 0.5 IU / ml and 12.0 IU / ml are also provided to verify the low-concentration and high-concentration detection sensitivities of the kit.

[0020] The volume ratio of reagent R1 to reagent R2 is 5:1.

[0021] The preparation method is as follows: 1) Preparation of reagent R1: Weigh 6.057 g of Tris-HCl buffer and 4.3 g of sodium chloride, dissolve them in 800 ml of deionized water, adjust the pH to 7.4 with 1 mol / L hydrochloric acid, add 20 ml of propanol and 5 g of BSA, stir until completely dissolved, add 0.2 g of sodium azide, make up the volume to 1000 ml with deionized water, sterilize by passing through a 0.22 μm filter membrane, dispense into brown bottles, and store at 4°C; 2) Preparation of reagent R2: Take 15 U of heparin-AT complex and 10 U of thrombin, dissolve them in 80 ml of deionized water, add 5 g of the chromogenic substrate S-2238 powder, slowly add 10 g of stable peptide A and a special stabilizer mixture (0.05 g of polyethylene glycol 6000, 0.05 g of glycerol, 0.05 g of dithiothreitol and 0.05 g of sodium EDTA) under continuous stirring to ensure the optimal stability of the chromogenic substrate and the enzyme, adjust the pH to 7.2 ± 0.1, make up the volume to 100 ml with deionized water, sterilize by passing through a 0.22 μm filter membrane, dispense into brown bottles, and store at 4°C in the dark; 3) Preparation of the standard: Take the pooled human plasma with a confirmed AT-III activity of 5.0 IU / ml, filter it through a 0.22 μm filter membrane and then perform lyophilization, dispense it into glass bottles, seal, and store at 4°C.

[0022] Example 2 Method for Determining Antithrombin III Activity The method for determining AT-III activity using the kit prepared in Example 1 is as follows: 1) Sample collection and processing: Collect 3 ml of venous blood from the subject, add 3.8% sodium citrate (the ratio of blood to anticoagulant is 9:1) for anticoagulation, mix well, centrifuge at 3000 rpm for 15 min, and take the supernatant plasma as the test sample; 2) Reagent preparation: Take out reagent R1, reagent R2, and the standard from the refrigerator and place them at room temperature (15 - 25°C) for 30 min; dissolve the standard with the diluent provided with the kit and let it stand for 10 min; 3) Instrument settings: Taking the Sysmex CA-7000 fully automatic coagulation analyzer as an example, set the following parameters according to the instrument operation manual: Test item: AT-III activity; Detection wavelength: 405 nm; Reaction temperature: 37°C; Determination type: kinetic method; 4) Determination steps: a) Place reagent R1, reagent R2, the standard, and the sample at the designated positions on the instrument; b) The instrument automatically aspirates 100 μl of reagent R1 and 20 μl of the sample (or the standard), mixes well, and incubates at 37°C for 3 minutes; c) The instrument automatically adds 20 μl of reagent R2, mixes well, and incubates at 37°C for exactly 3 minutes; d) The instrument continuously measures the change in absorbance at a wavelength of 405 nm and calculates △A / min; e) Establish a standard curve based on the known activity of the standard and the △A / min value, and calculate the AT-III activity of the sample through the △A / min value of the sample.

[0023] Example 3 Performance Verification of the Kit Perform the following performance verification on the antithrombin III determination kit prepared in Example 1: 1) Precision test: Intra-batch precision: Continuously measure the same normal plasma sample 20 times, CV < 3.0%; The specific data is as follows: Table 1 Results of Intra-batch Precision Experiment

[0024] Inter-batch precision: Continuously measure the same normal plasma sample 2 times every day for 10 days, CV < 5.0%; The specific data is as follows: Table 2 Results of Inter-batch Precision Experiment

[0025] 2) Accuracy test: The AT-III international standard (WHO IS 96 / 854) was determined, and the relative deviation between the measurement result and the labeled value of the standard was < 10%; Table 3 Results of accuracy experiment

[0026] 3) Linear range: In the range of 0.5 - 12 IU / ml, the measurement result showed a good linear relationship with the theoretical activity of the sample, r > 0.998; Table 4 Results of linear experiment

[0027] 4) Reference interval verification: Plasma samples of 200 healthy individuals were measured. The reference interval for AT-III activity was 2.5 - 3.7 IU / ml, which was consistent with the literature reports; Table 5 Results of reference interval verification experiment

[0028] Continued Table 5

[0029] The results analysis is shown in Table 6: Table 6 Results analysis of verification

[0030] 5) Interference test: The following substances were added to normal plasma respectively, and the AT-III activity was measured: a) Hemoglobin (final concentration 7 g / L): The influence on the measurement result was < 10%; b) Bilirubin (final concentration 200 mg / L): The influence on the measurement result was < 8%; c) Fat emulsion (final concentration 6.5 g / L): The influence on the measurement result was < 12%; The results are as follows: Table 7 Results of interference test

[0031] 6) Stability test: a) Reagents R1 and R2 were stored at 2 - 8 °C for 12 months, and the activity retention rate was > 90%; b) The standard was stored at 2 - 8 °C for 24 months, and the activity retention rate was > 95%; c) The reagent was stable for 45 days at 2 - 8 °C after opening the bottle;

[0032] Table 8 Results of stability test

[0033] The test results show that the antithrombin III assay kit provided by the present invention has good precision, accuracy, linear range, anti-interference ability and stability, meeting the clinical detection requirements.

[0034] Example 4 Clinical Application Verification Collect plasma samples from 100 healthy physical examination subjects and 100 patients diagnosed with deep vein thrombosis (DVT). Use the kit of the present invention and a certain imported commercial kit (Anti-Thrombin III Assay Kit (Chromogenic Substrate Method) Berichrom Antithrombin Ⅲ (A) registered by Siemens Medical Solutions Diagnostics GmbH, Germany, registration number: CFDA import registration number 20152401859) to measure the activity of AT-III, as shown in Figure 3 and Table 9. The results are analyzed as follows: 1) From Figure 3 it can be seen that the correlation between the two methods is: r = 0.989, indicating that the kit of the present invention has a good correlation with the imported commercial kit; Table 9 Comparison between the kit of the present invention and the imported commercial kit

[0035] Continued Table 9

[0036] Continued Table 9

[0037] 2) Comparison of AT-III activity between the healthy group and the DVT patient group: Healthy group: 3.17 ± 0.32 IU / ml; DVT patient group: 1.44 ± 0.53 IU / ml; The difference between the two groups was statistically significant (P < 0.01), proving that the measurement of AT-III activity has clinical significance for the diagnosis of DVT; 3) Comparison of measurement time: Kit of the present invention: average 7.5 minutes / sample; Imported commercial kit: average 12.3 minutes / sample; The kit of the present invention can save about 40% of the measurement time.

[0038] The above examples prove that the anti-thrombin III assay kit provided by the present invention has good diagnostic performance in clinical applications, is easy to operate, has a short measurement time, and is suitable for the needs of clinical routine tests.

[0039] Example 5 Influence of the novel stabilizing peptide A on the stability of the reagent To verify the influence of the novel stabilizing peptide A on the stability of the reagent, the following comparative tests were carried out: 1) Preparation of the test group and the control group: a) Test group: Prepare a kit containing 10% novel stabilizing peptide A according to the method of Example 1; b) Control group: Prepare a kit without novel stabilizing peptide A, and other components are the same as those of the test group; 2) Stability investigation method: The reagents of the test group and the control group were stored under the following conditions respectively: a) stored sealed at 2 - 8°C; b) accelerated test at 37°C; c) stability test after opening at room temperature (25°C). 3) Test results: a) Stability under the condition of storing sealed at 2 - 8°C: Test group: The activity retention rate > 90% after 12 months; Control group: The activity retention rate decreased to 75% after 6 months and decreased to 60% after 12 months. b) Results of the accelerated test at 37°C: Test group: The activity retention rate > 85% after 7 days; Control group: The activity retention rate decreased to 65% after 7 days. c) Stability after opening at room temperature: Test group: The activity retention rate > 85% after 45 days; Control group: The activity retention rate decreased to 70% after 15 days. 4) Conclusion: The addition of the novel stable peptide A significantly improved the stability of the reagent, which was mainly reflected in: a) extending the shelf life of the reagent from 6 months to 12 months; b) improving the stability of the reagent under stress conditions; c) significantly improving the stability after opening, and the service life was extended from 15 days to 45 days. These improvements significantly enhanced the practicality and economy of the kit.

Claims

1. An antithrombin III assay kit, characterized in that, Comprising: Reagent R1, reagent R2 and standard solution; Reagent R1 is a buffer solution: containing 50 mM Tris-HCl buffer, pH 7.4 ± 0.2, 7.3 g / L sodium chloride, 2% propanol, 0.5% BSA and 0.02% preservative; Reagent R2 is a chromogenic substrate: containing 0.3 U / ml heparin-AT complex, 0.2 U / ml thrombin, 5% specific chromogenic substrate S-2238, 10% stable peptide A and 0.2% stabilizer; The stable peptide A in reagent R2 has the sequence: LFEGLRPLFEKKSLEDKTERELLRELRED; Among them, the volume ratio of reagent R1 to reagent R2 is 5:1, and the percentages in reagent R1 and reagent R2 are mass percentages; Standard solution: Antithrombin III, with an activity range of 0.5 - 12 IU / ml.

2. The antithrombin III assay kit according to claim 1, wherein The preservative in reagent R1 is sodium azide; the stabilizer in reagent R2 is a mixture, containing polyethylene glycol 6000, glycerol, dithiothreitol and sodium EDTA.

3. The antithrombin III assay kit according to claim 1, characterized in that, The specific chromogenic substrate S-2238 is D-phenylalanine-piperidine-arginine-p-nitroaniline.

4. The antithrombin III assay kit according to claim 1, wherein The standard solution is a freeze-dried human plasma standard.

5. A method for preparing an antithrombin III assay kit according to any one of claims 1-4, characterized in that, Including the following steps: (1) Preparation of reagent R1: Dissolve Tris-HCl in deionized water, adjust the pH value to 7.4 ± 0.2, add sodium chloride, propanol, BSA and preservative, make up the volume with deionized water, mix well, filter through bacteria, and dispense; (2) Preparation of reagent R2: Dissolve the heparin-AT complex in an appropriate amount of deionized water, add thrombin and chromogenic substrate S-2238, add stable peptide A and stabilizer, make up the volume with deionized water, mix well, filter through bacteria, and dispense; (3) Preparation of standard solution: Dissolve the freeze-dried powder of human plasma AT-III with known activity in deionized water to the required concentration, dispense, and perform freeze-drying treatment.

6. Use of an antithrombin III assay kit according to any one of claims 1-4 for determining the activity of antithrombin III in human plasma, characterized in that, Including the following determination steps: (1) Sample collection and treatment: Collect venous blood from the subject, anticoagulate with 3.8% sodium citrate, centrifuge at 3000 rpm for 15 min, and take the upper plasma as the test sample; (2) Preparation before determination: Warm reagent R1, reagent R2 and the sample to room temperature; (3) Determination operation: a) Add an appropriate amount of reagent R1, sample and standard to the determination well, mix well, incubate at 37°C for 3 minutes; b) Add an appropriate amount of reagent R2, mix well, incubate at 37°C for 3 minutes; c) Measure the absorbance at a wavelength of 405 nm; (4) Result calculation: Draw a standard curve based on the known activity and absorbance value of the standard, and find the corresponding AT-III activity from the absorbance value of the sample on the standard curve.

7. The application according to claim 6, wherein The determination operation can be carried out on a fully automatic coagulation analyzer, and the fully automatic coagulation analyzer is selected from one of the Sysmex CA series, STAGO series, ACL series or Behnk Elektronik Thrombolyzer series.

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