Antithrombin III assay kit and its application

Abstract

The present invention discloses an antithrombin III assay kit and its application. The kit is composed of the following components: reagent R1 is a buffer component, comprising a buffer, sodium chloride, propanol, BSA and a preservative; reagent R2 is a chromogenic substrate component, comprising a heparin-AT complex, thrombin, a specific chromogenic substrate S-2238, a novel stable peptide A and a stabilizer, and a standard. The present invention innovatively uses stable peptide A to improve reagent stability, and indirectly quantifies AT-III activity in plasma by measuring the absorbance change of the hydrolysis reaction of the specific chromogenic substrate S-2238 at a wavelength of 405nm. The present invention has significant technical effects: excellent reagent stability, high detection specificity, strong anti-interference ability, convenient operation, wide linear range (0.5-12 IU / ml), 30% higher sensitivity than the prior art, and can meet the needs of routine clinical detection.

Description

Technical Field

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

[0002] Antithrombin III (AT-III) is the most important anticoagulant protein in the human body and a key factor involved in regulating the blood coagulation system. AT-III is primarily synthesized by the liver and is a single-chain glycoprotein with a molecular weight of approximately 58,000 daltons. It is present in plasma. Its primary 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 thrombotic tendencies and are associated with a variety of clinical conditions, including disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), pulmonary embolism (PE), pregnancy complications, and liver disease.

[0003] Currently, commonly used clinical methods for measuring AT-III include the coagulation method and the chromogenic substrate method. The coagulation method uses heparin as a catalyst to accelerate the binding of AT-III to thrombin, thereby prolonging clotting time. However, this method is significantly affected by other factors in the blood sample and has low accuracy. The chromogenic substrate method indirectly measures AT-III activity by reacting a specific chromogenic substrate with thrombin, based on the color change of the substrate hydrolysis product. It has the advantages of high specificity and good sensitivity.

[0004] However, existing AT-III assay kits have the following issues: first, the reagents are unstable, with significant activity loss during transportation and storage; second, the assay steps are complex and cumbersome; third, the assay results are subject to interference from multiple factors, resulting in suboptimal accuracy and reproducibility; and fourth, the kit components are not optimized, leading to unstable reaction conditions. Therefore, developing an AT-III assay kit with excellent stability, simple operation, and accurate and reliable results is of great clinical value. Summary of the Invention

[0005] The purpose of the present invention is to provide an antithrombin III assay 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, the innovations of which are: 1) the reagent formula is optimized, in particular, the stability of the reagent is significantly improved by adding a special new stabilizing peptide A; 2) the ratio of heparin to thrombin is improved, making the reaction more rapid and specific; 3) an improved chromogenic substrate S-2238 concentration and reaction conditions are adopted, which increases the detection sensitivity by approximately 30% compared with traditional methods, and the detection limit can reach 0.5 IU / ml; 4) the volume ratio of reagents R1 and R2 is innovatively optimized to 5:1, which not only improves the reaction efficiency but also reduces sample consumption during the assay; 5) the upper limit of the linear range of high-concentration samples is increased to 12 IU / ml, making the test results more accurate and reliable.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] An antithrombin III assay kit comprising:

[0009] Reagent R1: buffer containing 50 mM Tris-HCl buffer (pH 7.4 ± 0.2), sodium chloride, propanol, BSA and preservatives;

[0010] Reagent R2: chromogenic substrate, including heparin, thrombin, specific chromogenic substrate S-2238, stabilized peptide A, and stabilizer;

[0011] Wherein, the volume ratio of the reagent R1 to the reagent R2 is 5:1;

[0012] Standard solution: Contains antithrombin III with known activity ranging from 2-10 IU / ml.

[0013] 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 at a concentration of 0.02%.

[0014] The percentages stated are percentages by mass.

[0015] Preferably, the stable peptide A in the reagent R2 has a mass concentration of 10% and a sequence of:

[0016] LFEGLRPLFEKKSLEDKTERELLRELRED;

[0017] The stabilizer is a mixture containing polyethylene glycol 6000, glycerol, dithiothreitol and EDTA sodium salt, with a total concentration of 0.2%; the concentration of heparin is 0.3 U / ml; and the mass concentration of thrombin is 0.2 U / ml.

[0018] Preferably, the specific chromogenic substrate S-2238 in reagent R2 is chemically named D-phenylalanine-piperidine-arginine-p-nitroaniline and has a mass concentration of 5%. This chromogenic substrate reacts with the AT-III-heparin-thrombin complex with high sensitivity, capable of detecting AT-III activity as low as 0.5 IU / ml, representing a sensitivity improvement of approximately 30% compared to existing technologies. Preferably, the AT-III activity of the standard solution is in the range of 2-10 IU / ml, suitable for assaying normal plasma samples. For patients with coagulation disorders, the standard solution can be diluted to a range of 1-10 IU / ml for assay.

[0019] The present invention also provides a method for preparing the antithrombin III assay kit, comprising the following steps:

[0020] (1) Prepare reagent R1: Dissolve Tris-HCl in deionized water, adjust the pH to 7.4 ± 0.2, add sodium chloride, propanol, BSA, and preservative, dilute to volume with deionized water, mix well, filter sterilize, and aliquot;

[0021] (2) Prepare reagent R2: Dissolve heparin in an appropriate amount of deionized water, add thrombin and chromogenic substrate S-2238, add stabilizer peptide A and stabilizer, dilute to volume with deionized water, mix well, filter sterilize, and aliquot;

[0022] (3) Preparation of standard solution: Dissolve the lyophilized powder of human plasma AT-III of known activity in deionized water to the required concentration and aliquot.

[0023] The present invention also provides the use of the above-mentioned antithrombin III assay kit for determining antithrombin III activity in human plasma, comprising the following assay steps:

[0024] (1) Sample collection and processing: Collect venous blood from the subjects, anticoagulate it with 3.8% sodium citrate (1:9), centrifuge it at 3000 rpm for 15 min, and take the upper layer of plasma as the test sample;

[0025] (2) Preparation before the test: Return reagent R1, reagent R2 and the sample to room temperature (15-25°C);

[0026] (3) Assay operation: a) Add appropriate amount of reagent R1, sample and standard to the assay well, mix well, and incubate at 37°C for 3 minutes; b) Add 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;

[0027] (4) Calculation of results: Draw a standard curve based on the known activity and absorbance value of the standard sample, and find the corresponding AT-III activity on the standard curve using the absorbance value of the sample.

[0028] Beneficial effects of the present invention:

[0029] 1) The kit provided by the present invention has good stability. The R1 and R2 reagents can be stored stably at 2-8°C for at least 12 months, and the lyophilized standard can be stored stably at 2-8°C for at least 24 months. Compared with traditional kits, the present invention improves the stability of the reagents by more than 40% by adding a special stabilizer formula.

[0030] 2) The present invention uses a specific chromogenic substrate, S-2238, which can directly measure AT-III activity through changes in absorbance at a wavelength of 405 nm, avoiding the interference factors of traditional coagulation methods, with high specificity and good sensitivity;

[0031] 3) The present invention has simple operation steps and a high degree of automation, and is applicable to a variety of fully automatic coagulation analyzers, such as the Sysmex CA series, STAGO series, and ACL series;

[0032] 4) By optimizing the ratio of reagent components, the present invention reduces the impact of possible interfering factors in plasma, such as hemoglobin (≤7g / L), bilirubin (≤200mg / L), and lipemia (≤6.5g / L), on the measurement results, and achieves good accuracy and repeatability;

[0033] 5) The method provided by this invention has a wide linear range (0.5-12 IU / ml), which can meet the needs of AT-III activity determination in normal people and patients with pathological conditions. Its sensitivity is approximately 30% higher than that of similar products on the market, and the upper limit of measurement for high-concentration samples is increased by 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of the novel stable peptide A binding to the thrombin heavy chain;

[0035] Figure 2 The conformational change caused by the binding of the novel stable peptide A to the thrombin heavy chain;

[0036] Figure 3 It is a comparison chart of the kit of the present invention and the imported kit. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1 Preparation of Antithrombin III Assay Kit

[0039] This embodiment provides an antithrombin III assay kit, comprising:

[0040] 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);

[0041] Reagent R2: heparin (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 EDTA sodium salt), 10% stabilizer peptide A sequence (LFEGLRPLFEKKSLEDKTERELLRELRED);

[0042] Figure 1 The binding pattern of the novel stabilizing peptide A (represented by the protein backbone) and the thrombin heavy chain (represented by the gray simulation) is shown. The red area indicates the binding region between the two.

[0043] Figure 2 This image shows the conformational changes of the novel stabilizing peptide A before and after binding to the thrombin heavy chain. Gray represents the unbound novel stabilizing peptide A and thrombin heavy chain. Red and yellow represent the conformational changes that occur after binding, respectively.

[0044] Standard solution: Freeze-dried human plasma standard with an AT-III activity of 5.0 IU / ml. Calibrators at 0.5 IU / ml and 12.0 IU / ml are also provided to verify the low- and high-concentration detection sensitivity of the kit.

[0045] The volume ratio of reagent R1 to reagent R2 is 5:1.

[0046] The preparation method is as follows:

[0047] 1) Preparation of Reagent R1: Weigh 6.057 g Tris-HCl buffer and 4.3 g sodium chloride, dissolve in 800 ml deionized water, adjust the pH to 7.4 with 1 mol / L hydrochloric acid, add 20 ml propanol and 5 g BSA, stir until completely dissolved, add 0.2 g sodium azide, and make up to 1000 ml with deionized water. Sterilize through a 0.22 μm filter, aliquot into brown bottles, and store at 4°C.

[0048] 2) Preparation of Reagent R2: Dissolve 15 units of heparin and 10 units of thrombin in 80 ml of deionized water. Add 5 g of the chromogenic substrate S-2238 powder. Slowly add 10 g of stabilized peptide A and a special stabilizer mixture (0.05 g polyethylene glycol 6000, 0.05 g glycerol, 0.05 g dithiothreitol, and 0.05 g EDTA sodium salt) while stirring to ensure optimal stability of the chromogenic substrate and enzyme. Adjust the pH to 7.2 ± 0.1, make up to 100 ml with deionized water, sterilize through a 0.22 μm filter, aliquot into brown bottles, and store at 4°C in the dark.

[0049] 3) Preparation of Standard: Collect pooled human plasma with an AT-III activity of 5.0 IU / ml, filter through a 0.22 μm filter, and lyophilize. Aliquot into glass bottles, seal, and store at 4°C.

[0050] Example 2 Antithrombin III Activity Assay

[0051] The method for measuring AT-III activity using the kit prepared in Example 1 is as follows:

[0052] 1) Sample Collection and Processing: Collect 3 ml of venous blood from the subject, add 3.8% sodium citrate (blood to anticoagulant ratio 9:1) for anticoagulation, mix thoroughly, centrifuge at 3000 rpm for 15 minutes, and collect the supernatant plasma as the test sample;

[0053] 2) Reagent Preparation: Remove reagent R1, reagent R2, and the standard from the refrigerator and place at room temperature (15-25°C) for 30 minutes. Dissolve the standard in the diluent provided with the kit and let it stand for 10 minutes.

[0054] 3) Instrument Settings: Taking the Sysmex CA-7000 fully automatic coagulation analyzer as an example, set the following parameters according to the instrument operating manual:

[0055] Test item: AT-III activity; Detection wavelength: 405 nm; Reaction temperature: 37°C; Assay type: kinetic method;

[0056] 4) Determination steps:

[0057] a) Place reagent R1, reagent R2, standards and samples in the designated positions on the instrument;

[0058] b) The instrument automatically aspirates 100 μl of reagent R1 and 20 μl of sample (or standard), mixes, and incubates at 37°C for 3 minutes;

[0059] c) The instrument automatically adds 20 μl of reagent R2, mixes well, and incubates at 37°C for exactly 3 minutes;

[0060] d) The instrument continuously measures the absorbance change at a wavelength of 405 nm and calculates ΔA / min;

[0061] e) Establish a standard curve based on the known activity and ΔA / min value of the standard. Calculate the AT-III activity of the sample using the ΔA / min value.

[0062] Example 3 Kit Performance Verification

[0063] The following performance verification was performed on the antithrombin III assay kit prepared in Example 1:

[0064] 1) Precision test:

[0065] Intra-batch precision: The same normal plasma sample was measured 20 times continuously, with a CV of <3.0%. The specific data are as follows:

[0066] Table 1 Results of intra-batch precision experiments

[0067]

[0068] Inter-assay precision: The same normal plasma sample was measured twice daily for 10 consecutive days with a CV < 5.0%; the specific data are as follows:

[0069] Table 2 Results of inter-batch precision experiments

[0070]

[0071] 2) Accuracy test: The relative deviation between the test result and the labeled value of the AT-III international standard (WHO IS 96 / 854) is less than 10%;

[0072] Table 3 Accuracy test results

[0073]

[0074] 3) Linear range: In the range of 0.5-12 IU / ml, the assay results showed a good linear relationship with the theoretical activity of the sample, with r>0.998;

[0075] Table 4 Linearity test results

[0076]

[0077] 4) Reference interval validation: Plasma samples from 200 healthy individuals were tested, and the reference interval for AT-III activity was 2.5-3.7 IU / ml, consistent with literature reports.

[0078] Table 5 Results of the reference interval validation experiment

[0079]

[0080] Table 5

[0081]

[0082] The results are analyzed in Table 6:

[0083] Table 6 Verification results analysis

[0084]

[0085] 5) Interference test: The following substances were added to normal plasma and the AT-III activity was measured:

[0086] a) Hemoglobin (final concentration 7 g / L): Impact on the test results <10%; b) Bilirubin (final concentration 200 mg / L): Impact on the test results <8%; c) Fat emulsion (final concentration 6.5 g / L): Impact on the test results <12%; The results are as follows:

[0087] Table 7 Interference test results

[0088]

[0089] 6) Stability test:

[0090] a) Reagents R1 and R2 retain >90% activity when stored at 2-8°C for 12 months; b) The standard retains >95% activity when stored at 2-8°C for 24 months; c) Once opened, the reagents are stable at 2-8°C for 45 days.

[0091] Table 8 Stability test results

[0092]

[0093] The test results show that the antithrombin III determination kit provided by the present invention has good precision, accuracy, linear range, anti-interference ability and stability, and meets the needs of clinical detection.

[0094] Example 4 Clinical application verification

[0095] Plasma samples were collected from 100 healthy subjects and 100 patients diagnosed with deep vein thrombosis (DVT). AT-III activity was determined using the kit of the present invention and an imported commercial kit (Berichrom Antithrombin III (A) assay kit (chromogenic substrate method) manufactured by Siemens Medical Diagnostics GmbH, Germany). See [see reference text for details]. Figure 3 And Table 9, the results are analyzed as follows:

[0096] 1) From Figure 3 As can be seen from the figure, 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;

[0097] Table 9 Comparison of the kit of the present invention and imported commercial kits

[0098]

[0099] Table 9

[0100]

[0101] Table 9

[0102]

[0103] 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), demonstrating that AT-III activity measurement is clinically meaningful for the diagnosis of DVT.

[0104] 3) Comparison of assay time: The kit of the present invention: an average of 7.5 minutes per sample; the imported commercial kit: an average of 12.3 minutes per sample; the kit of the present invention can save approximately 40% of the assay time.

[0105] The above examples demonstrate that the antithrombin III assay kit provided by the present invention has good diagnostic performance in clinical applications, is easy to operate, and has a short assay time, and is suitable for routine clinical testing needs.

[0106] Example 5 Effect of the Novel Stabilizing Peptide A on Reagent Stability

[0107] In order to verify the effect of the new stable peptide A on the stability of the reagent, the following comparative test was performed:

[0108] 1) Preparation of experimental and control groups: a) Experimental group: A kit containing 10% novel stable peptide A was prepared according to the method of Example 1; b) Control group: A kit without novel stable peptide A was prepared, with other components being the same as those of the experimental group;

[0109] 2) Stability test method: The test group and control group reagents were stored under the following conditions: a) sealed at 2-8°C; b) accelerated test at 37°C; c) stability test at room temperature (25°C) after opening the bottle;

[0110] 3) Test results:

[0111] a) Stability under sealed storage conditions at 2-8°C: Experimental group: Activity retention rate >90% after 12 months; Control group: Activity retention rate decreased to 75% after 6 months and to 60% after 12 months;

[0112] b) 37℃ accelerated test results: experimental group: 7-day activity retention rate >85%; control group: 7-day activity retention rate dropped to 65%;

[0113] c) Stability after opening the bottle at room temperature: experimental group: activity retention rate >85% after 45 days; control group: activity retention rate dropped to 70% after 15 days;

[0114] 4) Conclusion:

[0115] The addition of the new stabilizing peptide A significantly improves the stability of the reagent, 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 the bottle, extending the shelf life from 15 days to 45 days. These improvements significantly enhance the practicality and economy of the test kit.

Claims

1. An antithrombin III assay kit, characterized in that: include: Reagent R1, reagent R2 and standard solution; Reagent R1 is a buffer 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, 0.2 U / ml thrombin, 5% specific chromogenic substrate S-2238, 10% stabilized peptide A, and 0.2% stabilizer; The stabilizing peptide A in reagent R2 has the sequence: LFEGLRPLFEKKSLEDKTERELLRELRED; The volume ratio of the reagent R1 to the reagent R2 is 5:1, and the percentages of the reagent R1 to the reagent R2 are by mass. Standard solution: Antithrombin III, activity range 0.5-12 IU / ml.

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

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, characterized in that The standard solution is freeze-dried human plasma standard.

5. A method for preparing the antithrombin III assay kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Prepare reagent R1: Dissolve Tris-HCl in deionized water, adjust the pH to 7.4 ± 0.2, add sodium chloride, propanol, BSA, and preservative, dilute to volume with deionized water, mix well, filter sterilize, and aliquot; (2) Prepare reagent R2: Dissolve heparin in an appropriate amount of deionized water, add thrombin and chromogenic substrate S-2238, add stabilizer peptide A and stabilizer, dilute to volume with deionized water, mix well, filter sterilize, and aliquot; (3) Preparation of standard solution: Dissolve human plasma AT-III lyophilized powder of known activity in deionized water to the required concentration, divide into portions, and lyophilize.

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