Antithrombin III determination kit and preparation method thereof

Abstract

The invention relates to the field of biological medicine, in particular to an antithrombin III determination kit and a preparation method thereof. The antithrombin III determination kit comprises a reagent 1, a reagent 2, an ATIII calibration product and an ATIII quality control product. The reagent 1 is prepared from a Tris-HCl buffer solution, calcium chloride, bovine serum albumin, potassium ferrocyanide, gentamicin sulfate, ProClin 300, recombinant human thrombin and a nano blocking agent; the reagent 2 is prepared from a Tris-HCl buffer solution, S-2238, gentamicin sulfate and ProClin 300; the ATIII calibrator is prepared from a synthetic plasma matrix, recombinant human ATIII, natural ATIII, ProClin 300, a blood coagulation inhibitor, protamine and trehalose; the ATIII quality control product is prepared from a synthetic plasma matrix, recombinant human ATIII, natural ATIII, ProClin 300, a blood coagulation inhibitor, trehalose and mannitol, according to the scheme, the method is suitable for a full-automatic blood coagulation analysis platform and conforms to the development trend of in-vitro diagnostic reagents.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antithrombin III assay kit and a preparation method thereof. Background Art

[0002] Antithrombin III is an important indicator used clinically to assess blood coagulation status. Its measurement is often used to assist in determining whether patients have thrombotic diseases, abnormal liver function, hereditary antithrombin deficiency, etc. It is widely used in in vitro diagnosis, disease screening, and personalized treatment.

[0003] Currently, the widely used antithrombin III assay primarily utilizes an indirect chromogenic method for activity analysis. This typically involves reacting antithrombin with a known amount of thrombin, then detecting the remaining thrombin activity through a substrate colorimetric reaction, thereby inferring the antithrombin III content. Assay kits typically consist of two core reagents: a reagent solution containing thrombin and a buffer system, and a substrate solution containing a chromogenic substrate. Some protocols also incorporate preservatives such as heparin, bovine serum albumin, and biological extracts to improve stability and storage conditions. In specific applications, while these kits have achieved certain optimizations in component pairing, reaction efficiency, and reagent storage, they still face challenges such as complex formulations, insufficient precision in controlling component concentrations, and poor consistency between standard and ATIII quality control materials, which limit the accuracy and reproducibility of test results.

[0004] In response to the above-mentioned technical problems, there is an urgent need in this field to develop an antithrombin III assay kit with a simple formula, high reaction sensitivity, stable color development reaction, and easy standardized preparation and quality control. Summary of the Invention

[0005] In order to meet the requirements of an antithrombin III assay kit having a simple formula, high reaction sensitivity, stable color development reaction, and convenient standardized preparation and quality control, the present application provides an antithrombin III assay kit and a preparation method thereof.

[0006] The present application provides an antithrombin III assay kit, which comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0007] Preferably, the reagent 1 comprises the following raw materials in thousandths by weight: Tris-HCl buffer powder 3-7‰, calcium chloride 0.5-1.5‰, bovine serum albumin 0.5-1.5‰, potassium ferrocyanide 0.1-1.5‰, gentamicin sulfate 0.01-0.1‰, ProClin 300 0.1-0.5‰, recombinant human thrombin 0.001-0.01‰, and anti-heterophilic antibody nanoblocker 0.1-1‰.

[0008] By adopting the above scheme, calcium chloride is added to supplement the calcium ion in Tris-HCl buffer, which can activate the activity of recombinant human thrombin, replace the traditional EDTA anticoagulant, and avoid interfering with the coagulation reaction; the addition of bovine serum albumin can reduce nonspecific adsorption; potassium ferrocyanide oxidizes bilirubin to eliminate its interference, improves the accuracy of the reagent in jaundice samples, and can also inhibit the turbidity interference of lipemia samples, and synergistically stabilizes the liquid reagent with bovine serum albumin; the addition of ProClin 300 can effectively inhibit the nonspecific binding of rheumatoid factor and heterophilic antibodies, and enhance anti-interference. At the same time, ProClin 300 has antibacterial properties covering bacteria, fungi and yeasts, and gentamicin sulfate targets Gram-negative bacteria. The combination of ProClin 300 and gentamicin sulfate has a broader spectrum of antibacterial properties and a safer synergistic preservative effect; the anti-heterophilic antibody nanoblocker has a lower dosage and stronger specificity than traditional animal-derived blockers, and can inhibit interference such as rheumatoid factor; at the same time, Tris-HCl, ProClin 300. Bovine serum albumin, thrombin and other ingredients are compatible and do not affect enzyme activity.

[0009] Preferably, the reagent 1 comprises the following raw materials in thousandths by weight: Tris-HCl buffer powder 4-6‰, calcium chloride 0.7-0.13‰, bovine serum albumin 0.7-0.13‰, potassium ferrocyanide 0.4-1.2‰, gentamicin sulfate 0.03-0.09‰, ProClin 300 0.2-0.4‰, recombinant human thrombin 0.003-0.009‰, and anti-heterophilic antibody nanoblocker 0.3-0.9‰.

[0010] Preferably, the potassium ferrocyanide in the reagent 1 is potassium ferrocyanide powder with a purity of ≥99%.

[0011] Preferably, the recombinant human thrombin in the reagent 1 has a potency of 224 IU / mg.

[0012] Preferably, the anti-heterophilic antibody nanoblocker in the reagent 1 is HBR G0096R.

[0013] Preferably, the reagent 1 is prepared using the following technical solution: weigh Tris-HCl buffer powder, add the pre-dissolved volume of deionized water, stir magnetically until completely dissolved, add calcium chloride, stir until completely dissolved, then add bovine serum albumin and potassium ferrocyanide in sequence, and stir until a transparent solution is formed; add anti-heterophilic antibody nanoblocker, stir at 4°C for 10 minutes, slowly add recombinant human thrombin, and avoid vigorous stirring to avoid enzyme inactivation; then add gentamicin sulfate and ProClin 300, adjust the solution pH to 6-7, add deionized water to the final volume, filter, and then package for storage.

[0014] Preferably, in the preparation of the reagent 1, the Tris-HCl buffer powder is added to a pre-dissolved volume of 70-80% of the final volume.

[0015] Preferably, in the preparation of the reagent 1, 3.65% HCl or 1 mol / L NaOH is used for pH adjustment, and the pH value after adjustment is 6.7±0.1.

[0016] Preferably, the preparation of the reagent 1 is performed by filtering with a 0.22 μm PES filter membrane and storing the obtained product at 2-8° C. after packaging.

[0017] Preferably, the reagent 2 comprises the following raw materials in thousandths by weight: Tris-HCl buffer powder 3-7‰, S-2238 1-5‰, gentamicin sulfate 0.01-0.1‰, ProClin 300 0.1-0.3‰.

[0018] Preferably, the reagent 2 comprises the following raw materials in thousandths by weight: Tris-HCl buffer powder 4-6‰, S-2238 2-4‰, gentamicin sulfate 0.03-0.09‰, ProClin 300 0.15-0.25‰.

[0019] Preferably, the reagent 2, S-2238, is HD-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline.

[0020] Preferably, the reagent 2 is prepared using the following technical solution: weigh Tris-HCl buffer powder, add the pre-dissolved volume of deionized water, and stir magnetically until completely dissolved; add S-2238 chromogenic substrate and gentamicin sulfate in sequence, and stir until uniformly mixed; add ProClin 300 preservative, and stir for 30-60 minutes in the dark; adjust the pH of the solution to 6.0-7.0, and add deionized water to the final volume; filter with a filter membrane, divide into portions, and store in the dark.

[0021] Preferably, in the preparation of the reagent 2, Tris-HCl buffer powder is added to a pre-dissolved volume of 70-80% of the final volume.

[0022] Preferably, as described in the preparation of the reagent 2, the pH is adjusted using 3.65% HCl or 1 mol / L NaOH, and the pH value after adjustment is 6.7±0.1.

[0023] Preferably, the preparation of the reagent 2 is performed by filtering with a 0.22 μm PES filter membrane, and the reagent is stored in a dark environment at 2-8° C. after packaging.

[0024] Preferably, the ATIII calibrator comprises the following raw materials in thousandths by weight: synthetic plasma matrix 700-850‰, recombinant human ATIII 50-120‰, natural ATIII 10-20‰, ProClin 300 0.5-1.5‰, coagulation inhibitor 0.0001-0.0005‰, protamine 0.0003-0.0007‰, and trehalose 0.2-0.5‰.

[0025] Using this protocol, the ATIII calibrator utilizes a synthetic plasma matrix to simulate the human plasma environment. Recombinant human ATIII ensures traceability and batch-to-batch consistency, avoiding the introduction of additional buffering components. The degree of glycosylation of recombinant ATIII may differ from that of native ATIII, affecting its binding activity to heparin and thrombin. The addition of native ATIII effectively calibrates for glycosylation differences. The addition of a coagulation inhibitor prevents degradation of the calibrator, and the ProClin 300 concentration is optimized to minimize ATIII activity. For heparinized samples, protamine can be added as a heparin neutralizer to specifically neutralize heparin and prevent false elevations in heparinized samples.

[0026] Preferably, the ATIII calibrator comprises the following raw materials in thousandths by weight: synthetic plasma matrix 750-850‰, recombinant human ATIII 80-100‰, natural ATIII 13-18‰, ProClin 300 0.7-1.2‰, coagulation inhibitor 0.0002-0.0004‰, protamine 0.0004-0.0006‰, and trehalose 0.3-0.4‰.

[0027] Preferably, the ATIII calibrator is prepared using the following technical scheme: recombinant ATIII and natural ATIII are slowly added to a synthetic plasma matrix, gently mixed to avoid foaming, and then a coagulation inhibitor, ProClin 300, protamine, and trehalose are added; the target volume is adjusted with ultrapure water, filtered through a 0.22 μm PES filter for sterilization, and stored in a dark environment at 2-8°C after aliquoting.

[0028] Preferably, the ATIII quality control product comprises the following raw materials in thousandths by weight: synthetic plasma matrix 700-850‰, recombinant human ATIII 50-120‰, natural ATIII 10-20‰, ProClin 300 0.5-1.5‰, coagulation inhibitor 0.0001-0.0005‰, trehalose 0.3-1‰, and mannitol 0.05-0.15‰.

[0029] Preferably, the ATIII quality control product comprises the following raw materials in thousandths by weight: synthetic plasma matrix 750-850‰, recombinant human ATIII 80-100‰, natural ATIII 13-18‰, ProClin 300 0.7-1.2‰, coagulation inhibitor 0.0002-0.0004‰, trehalose 0.5-0.9‰, and mannitol 0.08-0.13‰.

[0030] Preferably, the ATIII quality control product is prepared using the following technical solution: recombinant ATIII and natural ATIII are slowly added to a synthetic plasma matrix, gently mixed to avoid foaming, to obtain a plasma base solution, which is diluted with PBS buffer to obtain high-level plasma, medium-level plasma, and low-level plasma; pre-dissolved volumes of high-level plasma, medium-level plasma, and low-level plasma are respectively taken, and coagulation inhibitors, ProClin 300, trehalose, and mannitol are added; the target volume is adjusted with high-level plasma, medium-level plasma, and low-level plasma, filtered and sterilized with a 0.22μm PES filter membrane, and stored in a dark environment at 2-8°C after aliquoting to obtain high / medium / low-level ATIII quality control products.

[0031] Preferably, the synthetic plasma matrix is ​​one of Feipeng Bio FPHM001 and Merck Human Plasma-LikeMatrix.

[0032] Preferably, the recombinant human ATIII is one of HyTest 8AT10, Molecular Innovations HCATIII, Sigma-Aldrich SRP6379, R&D Systems 3658-AT and Abcam ab152161.

[0033] Preferably, the native ATIII is one of Sekisui Diagnostics, Hyphen BioMed and Sigma-Aldrich.

[0034] Preferably, the coagulation inhibitor is one of an argatroban preparation and a rivaroxaban solution.

[0035] Preferably, the natural ATIII has a purity greater than 95%.

[0036] Preferably, the PBS buffer has a phosphate concentration of 10 mM and a pH of 7.2-7.4.

[0037] Preferably, the ATIII quality control product is high-level plasma, the plasma base solution and the PBS buffer solution are in a mass ratio of 1:1, and the expected activity range after dilution is 80-120%.

[0038] Preferably, the mass ratio of the ATIII quality control product is 1:2 among plasma, plasma base solution and PBS buffer, and the expected activity range after dilution is 30-50%.

[0039] Preferably, the ATIII quality control product is low-level plasma, the plasma base solution and PBS buffer solution are in a mass ratio of 1:4, and the expected activity range after dilution is <30%.

[0040] By adopting the above scheme, through precise component ratios and the application of recombinant proteins, it is suitable for a fully automatic coagulation analysis platform. The use of recombinant human ATIII avoids the potential viral risks of plasma-derived substances, which is in line with the development trend of in vitro diagnostic reagents.

[0041] In summary, this application has the following beneficial effects: 1. The present application discloses an antithrombin III assay kit, which utilizes precise component ratios and recombinant proteins for use on fully automated coagulation analysis platforms. The use of recombinant human ATIII avoids the potential viral risks of plasma-derived materials and is consistent with the development trend of in vitro diagnostic reagents.

[0042] 2. An antithrombin III assay kit of the present application, wherein the reagent 1, by adding calcium chloride to supplement the calcium ions in the Tris-HCl buffer, can activate the activity of recombinant human thrombin, replace the traditional EDTA anticoagulant, and avoid interfering with the coagulation reaction; the addition of bovine serum albumin can reduce nonspecific adsorption; potassium ferrocyanide oxidizes bilirubin to eliminate its interference, thereby improving the accuracy of the reagent in jaundice samples, and can also inhibit the turbidity interference of lipemia samples, and synergistically stabilize the liquid reagent with bovine serum albumin; the addition of ProClin 300 can effectively inhibit the nonspecific binding of rheumatoid factor and heterophilic antibodies, and enhance anti-interference. At the same time, ProClin 300 has antibacterial properties covering bacteria, fungi and yeasts, and gentamicin sulfate targets Gram-negative bacteria. The combination of ProClin 300 and gentamicin sulfate has a broader spectrum of antibacterial properties and a safer synergistic preservative effect; the anti-heterophilic antibody nanoblocker has a lower dosage and stronger specificity than traditional animal-derived blockers, and can inhibit interference such as rheumatoid factor; at the same time, Tris-HCl, ProClin 300. Bovine serum albumin, thrombin and other ingredients are compatible and do not affect enzyme activity.

[0043] 3. This application discloses an antithrombin III assay kit and an ATIII calibrator. Both the ATIII calibrator and ATIII quality control utilize a synthetic plasma matrix to simulate the human plasma environment. Recombinant human ATIII ensures traceability and batch-to-batch consistency, avoiding the introduction of additional buffering components. The addition of a coagulation inhibitor prevents degradation of the calibrator itself, and the ProClin 300 concentration is optimized to minimize impact on ATIII activity. DETAILED DESCRIPTION

[0044] The technical solution of the present application is further illustrated below through specific embodiments. The specific embodiments do not limit the scope of protection of the present application; some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the scope of protection of the present application.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. The reagents, enzyme preparations, and materials described are all commercially available products.

[0046] The present application is further described in detail below with reference to the following examples and comparative examples.

[0047] Preparation Example Preparation of Preparation Example 1-6 Reagent 1 Preparation of reagent 1 includes the following steps: weighing Tris-HCl buffer powder, pre-dissolving it in 700 mL of deionized water, magnetically stirring until completely dissolved, adding calcium chloride, stirring until completely dissolved, then sequentially adding bovine serum albumin and potassium ferrocyanide, and stirring until a transparent solution is formed; adding an anti-heterophilic antibody nanoblocker, stirring at 4°C for 10 minutes, slowly adding recombinant human thrombin to avoid enzyme inactivation due to vigorous stirring; then adding gentamicin sulfate and ProClin 300, adjusting the pH to 6.7 using 3.65% HCl or 1 mol / L NaOH, adding deionized water to make the volume 1000 mL, filtering using a 0.22 μm PES filter, and storing at 2-8°C after aliquoting.

[0048] Preferably, the recombinant human thrombin in the reagent 1 has a potency of 224 IU / mg.

[0049] Preferably, the anti-heterophilic antibody nanoblocker in the reagent 1 is HBR G0096R.

[0050] Preferably, the potassium ferrocyanide in the reagent 1 is potassium ferrocyanide powder with a purity of ≥99%.

[0051] The different addition amounts of each component are shown in Table 1.

[0052] Table 1 Addition amount of each component of Preparation Example 1-6 Reagent 1 (g / L) Preparation Example 7-12 Preparation of Reagent 2 Preparation of reagent 2 includes the following steps: weighing Tris-HCl buffer powder, adding 700 mL of deionized water to pre-dissolve it, and magnetically stirring until completely dissolved; sequentially adding S-2238 chromogenic substrate and gentamicin sulfate, and stirring until uniformly mixed; adding ProClin 300 preservative, and stirring for 30-60 minutes in the dark; adjusting the pH to 6.7 using 3.65% HCl or 1 mol / L NaOH, adding deionized water to the volume of 1000 mL, filtering using a 0.22 μm PES filter, and storing the aliquoted solution at 2-8°C in the dark.

[0053] The different addition amounts of each component are shown in Table 2.

[0054] Table 2 Addition amount of each component of reagent 2 in preparation examples 7-12 (g / L) Preparation Example 13-18 Preparation of ATIII Calibrator Preparation of the ATIII calibrator involves the following steps: slowly adding recombinant ATIII to a synthetic plasma matrix, gently mixing to avoid foaming, and then adding the coagulation inhibitor, ProClin 300. Adjust the volume to the target volume with ultrapure water, sterilize by filtration through a 0.22 μm PES filter, and store in aliquots at 2–8°C in the dark.

[0055] The synthetic plasma matrix is ​​Feipeng Bio FPHM001, the recombinant human ATIII is HyTest 8AT10, and the coagulation inhibitor is argatroban preparation.

[0056] The different addition amounts of each component are shown in Table 3.

[0057] Table 3 Addition amount of each component of ATIII calibrator in preparation examples 13-18 (g / L) Preparation Example 19-24 Preparation of ATIII Quality Control Preparation of the ATIII quality control product, comprising the following steps: preferably, the ATIII quality control product is prepared by the following technical scheme: slowly adding recombinant ATIII into a synthetic plasma matrix, gently mixing, avoiding foaming, obtaining a plasma base solution, diluting with PBS buffer to obtain high-level plasma, medium-level plasma and low-level plasma, respectively taking a pre-dissolved volume of high-level plasma, medium-level plasma and low-level plasma, adding coagulation inhibitors, ProClin 300, trehalose and mannitol; adjusting to the target volume with high-level plasma, medium-level plasma and low-level plasma, filtering and sterilizing with a 0.22 μm PES filter, and storing in a light-proof environment at 2-8℃ after dispensing, to obtain high-level ATIII quality control product, medium-level ATIII quality control product and low-level ATIII quality control product.

[0058] The PBS buffer has a phosphate concentration of 10 mM and a pH of 7.2-7.4.

[0059] The synthetic plasma matrix is FPHM001 from Feipeng Biology, the recombinant human ATIII is HyTest 8AT10, and the coagulation inhibitor is an argatroban preparation.

[0060] The high / medium / low level diluted plasma in the ATIII quality control product is shown in Table 4.

[0061] Table 4 High / medium / low level diluted plasma in the ATIII quality control product Quality control product level Dilution ratio (plasma base solution: PBS buffer) Activity range (%) high level 1:1 80-120％ Intermediate level 1:2 30-50％ Low level 1:4 ＜30％ The different addition amounts of each component in Preparation Examples 19-24 are shown in Table 5.

[0062] Table 5 Addition amounts (g / L) of each component in the ATIII quality control product of Preparation Examples 19-24 Embodiment

[0063] Embodiment 1 The present application provides an antithrombin III assay kit, which comprises reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0064] Reagent 1 is obtained by Preparation Example 1, reagent 2 is obtained by Preparation Example 7, ATIII calibrator is obtained by Preparation Example 13, and ATIII quality control product is obtained by Preparation Example 19.

[0065] Embodiment 2 The present application provides an antithrombin III assay kit, which comprises reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0066] Reagent 1 was obtained by Preparation Example 2, reagent 2 was obtained by Preparation Example 8, ATIII calibrator was obtained by Preparation Example 14, and ATIII quality control was obtained by Preparation Example 20.

[0067] Example 3 The present application provides an antithrombin III assay kit, which comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0068] Reagent 1 was obtained by Preparation Example 3, reagent 2 was obtained by Preparation Example 9, ATIII calibrator was obtained by Preparation Example 15, and ATIII quality control was obtained by Preparation Example 21.

[0069] Example 4 The present application provides an antithrombin III assay kit, which comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0070] Reagent 1 was obtained by Preparation Example 4, reagent 2 was obtained by Preparation Example 10, ATIII calibrator was obtained by Preparation Example 16, and ATIII quality control was obtained by Preparation Example 22.

[0071] Example 5 The present application provides an antithrombin III assay kit, which comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0072] Reagent 1 was obtained by Preparation Example 5, reagent 2 was obtained by Preparation Example 11, ATIII calibrator was obtained by Preparation Example 17, and ATIII quality control was obtained by Preparation Example 23.

[0073] Example 6 The present application provides an antithrombin III assay kit, which comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

[0074] Reagent 1 was obtained by Preparation Example 6, reagent 2 was obtained by Preparation Example 12, ATIII calibrator was obtained by Preparation Example 18, and ATIII quality control was obtained by Preparation Example 24.

[0075] Comparative Example Comparative Example 1 The same as Example 1, except that calcium chloride is not added to Reagent 1 and Reagent 2.

[0076] Comparative Example 2 Same as Example 1, except that potassium ferrocyanide is not added to the reagents 1 and 2 Comparative Example 3 The same as Example 1, except that the anti-heterophilic antibody nanoblocker in Reagent 1 and Reagent 2 is replaced by aprotinin.

[0077] Comparative Example 4 The same as Example 1, except that the anti-heterophilic antibody nanoblocker and heparinase are not added to the reagents 1 and 2.

[0078] Comparative Example 5 The same as Example 1, except that the anti-heterophilic antibody nanoblocker in Reagent 1 and Reagent 2 is replaced by ethylenediaminetetraacetic acid.

[0079] Comparative Example 6 The same as Example 1, except that the recombinant human thrombin in Reagent 1 and Reagent 2 is replaced by bovine thrombin.

[0080] Comparative Example 7 The method is the same as Example 1, except that the synthetic plasma matrix and recombinant human ATIII in the ATIII calibrator and ATIII quality control are replaced with normal human plasma and diluted with PBS buffer.

[0081] Performance testing The test was performed using a Sysmex CS-5100 coagulometer. The test steps are shown in Table 6: Table 6 Antithrombin III assay kit detection steps The antithrombin III assay kits of Examples 1-6 and Comparative Examples 1-7 were tested for linear range, accuracy, and stability.

[0082] 1. Accuracy Test Results According to the test steps described above, the antithrombin III activity of the international standard material (WHO International Standard NIBSC code: 08 / 258d) was measured. The test results are shown in Table 7.

[0083] Table 7 Antithrombin III activity % detected in Examples 1-6 and Comparative Examples 1-7 As can be seen from Table 7, the relative deviations of Examples 1-6 are between 0.51% and 0.99%, which are relatively small, indicating that the accuracy is higher; the relative deviations of Comparative Examples 1-7 are relatively large, between 1.93% and 3.5%, indicating that the accuracy is not as high as the antithrombin III assay kit prepared in Examples 1-6.

[0084] 2. Linear range test and results Following the above-described assay procedure, a high-activity sample (140%) near the upper limit of the linear range and a low-activity sample (20%) near the lower limit of the linear range were selected. Plasma samples were assayed at concentrations of 20%, 40%, 60%, 80%, 100%, 120%, and 140% of the theoretical concentration. Linear regression values ​​were calculated, with the assay results plotted on the ordinate and the theoretical values ​​plotted on the abscissa. The results are shown in Tables 8 and 9.

[0085] Table 8 Linear regression values ​​detected in Examples 1-6 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 <![CDATA[R 2 ]]> 0.999 0.999 0.999 0.999 0.999 0.999 Table 9 Linear regression values ​​detected in Comparative Examples 1-7 The linear regression values ​​of Examples 1-6 and Comparative Examples 1-7 were measured and shown in Tables 8 and 9. 2 It is an important indicator to measure the goodness of fit of the model, indicating the proportion of the variation of the dependent variable that can be explained by the independent variable. The closer the correlation coefficient is to 1, the better the linearity. 2 The values ​​of R are both 0.999, indicating that they have a good linear relationship in the range of 20-140%. 2 It is about 0.996%-0.998%, which is slightly worse than the linear relationship of Examples 1-6.

[0086] 3. Stability test and results The prepared antithrombin III assay kit was packaged in glass bottles, sealed with caps, and placed in a 37°C water bath. Antithrombin III activity was measured every two days using an international standard substance. Alternatively, the kit was stored in a 2-8°C refrigerator and antithrombin III activity was measured every two months using an international standard substance. Changes in the accuracy of the measurement results were observed.

[0087] Table 10 37℃ Storage Stability Accuracy Test As shown in Table 10, the antithrombin III assay kits prepared in Examples 1-6 showed a 4.08%-4.93% decrease in activity over 14 days at 37°C, with a calculated average daily decay rate of 0.29%-0.35% / day, indicating an expected activity decay trend. In contrast, the antithrombin III assay kits prepared in Comparative Examples 1-7 showed a 32.40%-43.80% decrease in activity over 14 days, with a calculated average daily decay rate of 2.31%-3.13% / day, representing 7-10 times that of the Example group. The antithrombin III assay kits prepared in Examples 1-6 showed greater stability than those in Comparative Examples 1-7.

[0088] Table 11 4℃ Storage Stability Accuracy Test As can be seen from Table 11, the antithrombin III assay kits prepared in Examples 1-6 retained >91.6% of their activity at 4°C for 14 months, with a total decrease of only 3.43%-4.49%. The shelf life is conservatively estimated to be at least 14 months at 25°C (activity retention >90%). Comparative Examples 1-7 all showed activity below 85% at 4 months, with activity decreasing by 11.26%-29.72% over 6 months, and an average monthly attenuation rate of 2.81%-7.43% / month. This represents a failure rate nine times that of the Example group, exceeding the clinically acceptable range.

[0089] The above test results demonstrate that the antithrombin III assay kit prepared in this application, through precise component ratios and the use of recombinant proteins, is suitable for use on fully automated coagulation analysis platforms. The use of recombinant human ATIII avoids the potential viral risks of plasma-derived materials and is in line with the development trend of in vitro diagnostic reagents. Reagent 1, by adding calcium chloride to the Tris-HCl buffer to supplement calcium ions, activates the activity of recombinant human thrombin, replacing the traditional EDTA anticoagulant and preventing interference with the coagulation reaction. Bovine serum albumin reduces nonspecific adsorption. Potassium ferrocyanide oxidizes bilirubin to eliminate its interference, improving the reagent's accuracy in icteric samples and inhibiting turbidity interference in lipemic samples, synergistically stabilizing the liquid reagent with bovine serum albumin. The addition of ProClin 300 effectively inhibits the nonspecific binding of rheumatoid factor and heterophilic antibodies, enhancing anti-interference properties. ProClin 300 also has antibacterial properties covering bacteria, fungi, and yeasts, while gentamicin sulfate targets Gram-negative bacteria. The combination of ProClin 300 and gentamicin sulfate exhibits a broader spectrum of antibacterial activity and a safer synergistic preservative effect. The antibody nanoblocker requires less dosage and is more specific than traditional animal-derived blockers, inhibiting interference from rheumatoid factor and other factors. Furthermore, Tris-HCl, ProClin 300, bovine serum albumin, and thrombin are compatible and do not affect enzyme activity. Both the calibrator and ATIII quality control utilize a synthetic plasma matrix to simulate the human plasma environment. Recombinant human ATIII ensures traceability and batch-to-batch consistency, eliminating the need for additional buffering components. The addition of a coagulation inhibitor prevents degradation of the calibrator, and the ProClin 300 concentration is optimized to minimize impact on ATIII activity.

[0090] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as they are within the scope of the present invention, they are protected by patent law.

Claims

1. An antithrombin III assay kit, characterized in that: The kit comprises: reagent 1, reagent 2, ATIII calibrator and ATIII quality control product.

2. The antithrombin III assay kit according to claim 1, characterized in that The reagent 1 comprises the following raw materials in thousandths by weight: 3-7‰ Tris-HCl buffer powder, 0.5-1.5‰ calcium chloride, 0.5-1.5‰ bovine serum albumin, 0.1-1.5‰ potassium ferrocyanide, 0.01-0.1‰ gentamicin sulfate, 0.1-0.5‰ ProClin 300, 0.001-0.01‰ recombinant human thrombin, 0.1-1‰ anti-heterophilic antibody nanoblocker, and deionized water to make up the balance.

3. The antithrombin III assay kit according to claim 1, characterized in that Reagent 1 is prepared using the following technical solution: weigh Tris-HCl buffer powder, add the pre-dissolved volume of deionized water, and stir magnetically until completely dissolved; add calcium chloride and stir until completely dissolved; then add bovine serum albumin and potassium ferrocyanide in sequence and stir until a transparent solution is formed; add anti-heterophilic antibody nanoblocker and stir at 4°C for 10 minutes; then slowly add recombinant human thrombin, avoiding vigorous stirring; then add gentamicin sulfate and ProClin 300, adjust the solution pH to 6-7, add deionized water to the final volume, filter, and store in aliquots.

4. The antithrombin III assay kit according to claim 1, characterized in that The reagent 2 comprises the following raw materials, calculated by weight per thousand: 3-7‰ Tris-HCl buffer powder, 1-5‰ S-2238, 0.01-0.1‰ gentamicin sulfate, 0.1-0.3‰ ProClin 300, and the balance is made up with deionized water.

5. The antithrombin III assay kit according to claim 1, characterized in that Reagent 2 is prepared using the following technical solution: weigh Tris-HCl buffer powder, add the pre-dissolved volume of deionized water, and stir magnetically until completely dissolved; add S-2238 chromogenic substrate and gentamicin sulfate in sequence, and stir until uniformly mixed; add ProClin 300 preservative, and stir for 30-60 minutes in the dark; adjust the pH of the solution to 6.0-7.0, and add deionized water to the final volume; filter with a filter membrane, aliquot, and store in the dark.

6. The antithrombin III assay kit according to claim 1, characterized in that: The ATIII calibrator comprises the following raw materials, calculated by weight per thousand: synthetic plasma matrix 700-850‰, recombinant human ATIII 50-120‰, natural ATIII 10-20‰, ProClin 300 0.5-1.5‰, coagulation inhibitor 0.0001-0.0005‰, protamine 0.0003-0.0007‰ and trehalose 0.2-0.5‰.

7. The antithrombin III assay kit according to claim 1, characterized in that The ATIII calibrator is prepared using the following technical scheme: recombinant ATIII is slowly added to a synthetic plasma matrix, gently mixed to avoid foaming, and then a coagulation inhibitor, ProClin 300, is added; the target volume is adjusted with ultrapure water, filtered through a 0.22 μm PES filter for sterilization, and stored in a dark environment at 2-8°C after aliquoting.

8. The antithrombin III assay kit according to claim 1, characterized in that: The ATIII quality control product includes the following raw materials in thousandths by weight: synthetic plasma matrix 700-850‰, recombinant human ATIII 50-120‰, natural ATIII 10-20‰, ProClin 300 0.5-1.5‰, coagulation inhibitor 0.0001-0.0005‰, trehalose 0.3-1‰ and mannitol 0.05-0.15‰.

9. The antithrombin III assay kit according to claim 1, characterized in that: The ATIII quality control product is prepared using the following technical scheme: recombinant human ATIII and natural ATIII are slowly added to a synthetic plasma matrix, gently mixed to avoid foaming, to obtain a plasma base solution, which is diluted with PBS buffer to obtain high-level plasma, intermediate-level plasma, and low-level plasma. The pre-dissolved volumes of high-level plasma, intermediate-level plasma, and low-level plasma are respectively taken, and a coagulation inhibitor, ProClin 300, trehalose, and mannitol are added; the target volumes are adjusted with high-level plasma, intermediate-level plasma, and low-level plasma, and the volumes are sterilized by filtration with a 0.22 μm PES filter membrane. After aliquoting, the products are stored in a dark environment at 2-8°C to obtain high / intermediate / low-level ATIII quality control products.

10. The antithrombin III assay kit according to claim 1, characterized in that: The recombinant human ATIII is one of HyTest 8AT10, Molecular Innovations HCATIII, Sigma-Aldrich SRP6379, R&D Systems 3658-AT, and Abcam ab152161.

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