CN114755427B - Anti Xa activity assay kit of external source addition antithrombin - Google Patents
Anti Xa activity assay kit of external source addition antithrombin Download PDFInfo
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Abstract
The invention discloses an anti-Xa activity assay kit with exogenously added antithrombin, which is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components: a first salt ion, a first stabilizing agent, a first preservative, a first buffer solution and antithrombin; the pH value of the first reagent is 7.2 to 7.6; the second reagent comprises the following components: a second chloride ion, a second stabilizing agent, a second preservative, a second buffer solution and a coagulation factor; the pH value of the second buffer solution is 7.8 to 8.2; the third reagent comprises the following components: a third salt ion, a third stabilizing agent, a third preservative, a third buffer solution and a chromogenic substrate; and the pH value of the third buffer solution is 7.8 to 8.2. The invention has more accurate detection on the sample with low antithrombin activity, wide linear range and good reagent stability.
Description
Technical Field
The invention relates to the technical field of medical in-vitro diagnosis, in particular to an anti-Xa activity assay kit with exogenous antithrombin.
Background
In recent decades, heparin in general (UFH), low Molecular Weight Heparin (LMWH), heparin derivatives and direct oral anticoagulants have been approved for the prevention and treatment of thromboembolic diseases. Among them, common heparin and low molecular heparin are the most widely used anticoagulant drugs at present, and are mainly used in departments such as ICU, cardiac surgery, cardiology, hemodialysis, orthopedics and the like. Due to the complex pharmacokinetic and pharmacodynamic properties of heparin, resulting in narrow therapeutic concentration ranges and large individual differences, frequent laboratory monitoring and dose adjustments are required. The Activated Partial Thromboplastin Time (APTT) assay is a traditional method of monitoring UFH, and although simple, rapid, and inexpensive, it is difficult to standardize, varies from laboratory to laboratory by as much as 4-fold, and its associated clinical treatment scope is not clear. At present, an anti-Xa (blood coagulation factor) activity determination kit which can standardize the result, has stable measurement result and reliably reflects the treatment range becomes a more preferable choice for UFH and LMWH monitoring. The anticoagulation effect of heparin depends on the Antithrombin (AT) activity, and when the in vivo antithrombin activity is lower than 50%, the heparin effect is obviously reduced; below 30%, heparin hardly exerts an anticoagulation effect. In order to detect the heparin content of patients with low AT more accurately, antithrombin is exogenously added.
Most of the externally added thrombin Xa activity assay kits sold in the market are still in the form of freeze-dried powder, and the kit needs to be redissolved before use and can be used after standing for a period of time, which is not beneficial to the operation of customers. The anti-Xa activity determination kit does not contain a cosolvent, the quality of the cosolvent selected by a customer cannot be controlled, and certain influence is brought to a test result.
Disclosure of Invention
The invention aims to solve the technical problem that most of the commercially available anti-Xa activity assay kits with exogenously added antithrombin are still in the form of freeze-dried powder and need to be redissolved before use in the prior art, and provides a liquid anti-Xa activity assay kit with exogenously added antithrombin.
The technical scheme adopted by the invention for solving the technical problem is as follows: an anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components by taking the total volume of the first reagent as 1L: 10 to 30g of first salt ion, 56 to 130g of first stabilizer, 0.1 to 1g of first preservative, 50 to 100mmol of first buffer solution and 3000 to 5000IU of antithrombin; the pH value of the first reagent is 7.2 to 7.6; the second reagent comprises the following components in a total volume of 1L: 5 to 15g of second dihydrochloride, 67 to 165g of a second stabilizer, 0.1 to 1g of a second preservative, 50 to 100mmol of a second buffer solution, and 1000 to 5000IU of a blood coagulation factor; the pH value of the second buffer solution is 7.8 to 8.2; the third reagent comprises the following components by the total volume of 1L: 5 to 15g of third salt ion, 46 to 100100 g of third stabilizer, 0.1 to 1g of third preservative, 50 to 100100mmol of third buffer solution and 0.5 to 1.5mmol of chromogenic substrate; and the pH value of the third buffer solution is 7.8 to 8.2.
Preferably, the first stabilizer comprises a polyhydric alcohol, a saccharide, a surfactant, and a protein; the second stabilizer comprises polyhydric alcohol, saccharide, amino acid, surfactant and protein; the third stabilizer includes a polyhydric alcohol, a saccharide, and a surfactant.
Preferably, the first stabilizer comprises mannitol, sucrose, PEG-8000, tween-20 and BSA; the second stabilizer comprises mannitol, sucrose, glycine, PEG-8000, tween-20, BSA and dextran sulfate; the third stabilizer includes mannitol, sucrose, PVP and Tween-20.
Preferably, the first salt ion, the second salt ion and the third salt ion are respectively one of sodium chloride, calcium chloride and ammonium sulfate.
Preferably, the first salt ion, the second salt ion, and the third salt ion are all sodium chloride.
Preferably, the first preservative, the second preservative and the third preservative are respectively one of sodium azide, proClin 300 and ProClin 150.
Preferably, the first preservative, the second preservative and the third preservative are all sodium azide.
Preferably, the first buffer, the second buffer and the third buffer are one of Tris buffer, HEPES buffer and PBS buffer respectively.
Preferably, the first buffer, the second buffer and the third buffer are all Tris buffers.
Preferably, the antithrombin is one of human antithrombin, porcine antithrombin and bovine antithrombin.
Preferably, the antithrombin is human antithrombin.
Preferably, the blood coagulation factor is one of bovine factor Xa, porcine factor Xa, and human factor Xa.
Preferably, the coagulation factor is bovine factor Xa.
Preferably, the chromogenic substrate is s-5288.
The invention has the beneficial effects that:
1. the invention provides an anti-Xa activity assay kit (color substrate method) with exogenously added antithrombin, which is fully combined with UFH or LMWH in plasma of a patient with low antithrombin activity by exogenously added antithrombin, and then the concentration of UFH or LMWH is detected by using bovine Xa factor and a color substrate s-5288, thereby solving the problem that the result of detecting heparin by using the anti-Xa activity assay kit for patients with low antithrombin activity such as congenital antithrombin deficiency, newborn and the like is low, and filling the blank of the kit in domestic market.
2. The kit is a full liquid reagent, and has the advantages of convenient use, low cost, wide linear range and good stability. Can realize the substitution of the imported anti-Xa activity assay kit with the externally added antithrombin, fully meet the requirements of clinical examination, reduce the cost of purchasing detection reagents in hospitals and reduce the detection cost of patients.
Drawings
Figure 1 is a linear correlation of a reference kit and the UFH sample tested in example 1;
FIG. 2 is a linear correlation of the reference kit and the LMWH sample tested in example 1;
figure 3 is a linear correlation of the reference kit and the UFH sample tested in example 2;
figure 4 is a linear correlation of the reference kit and the LMWH sample tested in example 2;
figure 5 is a linear correlation of the reference kit and the UFH sample tested in example 3;
FIG. 6 is a linear correlation of the reference kit and the LMWH sample tested in example 3;
figure 7 is a linear correlation of the reference kit and the UFH sample tested in comparative example 1;
figure 8 is a linear correlation of the reference kit and the LMWH sample tested in comparative example 1.
Detailed Description
In order to clearly understand the technical features, objects and effects of the present invention, the present invention will be further described in detail with reference to the following embodiments, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components by taking the total volume of the first reagent as 1L: 10 to 30g of first salt ion, 56 to 130g of first stabilizer, 0.1 to 1g of first preservative, 50 to 100mmol of first buffer solution and 3000 to 5000IU of antithrombin; the pH value of the first reagent is 7.2 to 7.6; the second reagent comprises the following components in a total volume of 1L: 5 to 15g of second dihydrochloride, 67 to 165g of a second stabilizer, 0.1 to 1g of a second preservative, 50 to 100mmol of a second buffer solution, and 1000 to 5000IU of a blood coagulation factor; the pH value of the second buffer solution is 7.8 to 8.2; the third reagent comprises the following components by the total volume of 1L: 5 to 15g of third salt ion, 46 to 100100 g of third stabilizer, 0.1 to 1g of third preservative, 50 to 100100mmol of third buffer solution and 0.5 to 1.5mmol of chromogenic substrate; and the pH value of the third buffer solution is 7.8 to 8.2.
The first stabilizer includes polyhydric alcohol, saccharide, surfactant and protein, the polyhydric alcohol: saccharides: surfactant (b): the mass ratio of the proteins is as follows: (3.5-6.5): (1-3): (0.1-0.5): (1-3), preferably mannitol, sucrose, PEG-8000, tween-20 and BSA; the second stabilizer includes polyhydric alcohol, saccharide, amino acid, surfactant and protein, the polyhydric alcohol: saccharides: amino acids: surfactant (b): the mass ratio of the proteins is as follows: (3.5-6.5): (1.1-3.5): (1-3): (0.1-0.5): (1-3), preferably mannitol, sucrose, glycine, PEG-8000, tween-20, BSA and dextran sulfate; the third stabilizer includes a polyhydric alcohol, a saccharide and a surfactant, the polyhydric alcohol: saccharides: the mass ratio of the surface active agent is as follows: (3-5): (1-3): (0.6-2), preferably mannitol, sucrose, PVP and Tween-20. Among them, dextran sulfate is mainly used in the present invention to neutralize free PF4 released by platelets in plasma, reduce its interference, and avoid inaccuracy of plasma heparin test result.
The first salt ion, the second salt ion and the third salt ion are respectively one of sodium chloride, calcium chloride and ammonium sulfate, and sodium chloride is preferred in the invention. The salt ion with a proper content improves OD signals in the reaction process, and the resolution of a reagent can be improved, so that the content of the first salt ion is preferably 10 to 30g, and the content of the second salt ion and the third salt ion is preferably 5 to 15g; in view of cost and final effect, the effect of sodium chloride is the best, and therefore, the first salt ion, the second salt ion and the third salt ion are preferably sodium chloride in the invention.
The first preservative, the second preservative and the third preservative are respectively one of sodium azide, proClin 300 and ProClin 150, and the preferred preservative is sodium azide.
The first buffer solution, the second buffer solution and the third buffer solution are respectively one of Tris buffer solution, HEPES buffer solution and PBS buffer solution, and Tris buffer solution is preferably selected in the invention.
The antithrombin is one of human antithrombin, porcine antithrombin and bovine antithrombin, and human antithrombin is preferred in the invention.
The blood coagulation factor is one of bovine factor Xa, porcine factor Xa and human factor Xa, and bovine factor Xa is preferable in the present invention.
Chromogenic substrates are preferred in the present invention as s-5288.
The kit for measuring the anti-Xa activity of the exogenously added antithrombin accurately and sensitively measures the concentration of heparin in plasma through a two-step method. Firstly, heparin in plasma to be detected is combined with antithrombin in the presence of exogenous antithrombin to form a compound, and bovine Xa factor with constant quantity is inhibited; then, excessive bovine Xa hydrolyzes Xa factor specific chromogenic substrate (s-5288) to release a chromogenic group p-nitroaniline (pNA), and the pNA has a maximum absorption peak at the wavelength of 405nm, and the chromogenic degree of the pNA is positively correlated with the amount of the residual bovine Xa factor and negatively correlated with the content of heparin in a plasma sample. The antithrombin is added into the kit, so that the problem of low detection result in the detection of a sample with low antithrombin activity can be solved, and a more accurate detection result is provided for a patient with low antithrombin activity.
The kit is a full liquid reagent and is taken out from a refrigerator at 2 to 8 ℃ for direct use. The kit is a full liquid reagent, and has the advantages of convenient use, low cost, wide linear range and good stability. Can realize the substitution of an imported exogenous anti-Xa activity assay kit, fully meet the requirements of clinical examination, reduce the cost of purchasing detection reagents in hospitals and reduce the detection cost of patients.
The following is illustrated by specific examples:
example 1
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components by taking the total volume of the first reagent as 1L: tris buffer solution with the content of 50mmol, 10g of sodium chloride, 30g of mannitol, 10g of sucrose, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA, human antithrombin with the content of 3000IU, 0.1g of sodium azide, and the pH value of the first reagent is 7.2; the second reagent comprises the following components in a total volume of 1L: tris buffer solution with the content of 50mmol, 5g of sodium chloride, 30g of mannitol, 10g of sucrose, 10g of glycine, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA, 1g of dextran sulfate, 0.1g of sodium azide and bovine factor Xa with the content of 1000IU, and the pH value of the second buffer solution is 7.8; the third reagent comprises the following components by the total volume of 1L: tris buffer with the content of 50mmol, 5g of sodium chloride, 30g of mannitol, 10g of sucrose, 5g of PVP, 1mL of Tween-20, 0.1g of sodium azide, chromogenic substrate s-5288 with the content of 0.5mmol, and the pH value of the third buffer is 7.8.
The preparation method of each reagent in the kit comprises the following steps:
1. preparing a Tris buffer solution with the concentration of 50mmol/L, adding 10g of sodium chloride, 30g of mannitol, 10g of sucrose, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA, 3000IU of human antithrombin and 0.1g of sodium azide into the Tris buffer solution, supplementing the Tris buffer solution to 1L, uniformly mixing, and adjusting the pH value of the solution to 7.2 to obtain a first reagent.
Since the content of the first buffer (Tris buffer) is much larger than that of the other components, the concentration and content of the first buffer in the prepared first reagent are changed little, and it can be considered that the original concentration and content are maintained. The second buffer and the third buffer in steps 2-3 are the same.
2. Preparing a Tris buffer solution with the concentration of 50mmol/L, adding 5g of sodium chloride, 30g of mannitol, 10g of sucrose, 10g of glycine, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA, 1g of dextran sulfate, 0.1g of sodium azide and bovine factor Xa with the content of 1000IU into the Tris buffer solution, supplementing the Tris buffer solution to 1L, uniformly mixing, and adjusting the pH value of the solution to be 7.8 to obtain a second reagent.
3. Preparing a Tris buffer solution with the concentration of 50mmol/L, adding 5g of sodium chloride, 30g of mannitol, 10g of cane sugar, 5g of PVP, 1mL of Tween-20, 0.1g of sodium azide and 0.5mmol of chromogenic substrate s-5288 into the Tris buffer solution, supplementing the Tris buffer solution until 1L is mixed uniformly, and adjusting the pH value of the solution to be 7.8 to obtain a third reagent.
The following examples were prepared in the same manner.
Example 2
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components in a total volume of 1L: HEPES buffer solution with the content of 75mmol, 20g of calcium chloride, 40g of mannitol, 20g of sucrose, 10g of PEG-8000, 2.5mL of Tween-20, 20g of BSA, porcine antithrombin with the content of 4000IU, 0.5g of ProClin 300, and the pH value of the first reagent is 7.4; the second reagent comprises the following components by taking the total volume of the second reagent as 1L: HEPES buffer solution with the content of 75mmol, 10g of calcium chloride, 40g of mannitol, 20g of sucrose, 20g of glycine, 10g of PEG-8000, 2.5mL of Tween-20, 20g of BSA, 3g of dextran sulfate, 0.5g of ProClin 300 and pig factor Xa with the content of 3000IU, and the pH value of the second buffer solution is 8.0; the third reagent comprises the following components by taking the total volume of the third reagent as 1L: HEPES buffer solution with the content of 75mmol, 10g of calcium chloride, 40g of mannitol, 20g of sucrose, 10g of PVP, 2.5mL of Tween-20, 0.5g of ProClin 300, chromogenic substrate s-5288 with the content of 1.0mmol, and the pH value of the third buffer solution is 8.0.
Example 3
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components in a total volume of 1L: PBS buffer solution with the content of 100mmol, 30g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 15g of PEG-8000, 4.5mL of Tween-20, 30g of BSA, bovine antithrombin with the content of 5000IU, and 1g of ProClin 150, wherein the pH value of the first reagent is 7.6; the second reagent comprises the following components by taking the total volume of the second reagent as 1L: PBS buffer solution with the content of 100mmol, 15g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 30g of glycine, 15g of PEG-8000, 4.5mL of Tween-20, 30g of BSA, 5g of dextran sulfate, 1g of ProClin 150, human Xa factor with the content of 5000IU, and the pH value of the second buffer solution is 8.2; the third reagent comprises the following components by taking the total volume of the third reagent as 1L: PBS buffer with a content of 100mmol, 15g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 15g of PVP, 4.5mL of Tween-20, 1g of ProClin 150, chromogenic substrate s-5288 with a content of 1.5mmol, and the pH value of the third buffer is 8.2.
Comparative example 1
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components in a total volume of 1L: tris buffer solution with the content of 50mmol, 10g of sodium chloride, 30g of mannitol, 10g of sucrose, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA and 0.1g of sodium azide, and the pH value of the first reagent is 7.2; the second reagent comprises the following components in a total volume of 1L: tris buffer solution with the content of 50mmol, 5g of sodium chloride, 30g of mannitol, 10g of sucrose, 10g of glycine, 5g of PEG-8000, 1mL of Tween-20, 10g of BSA, 1g of dextran sulfate, 0.1g of sodium azide and bovine factor Xa with the content of 1000IU, and the pH value of the second buffer solution is 7.8; the third reagent comprises the following components by the total volume of 1L: tris buffer with 50mmol content, 5g sodium chloride, 30g mannitol, 10g sucrose, 5g PVP, 1mL Tween-20, 0.1g sodium azide, chromogenic substrate s-5288 with 0.5mmol content, and the pH value of the third buffer is 7.8.
This example is a comparative example to example 1, except that the first reagent does not contain human antithrombin.
Comparative example 2
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components by taking the total volume of the first reagent as 1L: HEPES buffer solution with the content of 75mmol, 20g of calcium chloride, 40g of mannitol, 20g of sucrose, 10g of PEG-8000, 2.5mL of Tween-20, 20g of BSA and 0.5g of ProClin 300, and the pH value of the first reagent is 7.4; the second reagent comprises the following components in a total volume of 1L: HEPES buffer solution with the content of 75mmol, 10g of calcium chloride, 40g of mannitol, 20g of sucrose, 20g of glycine, 10g of PEG-8000, 2.5mL of Tween-20, 20g of BSA, 3g of dextran sulfate, 0.5g of ProClin 300, and porcine factor Xa with the content of 3000IU, wherein the pH value of the second buffer solution is 8.0; the third reagent comprises the following components by taking the total volume of the third reagent as 1L: HEPES buffer solution with the content of 75mmol, 10g of calcium chloride, 40g of mannitol, 20g of sucrose, 10g of PVP, 2.5mL of Tween-20, 0.5g of ProClin 300, chromogenic substrate s-5288 with the content of 1.0mmol, and the pH value of the third buffer solution is 8.0.
This example is a comparative example to example 2, with the difference that the first reagent does not contain antithrombin.
Comparative example 3
An anti-Xa activity assay kit with exogenously added antithrombin is a full liquid reagent and comprises a first reagent, a second reagent and a third reagent; the first reagent comprises the following components in a total volume of 1L: PBS buffer solution with the content of 100mmol, 30g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 15g of PEG-8000, 4.5mL of Tween-20, 30g of BSA, 1g of ProClin 150, and the pH value of the first reagent is 7.6; the second reagent comprises the following components by taking the total volume of the second reagent as 1L: PBS buffer solution with the content of 100mmol, 30g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 30g of glycine, 15g of PEG-8000, 4.5mL of Tween-20, 30g of BSA, 5g of dextran sulfate, 1g of ProClin 150, bovine factor Xa with the content of 5000IU, and the pH value of the second buffer solution is 8.2; the third reagent comprises the following components by the total volume of 1L: PBS buffer with the content of 100mmol, 30g of ammonium sulfate, 50g of mannitol, 30g of sucrose, 15g of PVP, 4.5mL of Tween-20, 1g of ProClin 150, chromogenic substrate s-5288 with the content of 1.5mmol, and the pH value of the third buffer is 8.2.
This example is a comparative example to example 3, with the difference that the first reagent does not contain antithrombin.
Performance test:
the accuracy and clinical relevance of the Xa-resistant activity assay kits of examples 1 to 3 and comparative examples 1 to 3 were compared, and the linear range and accelerated stability of examples 1 to 3 were evaluated. The samples used in the test are all samples with low antithrombin activity, i.e. antithrombin activity is less than 50%, unless otherwise specified.
1. Accuracy test
The test instrument: full-automatic blood coagulation analyzer
The test steps are as follows: after calibration, the kit of examples 1 to 3 and comparative examples 1 to 3 was used to measure the concentrations of the UFH and LMWH accuracy control products, the test was repeated 3 times for each accuracy control product, the relative deviation was calculated according to the formulas (1) and (2), and the test results are shown in tables 1 to 6. The test result is required to be: the relative deviation should be within + -10.00%.
Formula (1):
formula (2):
；
in the formula:
n is the number of tests;
i-serial number of test;
b-relative deviation;
t-accuracy control index value.
TABLE 1 results of accuracy test of the kit for measuring the anti-Xa activity of exogenously added antithrombin in example 1
TABLE 2 results of accuracy test of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 2
TABLE 3 accuracy test results of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 3
TABLE 4 results of accuracy test of the anti-Xa activity assay kit of comparative example 1
TABLE 5 results of accuracy test of anti-Xa activity assay kit of comparative example 2
TABLE 6 results of accuracy test of anti-Xa activity assay kit of comparative example 3
The test results in tables 1 to 6 show that the relative deviations of the examples 1 to 3 are relatively low and meet the requirement of being within the range of +/-10.0%, and the result shows that the Xa activity assay kit with the externally added antithrombin has good accuracy for the assay of the sample with low antithrombin activity; comparative examples 1 to 3 are all high in relative deviation and do not meet the requirement of being within the range of. + -. 10.0%, and when the anti-Xa activity assay kit without the exogenously added antithrombin is used for the assay, since the antithrombin activity in the sample is low, heparin resistance occurs, the assay result of the sample is lower than the indicated value, and the heparin test result is inaccurate. Therefore, the accuracy of the kit for measuring the anti-Xa activity of the exogenously added antithrombin is higher.
2. Clinical relevance testing
The test instrument: full-automatic blood coagulation analyzer
The test steps are as follows: a group of clinical samples covering a linear range (40 samples each having an antithrombin activity of 50% or less) were simultaneously tested using the reference kit, the measurement kit of examples 1 to 3 and comparative example 1, and a linear regression analysis was performed using the measured value of the reference kit as the x-axis and the measured value of the kit of examples 1 to 3 or comparative example 1 as the y-axis, and the test results are shown in tables 7 to 10 and FIGS. 1 to 8. The test result is required to be: the linear regression analysis should conform to the linear regression equation, wherein the slope k is between 0.9 and 1.1, and the correlation coefficient r is more than or equal to 0.975.
TABLE 7 results of clinical relevance test of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 1
TABLE 8 results of clinical relevance test of the kit for measuring the anti-Xa activity of exogenously added antithrombin in example 2
TABLE 9 results of clinical relevance test of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 3
TABLE 10 results of clinical relevance test of anti-Xa activity assay kit of comparative example 1
From the test results in tables 13-16, the results of the clinical sample test of the Xa activity assay kit with exogenous antithrombin and the reference kit are subjected to linear regression analysis, the slope k of the linear regression equation is 0.9 to 1.1, and the correlation coefficient r is more than or equal to 0.975; and the slope k in the linear regression equation of the anti-Xa activity assay kit without exogenous added antithrombin and the reference kit is less than 0.9, which does not meet the correlation requirement, and the accuracy of the test result is low. Therefore, the test results of the external source addition antithrombin Xa activity test kit and the reference kit have good correlation, and the measurement of the concentration of the low antithrombin heparin is more accurate.
3. Linear range test
The test instrument: full-automatic blood coagulation analyzer
The testing steps are as follows: the UFH high value sample and the LMWH high value sample which are close to the upper limit of the linear range of the kit are respectively diluted into 5 samples with different concentrations, the sample with each concentration is tested for 3 times by using the externally added antithrombin and Xa activity assay kit, a linear regression equation is solved by taking the theoretical value of the sample concentration as an x axis and the mean value of measured values as a y axis, and the correlation coefficient (r) is calculated, wherein the test results are shown in tables 11 to 13. The test result is required to be: when the concentration of the UFH high value sample is 0.1-1.5 IU/mL, and the concentration of the LMWH high value sample is in the linear range of 0.1-2.0 IU/mL, the linear correlation coefficient r is more than or equal to 0.990.
TABLE 11 results of the Linear Range test of the kit for measuring the anti-Xa Activity of exogenously added antithrombin in example 1
TABLE 12 results of the Linear Range test of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 2
TABLE 13 results of the Linear Range test of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 3
The test results in tables 11 to 13 show that when the concentration of UFH is 0.1-1.5 IU/mL and the concentration of LMWH is 0.1-2.0 IU/mL, the linear correlation coefficient r of the kit for determining the anti-Xa activity of the exogenously added antithrombin is more than 0.990, and the linear correlation is better. Therefore, the kit for measuring the anti-Xa activity of the exogenously added antithrombin has a wider linear range for measuring the concentration of the sample.
4. Accelerated stability testing
The test instrument: full-automatic blood coagulation analyzer
The testing steps are as follows: the reagents in the reagent kits for measuring the anti-Xa activity of exogenously added antithrombin of examples 1 to 3 were stored at 37 ℃ in an unopened state, accuracy tests were performed on days 0, 3, 6, 7, and 8, the concentrations of the UFH accuracy control product and the LMWH accuracy control product were measured, relative deviations were calculated, and the test results are shown in tables 14 to 16. The test result is required to be: the components of the kit can be stored at 37 ℃ for 7 days in an unopened state, namely, the relative deviation is within +/-10%.
TABLE 14 accelerated stability test results of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 1
TABLE 15 accelerated stability test results of the kit for measuring the anti-Xa activity of exogenously added antithrombin in example 2
TABLE 16 accelerated stability test results of the kit for measuring anti-Xa activity of exogenously added antithrombin in example 3
From the test results of tables 14 to 16, the external addition antithrombin Xa activity assay kit disclosed by the invention has the advantages that the relative deviation of the assay result of the sample is within +/-10% within 8 days, and the assay accuracy is higher. Therefore, the kit for measuring the anti-Xa activity of the exogenously added antithrombin can be stably stored for 7 days under accelerated conditions in an unopened state, has better stability and is equivalent to the kit for measuring the anti-Xa activity in the prior art.
The test results show that the exogenous antithrombin is added into the determination kit, so that a more accurate result can be obtained when the concentration of heparin in a sample with low antithrombin activity is detected; the kit is a full liquid reagent, and has a wider linear range of detection concentration and better stability.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (13)
1. The kit for measuring the anti-Xa activity of the exogenously added antithrombin is characterized by being a full liquid reagent and comprising a first reagent, a second reagent and a third reagent;
the first reagent comprises the following components in a total volume of 1L: 10 to 30g of first salt ion, 56 to 130g of first stabilizer, 0.1 to 1g of first preservative, 50 to 100mmol of first buffer solution and 3000 to 5000IU of antithrombin; the pH value of the first reagent is 7.2 to 7.6; the first stabilizer comprises a polyhydric alcohol, a saccharide, a first surfactant, and a protein; polyhydric alcohol: saccharides: first surfactant: the mass ratio of the proteins is as follows: (3.5-6.5): (1-3): (0.1-0.5): (1-3); the first surfactant is Tween-20;
the second reagent comprises the following components in a total volume of 1L: 5 to 15g of second dihydrochloride, 67 to 165g of a second stabilizer, 0.1 to 1g of a second preservative, 50 to 100mmol of a second buffer solution, and 1000 to 5000IU of a blood coagulation factor; the pH value of the second buffer solution is 7.8 to 8.2; the second stabilizer comprises a polyhydric alcohol, a second saccharide, an amino acid, a second surfactant, and a protein; polyhydric alcohol: a second saccharide: amino acid (b): second surfactant: the mass ratio of the proteins is as follows: (3.5-6.5): (1.1-3.5): (1-3): (0.1-0.5): (1-3); the second surfactant is Tween-20; the second saccharide comprises sucrose and dextran sulfate;
the third reagent comprises the following components in a total volume of 1L: 5 to 15g of third salt ion, 46 to 100100 g of third stabilizer, 0.1 to 1g of third preservative, 50 to 100100mmol of third buffer solution and 0.5 to 1.5mmol of chromogenic substrate; the pH value of the third buffer solution is 7.8 to 8.2; the third stabilizer comprises a polyhydric alcohol, a saccharide, and a third surfactant; polyhydric alcohol: saccharides: the third surfactant has the following mass ratio: (3-5): (1-3): (0.6-2); the third surfactant comprises Tween-20 and PVP.
2. The kit for measuring anti-Xa activity according to claim 1, wherein the first stabilizer includes mannitol, sucrose, PEG-8000, tween-20, and BSA; the second stabilizer comprises mannitol, sucrose, glycine, PEG-8000, tween-20, BSA and dextran sulfate; the third stabilizer includes mannitol, sucrose, PVP and Tween-20.
3. The kit for measuring anti-Xa activity according to claim 1, wherein the first salt ion, the second salt ion, and the third salt ion are each one of sodium chloride, calcium chloride, and ammonium sulfate.
4. The kit for measuring anti-Xa activity according to claim 3, wherein the first salt ion, the second salt ion, and the third salt ion are all sodium chloride.
5. The kit for measuring the anti-Xa activity according to claim 1, wherein the first preservative, the second preservative and the third preservative are each one of sodium azide, proClin 300 and ProClin 150.
6. The kit for measuring anti-Xa activity by exogenously adding antithrombin according to claim 5, wherein the first preservative, the second preservative, and the third preservative are all sodium azide.
7. The kit for measuring the anti-Xa activity, according to claim 1, wherein the first buffer, the second buffer, and the third buffer are each one of Tris buffer, HEPES buffer, and PBS buffer.
8. The kit for measuring anti-Xa activity according to claim 7, wherein the first buffer, the second buffer, and the third buffer are Tris buffers.
9. The kit for measuring anti-Xa activity according to claim 1, wherein the antithrombin is one of human antithrombin, porcine antithrombin, and bovine antithrombin.
10. The kit for measuring anti-Xa activity by exogenously adding antithrombin according to claim 1, wherein the antithrombin is human antithrombin.
11. The kit for measuring anti-Xa activity by exogenously adding antithrombin according to claim 1, wherein the blood coagulation factor is one of bovine factor Xa, porcine factor Xa and human factor Xa.
12. The kit for measuring anti-Xa activity according to claim 11, wherein the blood coagulation factor is bovine factor Xa.
13. The kit for measuring anti-Xa activity by exogenously adding antithrombin according to claim 1, wherein the chromogenic substrate is s-5288.
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| EP2818871A1 (en) * | 2013-06-28 | 2014-12-31 | Roche Diagniostics GmbH | Means and methods for universal calibration of anti-Factor Xa tests |
| CN104062243A (en) * | 2014-04-21 | 2014-09-24 | 上海贞元诊断用品科技有限公司 | FXa activity detection reagent, preparation method and application of FXa activity detection reagent |
| CN107153043A (en) * | 2017-06-23 | 2017-09-12 | 宁波艾科生物科技有限公司 | A kind of liquid instant Antiprothrombin antibodies determine reagent |
| CN107091814A (en) * | 2017-06-23 | 2017-08-25 | 宁波艾科生物科技有限公司 | A kind of detection reagent of liquid instant blood heparin concentration |
| CN107727872A (en) * | 2017-09-01 | 2018-02-23 | 上海太阳生物技术有限公司 | A kind of kit of heparin determination |
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| CN113341164A (en) * | 2021-07-21 | 2021-09-03 | 广州万孚生物技术股份有限公司 | Activated partial thromboplastin time determination reagent card and preparation method and application thereof |
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