CN114703252B - Kit for detecting content of hirudin, bivalirudin and dabigatran in blood plasma - Google Patents
Kit for detecting content of hirudin, bivalirudin and dabigatran in blood plasma Download PDFInfo
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Abstract
The invention discloses a kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma, which comprises an R1 reagent, an R2 reagent, a diluting reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator; the R1 reagent comprises thrombin, a first buffer solution and a first auxiliary material, the R2 reagent comprises a chromogenic substrate, a second buffer solution and a second auxiliary material, and the diluting reagent comprises a third buffer solution and a third auxiliary material; the hirudin calibrator comprises hirudin, the bivalirudin calibrator comprises bivalirudin, and the dabigatran calibrator comprises dabigatran; the reagent disclosed by the invention is high in sensitivity and good in stability, can be used for quickly and accurately detecting the content of hirudin, bivalirudin and dabigatran in blood plasma, can be used for simultaneously detecting the medication conditions of hirudin, bivalirudin and dabigatran, can be used for quickly and effectively knowing the drug residue concentration of a patient, and can be used for accurately evaluating the bleeding or thrombus risk in the patient.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma.
Background
A plurality of commercial direct thrombin inhibitors (anti-IIa activity 'DTI') can be used for treating thrombotic diseases, but a unified, effective and convenient drug monitoring method does not exist, the residual concentration of drugs in a patient cannot be accurately known, the bleeding or thrombus risk of the patient cannot be accurately evaluated, and great medication uncertainty exists. The direct thrombin inhibitors which are commonly used clinically comprise hirudin, bivalirudin, dabigatran and other medicaments, and with the increasing popularization of the clinical application of the medicaments, the monitoring of the medicament concentration and the anticoagulation effect is carried out in a targeted manner in various clinical complex states (especially in combination of various diseases, old and critically ill patients), so that the improvement of the treatment safety and the improvement of the prognosis are facilitated. For different types of direct thrombin inhibitors, medication monitoring using various methods including PT, APTT, ACT, ECT, dTT and LC-MS/MS are routinely recommended, but all suffer from limitations of varying degrees. The anti-IIa activity determination is a recommended detection method for monitoring the hirudin, bivalirudin and dabigatran anticoagulation effects, and in the anti-IIa activity determination methodology, the sensitivity and accuracy of a chromogenic substrate method are high, the anti-IIa activity in a patient can be accurately reflected, and the method is suitable for various automatic analytical instruments and has a wide prospect in clinical application; however, the existing kits based on the chromogenic substrate method have the following problems: the problem that only a single type of direct thrombin inhibitor can be detected, and different types of direct thrombin inhibitors (hirudin, bivalirudin and dabigatran) cannot be detected simultaneously is that the detection effect is poor when the multiple types of direct thrombin inhibitors (hirudin, bivalirudin and dabigatran) are detected; the reagent has poor sensitivity and stability and is easily interfered by other substances in blood plasma.
Disclosure of Invention
The invention aims to solve the technical problems that the kit for detecting the contents of hirudin, bivalirudin and dabigatran in blood plasma is provided aiming at the defects of the prior art, the reagent has high sensitivity and good stability, and can quickly and accurately detect the contents of hirudin, bivalirudin and dabigatran in blood plasma.
The technical scheme adopted by the invention for solving the technical problems is as follows: a kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma comprises an R1 reagent, an R2 reagent, a diluting reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator;
the R1 reagent comprises thrombin, a first buffer solution and a first auxiliary material, the R2 reagent comprises a chromogenic substrate, a second buffer solution and a second auxiliary material, and the diluting reagent comprises a third buffer solution and a third auxiliary material;
the hirudin calibrator comprises hirudin, the bivalirudin calibrator comprises bivalirudin, and the dabigatran calibrator comprises dabigatran.
Further, preferably, the thrombin is at least one of bovine thrombin and recombinant human thrombin, and the concentration of the thrombin is 1-3IU/mL.
Further, preferably, the chromogenic substrate is at least one of chromogenic substrate S-2238 and chromogenic substrate PA2493, and the concentration of the chromogenic substrate is 0.5-1.5 mmol/L.
Further, preferably, the first buffer solution, the second buffer solution and the third buffer solution are at least one of Tris buffer solution, hepes buffer solution, MES buffer solution, MOPS buffer solution, citric acid buffer solution or PBS buffer solution, and the pH of the first buffer solution, the pH of the second buffer solution and the pH of the third buffer solution are all 7.2-7.6.
Further, preferably, the first auxiliary material comprises a first stabilizer, and the first stabilizer comprises one or more of sodium chloride, glycine, sucrose, galactose, polyethylene glycol 4000, span-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride.
Further, it is preferred that the first stabilizer comprises the components: sodium chloride, glycine, sucrose, PEG-4000 and Span-40, wherein the components are as follows in percentage by mass in the R1 reagent: 0.5-1.5% of sodium chloride, 2-6% of glycine, 3-5% of sucrose, 0.5-1.5% of PEG-4000 and 0.1-1% of Span-40.
Further, preferably, the second auxiliary material comprises a second stabilizer, and the second stabilizer comprises at least one of glycine, galactose, polyvinylpyrrolidone, span-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride.
Further, it is preferred that the second stabilizer comprises the components: glycine, galactose, polyvinylpyrrolidone and Span-40, wherein the mass percentages of the components in the R2 reagent are as follows: 2-6% of glycine, 3-5% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-1% of Span-40.
Further, preferably, the third auxiliary material comprises a third stabilizer, and the third stabilizer comprises one or more of sodium chloride, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride.
Further, preferably, the third stabilizer comprises sodium chloride, and the mass percentage of the third stabilizer in the diluent is 0.5-1.5%.
Further, it is preferable that the R1 reagent, the R2 reagent, and the diluting reagent each include a preservative, and the preservative is at least one of sodium benzoate, sodium azide, proclin-300, gentamicin, and nitrite.
Further, preferably, the R1 reagent comprises heparinase, and the concentration of the heparinase is 1-3 IU/mL.
Further, preferably, the R1 reagent and the R2 reagent both include a protease protection agent, the protease protection agent is at least one of BSA, HAS, and Prionex, and the mass percentages of the protease protection agent in the R1 reagent and the R2 reagent are both 1-3%.
Further, preferably the hirudin calibrator comprises a first excipient, a first protectant; the bivalirudin calibrator comprises a second excipient and a second protective agent; the dabigatran calibration product comprises a third excipient and a third protective agent.
Further, the first excipient, the second excipient and the third excipient are preferably at least one of hydroxyethyl starch, glycine, mannitol, proline and casein.
Further, the first protective agent, the second protective agent and the third protective agent are preferably at least one of maltose, sucrose, glucose, trehalose, arginine and lysine.
Further, preferably, the content of hirudin in the hirudin calibrator is 5 ug/mL, the content of bivalirudin in the bivalirudin calibrator is 5 ug/mL, and the content of dabigatran in the dabigatran calibrator is 500 ng/mL.
The invention has the beneficial effects that: the invention provides a kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma; the reagent kit is in a liquid state, is convenient to use, low in cost, high in sensitivity, wide in linear range, strong in anti-interference capability and good in stability, can replace an imported anti-IIa activity determination reagent kit, fully meets the requirement of clinical examination, can reduce the cost of purchasing detection reagents in hospitals, reduces the detection cost of patients and reduces the burden; the sensitivity of the reagent is improved by using a specific buffer system, the discrimination of samples with different concentrations is increased, and the calibration operation, the linear range establishment and the clinical sample test are facilitated; the kit comprises a thrombin-containing R1 reagent and a chromogenic substrate-containing R2 reagent, and is matched with a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, so that the reagent has high sensitivity and good stability, can quickly and accurately detect the contents of hirudin, bivalirudin and dabigatran in blood plasma, can simultaneously realize the detection of the administration conditions of hirudin, bivalirudin and dabigatran, can quickly and effectively know the drug residue concentrations of hirudin, bivalirudin and dabigatran in a patient, and can evaluate the bleeding or thrombus risk in the patient.
Drawings
The invention will be further described with reference to the following drawings and examples, in which:
FIG. 1 is a graph showing the correlation between example 1 of the present invention and clinical specimens of hirudin detection using a reference reagent;
FIG. 2 is a graph showing the correlation between example 1 of the present invention and a reference reagent for detecting bivalirudin clinical specimens;
FIG. 3 is a graph showing the correlation results between example 1 of the present invention and a reference reagent for detecting a clinical sample of dabigatran;
FIG. 4 is a graph showing the correlation between clinical specimens of example 2 of the present invention and a reference reagent for detecting hirudin;
FIG. 5 is a graph showing the correlation between the assay results of example 2 of the present invention and a reference reagent for bivalirudin clinical specimens;
FIG. 6 is a graph showing the correlation results between example 2 of the present invention and a clinical sample of dabigatran detected using a reference reagent;
FIG. 7 is a graph showing the correlation between the clinical samples of example 3 of the present invention and a reference reagent for detecting hirudin;
FIG. 8 is a graph showing the correlation between example 3 of the present invention and a reference reagent for assaying bivalirudin clinical specimens;
FIG. 9 is a graph showing the correlation between example 3 of the present invention and a clinical sample of dabigatran detected using a reference reagent.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described in detail.
A kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma comprises an R1 reagent, an R2 reagent, a diluting reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator; the R1 reagent comprises thrombin, a first buffer solution and a first auxiliary material, the R2 reagent comprises a chromogenic substrate, a second buffer solution and a second auxiliary material, and the diluting reagent comprises a third buffer solution and a third auxiliary material; the hirudin calibrator comprises hirudin, the bivalirudin calibrator comprises bivalirudin, and the dabigatran calibrator comprises dabigatran.
The working principle of the invention is as follows: the method comprises the steps of calibrating an R1 reagent, an R2 reagent and a diluting reagent by using a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, adding excessive thrombin IIa into plasma to be detected, consuming the added IIa in proportion by residual direct thrombin inhibitors (hirudin, bivalirudin and dabigatran) in patient plasma, reacting the residual IIa on a chromogenic substrate, cracking out chromogenic groups (the released pNA is detected within a certain range), positively correlating the chromogenic degree with the amount of the residual IIa, negatively correlating with the content of the direct thrombin inhibitors (hirudin, bivalirudin and dabigatran) in the plasma, and further realizing the rapid detection of the content of the hirudin, the bivalirudin and the dabigatran.
The kit reagent is in a liquid state, is convenient to use, low in cost, high in sensitivity, wide in linear range, strong in anti-interference capability and good in stability, can replace an imported anti-IIa activity determination kit, fully meets the requirement of clinical examination, can reduce the cost for purchasing detection reagents in hospitals, reduces the detection cost of patients and reduces the burden; the sensitivity of the reagent is improved by using a specific buffer system, the discrimination of samples with different concentrations is increased, and the calibration operation, the establishment of a linear range and the test of clinical samples are facilitated; the kit comprises a R1 reagent containing thrombin and a R2 reagent containing a chromogenic substrate, and is matched with a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, so that the reagent has high sensitivity and good stability, can quickly and accurately detect the contents of hirudin, bivalirudin and dabigatran in blood plasma, can simultaneously realize the detection of the medication conditions of the hirudin, the bivalirudin and the dabigatran, can quickly and effectively know the drug residue concentrations of the hirudin, the bivalirudin and the dabigatran in a patient, and can accurately evaluate the bleeding or thrombus risk in the patient.
The source of the thrombin is not limited, preferably, the thrombin is at least one of bovine thrombin and recombinant human thrombin, and the concentration of the thrombin is 1-3IU/mL; at least one of chromogenic substrate S-2238 and chromogenic substrate PA2493, wherein the concentration of the chromogenic substrate is 0.5-1.5 mmol/L; preferably, thrombin is recombinant human thrombin, preferably, a chromogenic substrate is a chromogenic substrate PA2493, the recombinant human thrombin and the chromogenic substrate PA2493 are used for preparing reagents, and hirudin calibrator, bivalirudin calibrator and dabigatran calibrator are respectively matched for detecting the concentration of a direct thrombin inhibitor (hirudin, bivalirudin and dabigatran) in plasma.
The first buffer solution, the second buffer solution and the third buffer solution are at least one of Tris buffer solution, hepes buffer solution, MES buffer solution, MOPS buffer solution and citric acid buffer solution, the pH of the first buffer solution, the pH of the second buffer solution and the pH of the third buffer solution are all 7.2-7.6, the types of the buffer solutions which can be adopted by the invention are not particularly limited, according to the specific example of the invention, the first buffer solution, the second buffer solution and the third buffer solution are preferably Tris buffer solution as a buffer system, the concentration of the Tris buffer solution is 60-100mmol/L, the R1 reagent, the R2 reagent and the R3 reagent can be effectively maintained in the preset range, namely 7.2-7.6, by adopting the buffer system, the content determination of hirudin, billuding and dabigatran in a sample to be detected can not be adversely affected, and the thrombin and the substrate can exert the maximum efficiency under the buffer system, the sensitivity of the chromogenic reagent is improved, the sensitivity of samples with different concentrations is increased, and the operation, the linear range is favorable for establishing a clinical sample test.
Furthermore, the first auxiliary material also comprises a first stabilizer, the first stabilizer comprises one or more of sodium chloride, glycine, sucrose, galactose, polyethylene glycol 4000, span-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride, and the addition of the first stabilizer can improve the stability of the reagent, inhibit the non-specific adsorption in the system on the premise of not influencing the sensitivity of the reaction system, and more importantly, can also effectively protect the activity of thrombin and improve the stability of the thrombin; inorganic salts such as sodium chloride and potassium chloride, and the addition of the inorganic salts can be used for adjusting the osmotic pressure of the thrombin solution, so that the efficiency of the thrombin in the determination of the content of hirudin, bivalirudin and dabigatran can be improved; the addition of Span-40, tween-20 and the like can play a role of a surfactant, so that the stability of the reagent is further improved; the addition of polyhydric alcohols such as polyethylene glycol 4000 can improve the viscosity of the reagent, so that thrombin can be uniformly suspended in the first buffer solution and is not easy to settle, and a good stability effect is achieved; meanwhile, the first stabilizer can also play a certain role in protecting the protease so as to improve the stability of the kit.
In some embodiments, the first stabilizer comprises sodium chloride, glycine, sucrose, PEG-4000, span-40, and the mass percentages of the above components in the R1 reagent are: 0.5-1.5% of sodium chloride, 2-6% of glycine, 3-5% of sucrose, 0.5-1.5% of PEG-4000 and 0.1-1% of Span-40. The reagent kit has high accuracy and good stability.
Further, the second auxiliary material also comprises a second stabilizing agent, and the second stabilizing agent comprises at least one of glycine, galactose, polyvinylpyrrolidone, span-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride. The addition of the second stabilizer can improve the stability of the reagent, inhibit the nonspecific adsorption in the system on the premise of not influencing the sensitivity of the reaction system, and more importantly, improve the stability of the reagent; the addition of inorganic salts such as potassium chloride can be used for adjusting the osmotic pressure of the solution with the chromogenic substrate, thereby improving the efficiency of the chromogenic substrate in determining the content of hirudin, bivalirudin and dabigatran.
In a specific embodiment, the second stabilizer comprises glycine, galactose, polyvinylpyrrolidone and Span-40, and the mass percentages of the components in the R2 reagent are as follows: 2-6% of glycine, 3-5% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-1% of Span-40. The reagent kit has high accuracy and good stability.
Furthermore, the third auxiliary material also comprises a third stabilizer, wherein the third stabilizer comprises one or more of sodium chloride, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride, so as to improve the stability of the kit.
In one embodiment, the third stabilizer comprises sodium chloride, and the mass percentage of the third stabilizer in the diluting agent is 0.5-1.5%.
Further, the R1 reagent, the R2 reagent and the diluting reagent comprise a preservative, and the preservative is at least one of sodium benzoate, sodium azide, proclin-300, gentamicin and nitrite. The preservative is added to achieve the preservative effect, so that the kit is prevented from losing efficacy due to microbial pollution, and the storage period of the kit is prolonged.
Further, the R1 reagent comprises heparinase, and the concentration of the heparinase is 1-3 IU/mL. The influence of residual heparin in plasma is reduced when the content of hirudin, bivalirudin and dabigatran is detected by adding heparinase, thrombin in a reagent is prevented from being inactivated by combining heparin and antithrombin III, the condition that the activity of antithrombin III is inaccurate is further avoided, the capability of inactivating thrombin by antithrombin III is enhanced, and the anti-interference capability of the kit is improved. In the kit, polybrene and protamine in the prior art are replaced by heparinase, and although the heparinase, the polybrene and the protamine can reduce the influence of residual heparin in blood plasma, the action mechanisms are different. Specifically, the mechanism by which polybrene and protamine reduce the effect of heparin is: polybrene and protamine are strongly basic cationic substances, and are combined with heparin anions in a ratio of 1. The mechanism of reducing heparin effect by heparinase is as follows: heparinases are a class of polysaccharide-cleaving enzymes that act on heparin or heparan molecules, selectively cleaving the alpha (1-4) glycosidic bond between glucosamine and uronic acid in sulfated heparan glycans, rendering heparin inactive. Therefore, the action mechanisms of heparinase, polybrene and protamine are different, and the effect of using heparinase to neutralize heparin is better than that of polybrene and protamine.
Furthermore, the R1 reagent and the R2 reagent both comprise protease protective agents, the protease protective agents are at least one of BSA, HAS and Prionex, and the protease protective agents are used for prolonging the stability of the R1 reagent and the R2 reagent in the kit, are favorable for long-term use and storage of the kit, are favorable for protecting the activity of enzyme and preventing thrombin from denaturation, and play a role in protecting thrombin by improving the concentration of protein in a solution. Prevent enzyme decomposition and nonspecific adsorption, and reduce denaturation of some enzymes and adverse environmental factors such as heat, surface tension and chemical factors. The mass percentages of the protease protective agent in the R1 reagent and the R2 reagent are both 1-3%, and the preferred protease protective agent is Prionex.
Further, the hirudin calibrator comprises a first excipient, a first protectant; the bivalirudin calibrator comprises a second excipient and a second protective agent; the dabigatran calibration product comprises a third excipient and a third protective agent. The first excipient, the second excipient and the third excipient are respectively used for maintaining the shapes of the hirudin calibrator, the bivalirudin calibrator and the dabigatran calibrator in the freeze-drying process, can bear large temperature range change, are favorable for maintaining a cake-shaped structure, and are very small in dosage, the first excipient, the second excipient and the third excipient can be at least one of hydroxyethyl starch, glycine, mannitol, proline and casein, in a specific embodiment, the first excipient, the second excipient and the third excipient are all hydroxyethyl starch, and the mass percentage is 3%. The first protective agent, the second protective agent and the third protective agent are respectively used for protecting main drug substances in the freeze-drying process of the hirudin calibrator, the bivalirudin calibrator and the dabigatran calibrator, so that the loss of the hirudin, the bivalirudin and the dabigatran in the freeze-drying process is reduced, the due contents and activities of the hirudin, the bivalirudin and the dabigatran are maintained, the detection result of the freeze-dried calibrator is in an expected acceptance range, and the requirement of the calibrator is met. Further, the first protective agent, the second protective agent and the third protective agent can be at least one of maltose, sucrose, glucose, trehalose, arginine and lysine, and in a specific embodiment, the first protective agent, the second protective agent and the third protective agent are all maltose, and the mass percentage is 3%.
Further, the content of hirudin in the hirudin calibrator is 5 ug/mL, the content of bivalirudin in the bivalirudin calibrator is 5 ug/mL, and the content of dabigatran in the dabigatran calibrator is 500 ng/mL; the reagent is matched with a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator for use, and can simultaneously realize the determination of the contents of hirudin, bivalirudin and dabigatran.
The present invention is further explained below by means of specific embodiments.
Example 1
A kit comprises an R1 reagent, an R2 reagent, a dilution reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, and the specific components are shown in the following table:
TABLE 1 reagent Components of the kit of example 1
Note: "/" indicates the absence of this component.
Example 2
A kit comprises an R1 reagent, an R2 reagent, a diluting reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, and the specific components are shown in the following table:
TABLE 2 reagent Components of the kit of example 2
Note: "/" indicates the absence of this component.
Example 3
A kit comprises an R1 reagent, an R2 reagent, a dilution reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator, and the specific components are shown in the following table:
TABLE 3 reagent Components of the kit of example 3
Note: "/" indicates the absence of this component.
Comparative example 1
In comparative example 1, the buffer solution in the R1 reagent, the R2 reagent, and the diluting reagent was PB buffer solution, and other components and amounts were the same as in example 1.
Comparative example 2
Comparative example 2 did not contain heparinase, and the other components and amounts were the same as in example 1.
Comparative example 3
In comparative example 3, heparinase in comparative example 1 was replaced by polybrene, and other components and amounts were the same as in example 1.
Comparative example 4
In comparative example 4, the heparinase of comparative example 1 was replaced with protamine, and the other components and the amount were the same as those of example 1.
Comparative example 5
In comparative example 5, the protease protective agent Prionex was not contained in the R1 reagent, the R2 reagent, and the diluting reagent, and the rest were the same as in example 1.
The anti-IIa activity assay kits of examples 1-3 and comparative examples 1-5 were subjected to calibration, blank limit, accuracy, linear range, reproducibility, stability and clinical relevance tests.
1. Scaling and results thereof
The anti-IIa activity assay kits of examples 1-3 and comparative example 1 were used to complete the calibration of hirudin, bivalirudin and dabigatran items on a fully automated coagulation analyzer in combination with a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator. Each item of the calibrator contained 5 concentration levels of C0, C1, C2, C3, C4, and C5 (with increasing concentrations of C0-C5 in order), and the calibration results are shown in tables 4-7. The calibration result should meet the requirements: the linear regression equation r of the calibration curve is more than or equal to 0.980.
TABLE 4 calibration results for the kit of example 1
TABLE 5 calibration results for the kit of example 2
TABLE 6 calibration results for the kit of example 3
TABLE 7 calibration results for the kit of comparative example 1
The test results in tables 4-7 show that r of the calibration curvilinear regression equation in the embodiment 1-3 is more than or equal to 0.980, which meets the requirements, and proves that the embodiment 1-3 can obtain a better calibration curve. Comparative example 1 the r of the calibration curvilinear regression equation is less than 0.980, which is not in accordance with the requirement, the difference of OD values of adjacent calibrators is small, the drawing of the sensitivity of the reagent and the calibration curve is influenced, further, the thrombin and the chromogenic substrate can exert the maximum efficiency in Tris buffer, the sensitivity of the reagent is improved, the discrimination of samples with different concentrations is increased, and the calibration operation, the establishment of linear range and the test of clinical samples are facilitated.
2. Margin test and results thereof
Blank samples were measured 20 times using the kits of examples 1 to 3, and the average value was calculated according to the following formulas (1) and (2) ((
) Standard Deviation (SD) and margin (b) ((b))
+2 SD), the test results are shown in tables 8-10. The test result is required to be: the blank limit of the hirudin and bivalirudin projects is less than or equal to 0.2 mug/mL, and the blank limit of the dabigatran projects is less than or equal to 20 ng/mL.
In the formula:
n-the number of tests;
i——the serial number of the test;
B-relative deviation;
SD-standard deviation.
TABLE 8 blank limit test results for the kit of example 1
TABLE 9 blank Limit test results for the kit of example 2
TABLE 10 blank limit test results for the kit of example 3
From the test results of tables 8-10, it was found that examples 1-3 all met the specified margin requirements.
3. Accuracy test and results thereof
Hirudin calibrants (C1, C4), bivalirudin calibrants (C1, C4), and dabigatran calibrants (C1, C4) containing exogenously added heparin were each assayed using the kits of examples 1-3 and comparative example 2, and the test was repeated 3 times for each calibrator, and the relative deviation was calculated according to the above equations (1) and (3), and the test results are shown in tables 11-16. The test result is required to be: the relative deviation should be within 15%.
Formula (3):
in the formula:
t-calibrator index value;
b-relative deviation.
TABLE 11 accuracy test results of the kit of example 1
TABLE 12 accuracy test results of the kit of example 2
TABLE 13 accuracy test results of the kit of example 3
TABLE 14 accuracy test results of the kit of comparative example 2
TABLE 15 accuracy test results of the kit of comparative example 3
TABLE 16 accuracy test results of the kit of comparative example 4
The test results in tables 11-16 show that the relative deviation of the examples 1-3 is relatively small, which proves that the examples 1-3 have good accuracy, and the relative deviation of the comparative examples 2-4 does not meet the requirement that the relative deviation is within the range of +/-15%, and the test results of the examples 1 and the comparative examples 2-4 show that the influence of the residual heparin in the plasma when the content of hirudin, bivalirudin and dabigatran is detected is reduced by adding heparinase, the thrombin in the reagent is prevented from being inactivated by combining the heparin and the antithrombin III, the condition that the measurement of the activity of the antithrombin III is inaccurate is avoided, the thrombin inactivation capability of the antithrombin III is enhanced, and the anti-interference capability of the kit is improved. Comparative examples 2-4 were disturbed by free heparin in the sample, resulting in less thrombin cleaving the PA2493 substrate, weaker chromogenic strength, and higher test results. Thus, comparative examples 2-4 are less accurate than examples 1-3, further demonstrating that the use of heparinase has a superior effect on reducing heparin interference compared to polybrene and protamine.
4. Linear range test and results thereof
The hirudin, bivalirudin and dabigatran high-value samples which are close to the upper limit of the linear range of the kit are respectively diluted into 5 samples with different concentrations, each sample with different concentrations is tested for 3 times, the theoretical concentration is (xi), the mean value of the actual measurement results is (yi), a linear regression equation is solved, and the correlation coefficient (r) of the linear regression is calculated, wherein the test results are shown in tables 17-19. The test result is required to be: when the hirudin is 0.2-5 mug/mL, the bivalirudin is 0.2-5 mug/mL and the dabigatran is in the linear range of 30-500 ng/mL, the linear correlation coefficient r is more than or equal to 0.990.
TABLE 17 Linear Range test results for the kit of example 1
TABLE 18 Linear Range test results for the kit of example 2
TABLE 19 results of the Linear Range test of the kit of example 3
From the test results of tables 17 to 19, it was found that examples 1 to 3 all meet the above-mentioned linear range requirement.
5. Repeatability tests and results thereof
The low-value and high-value quality control substances of hirudin, low-value and high-value quality control substances of bivalirudin, and low-value and high-value quality control substances of dabigatran were each tested 10 times using the kits of examples 1 to 3. Calculating the average value of the test results according to the formulas (1) and (2) ((
) And Standard Deviation (SD), coefficient of Variation (CV) was calculated according to equation (4), and the test results are shown in tables 20 to 22. The test result is required to be: the variation Coefficient (CV) of the hirudin low-value quality control product, the bivalirudin low-value quality control product and the dabigatran low-value quality control product is less than or equal to 10 percent; the Coefficient of Variation (CV) of the hirudin high-value quality control product, the bivalirudin high-value quality control product and the dabigatran high-value quality control product is less than or equal to 8 percent.
Formula (4):
in the formula: CV is the coefficient of variation.
TABLE 20 results of the reproducibility test of the kit of example 1
TABLE 21 results of the reproducibility tests of the kit of example 2
TABLE 22 results of the reproducibility test of the kit of example 3
From the test results of tables 20 to 22, it was found that examples 1 to 3 all met the above-mentioned reproducibility requirements.
6. Stability testing and results thereof
1) Bottle opening stability: the R1 reagent, the R2 reagent, and the diluting reagent in the kit of examples 1 to 3 were stored at 2 to 8 ℃ after decapping, and accuracy tests were performed on days 0, 30, 50, 55, 60, and 65, and the test results are shown in tables 23 to 25. The test result is required to be: the R1 reagent, the R2 reagent and the diluting reagent in the kit are placed at 2-8 ℃ after being unpacked and can be stored stably for 60 days, namely the relative deviation is within the range of +/-15%.
TABLE 23 open bottle stability test results of the kit of example 1
TABLE 24 open bottle stability test results of the kit of example 2
TABLE 25 open bottle stability test results of the kit of example 3
From the test results in tables 23 to 25, it was found that examples 1 to 3 meet the above-mentioned requirements for stability in opening bottles, that is, the reagent R1, the reagent R2 and the diluting reagent in the kit can be stored at 2 to 8 ℃ for 65 days after opening bottles.
2) Accelerated stability: the R1 reagent, the R2 reagent and the diluting reagent in the kits of examples 1 to 3 and comparative example 3 were stored at 37 ℃ in an unopened state, and accuracy tests were performed on days 0, 8, 10 and 12, and the test results are shown in tables 26 to 29. The test result meets the requirements: the reagent R1, the reagent R2 and the diluting reagent in the kit can be stored for 10 days at 37 ℃ in an unsealed state, namely, the relative deviation is within the range of +/-15%.
TABLE 26 accelerated stability test results for the kit of example 1
TABLE 27 accelerated stability test results for the kit of example 2
TABLE 28 accelerated stability test results for the kit of example 3
TABLE 29 accelerated stability test results for the kit of comparative example 3
From the test results of tables 26 to 29, it was found that examples 1 to 3 meet the above accelerated stability requirements, that is, the R1 reagent, the R2 reagent, and the diluting reagent in the kit can be stored at 37 ℃ in an unopened state for 10 days, comparative example 3 to which no protease inhibitor is added does not meet the above accelerated stability requirements, and the kits of examples 1 to 3 have higher accelerated stability than comparative example 3.
7. Clinical relevance test and results thereof
A group of clinical specimens covering a linear range (40 cases) were simultaneously tested using a reference kit (Hyphen, france) purchased from a third party and the kits of examples 1 to 3, and a linear regression analysis was performed using the measured value of the reference kit as the X-axis and the measured values of the kits of examples 1 to 3 as the Y-axis, and the test results are shown in tables 30 to 32 and attached FIGS. 1 to 9. The test result is required to be: the linear regression analysis should conform to the linear regression equation with a slope k between 0.9 and 1.1 and a correlation coefficient r of 0.975 or more.
TABLE 30 correlation results of example 1 test clinical specimens with reference reagents
TABLE 31 correlation results of example 2 with reference reagent test clinical specimens
TABLE 32 correlation results of example 3 test clinical specimens with reference reagents
From the test results shown in tables 30 to 32 and fig. 1 to 9, it can be seen that the results of the test clinical samples of the kits and reference kit reagents of examples 1 to 3 are subjected to linear regression analysis, and the slope k and the correlation r of the linear regression equation both meet the requirements, thus proving that the kits and reference reagents of examples 1 to 3 have good correlation.
The data and the accompanying drawings show that the results obtained by using the kit of the invention to carry out related performance tests all meet the acceptance standards. The feasibility and rationality of the invention was demonstrated by the specific examples described above, which are only illustrative of alternative individual embodiments of the invention, but not limiting thereto. All changes, modifications and substitutions that may be suggested to one skilled in the art and included within the scope of the appended claims are intended to be embraced by the invention.
Claims (5)
1. A kit for detecting the content of hirudin, bivalirudin and dabigatran in blood plasma is characterized by comprising an R1 reagent, an R2 reagent, a diluting reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator;
the R1 reagent is composed of thrombin, a first buffer solution, heparinase, a protease protective agent and a first auxiliary material, the thrombin is recombinant human thrombin, the concentration of the recombinant human thrombin is 1-3 IU/mL, the concentration of the heparinase is 1-3 IU/mL, the first auxiliary material is composed of sodium chloride, glycine, sucrose, PEG-4000 and Span-40, and the mass percentages of the components in the R1 reagent are as follows: 0.5-1.5% of sodium chloride, 2-6% of glycine, 3-5% of sucrose, 0.5-1.5% of PEG-4000 and 0.1-1% of Span-40;
the R2 reagent consists of a chromogenic substrate, a second buffer solution, a protease protective agent and a second auxiliary material, wherein the chromogenic substrate is a chromogenic substrate PA2493, the second auxiliary material consists of glycine, galactose, polyvinylpyrrolidone and Span-40, and the mass percentages of the components in the R2 reagent are as follows: 2-6% of glycine, 3-5% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-1% of Span-40;
The diluting reagent consists of a third buffer solution and a third auxiliary material, wherein the third auxiliary material is sodium chloride, and the mass percent of the sodium chloride in the diluting reagent is 0.5-1.5%;
the first buffer solution, the second buffer solution and the third buffer solution are Tris buffer solutions, the concentration of the Tris buffer solutions is 60 mmol/L-100 mmol/L, and the pH values of the first buffer solution, the second buffer solution and the third buffer solution are all 7.2-7.6;
the protease protective agent is any one of BSA, HSA and Prionix, and the mass percentages of the protease protective agent in the R1 reagent and the R2 reagent are both 1-3%;
the hirudin calibrator comprises hirudin, the bivalirudin calibrator comprises bivalirudin, and the dabigatran calibrator comprises dabigatran.
2. The kit according to claim 1, characterized in that the concentration of the chromogenic substrate PA2493 is between 0.5 and 1.5 mmol/L.
3. The kit of claim 1, wherein the R1 reagent, R2 reagent, and diluting reagent each comprise a preservative, and the preservative is sodium benzoate.
4. The kit of claim 1, wherein the hirudin calibrator comprises hydroxyethyl starch and maltose, and the bivalirudin calibrator comprises hydroxyethyl starch and maltose; the dabigatran calibration material comprises hydroxyethyl starch and maltose.
5. The kit according to claim 1, wherein the hirudin content in the hirudin calibrator is 5 ug/mL, the bivalirudin content in the bivalirudin calibrator is 5 ug/mL, and the dabigatran content in the dabigatran calibrator is 500 ng/mL.
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| CN107936115A (en) * | 2018-01-03 | 2018-04-20 | 浙江中医药大学 | Hirudin extracting method |
| CN114544977A (en) * | 2022-03-25 | 2022-05-27 | 武汉市长立生物技术有限责任公司 | Antithrombin III determination kit |
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| CN104965017B (en) * | 2015-06-17 | 2017-06-16 | 广西师范学院 | The assay method of hirudin anticoagulant hemase activity |
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| US20220146538A1 (en) * | 2020-11-12 | 2022-05-12 | Joseph Louis Spano | Method and device for detecting coagulation factor inhibitors and antiplatelets in a sample |
| CN114277089B (en) * | 2021-12-24 | 2023-05-02 | 北京赛科希德科技股份有限公司 | Detection reagent and kit for dabigatran |
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| CN107936115A (en) * | 2018-01-03 | 2018-04-20 | 浙江中医药大学 | Hirudin extracting method |
| CN114544977A (en) * | 2022-03-25 | 2022-05-27 | 武汉市长立生物技术有限责任公司 | Antithrombin III determination kit |
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