CN114736948B - Alpha 2-antifibrinolytic enzyme activity determination kit - Google Patents
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Abstract
The invention discloses an alpha 2-antifibrinolytic enzyme activity determination kit, which comprises R1, R2 and a diluting reagent; the R1 reagent comprises recombinant bovine plasmin, a first buffer solution and a first auxiliary material, and the concentration of the recombinant bovine plasmin is 5-7 IU/mL; the R2 reagent comprises a chromogenic substrate S-2403, a second buffer solution and a second auxiliary material, wherein the concentration of the chromogenic substrate S-2403 is 2-4 mmol/L; the diluting reagent comprises a third buffer solution and a third auxiliary material, and the pH values of the first buffer solution, the second buffer solution and the third buffer solution are 7.2-7.6; the recombinant bovine plasmin and the chromogenic substrate S-2403 are matched for use, so that the cutting efficiency is high, the reaction signal is strong, the reaction speed is high, the sensitivity is high, the linear range is wide, the reaction time is short, the discrimination of a sample is increased, and the establishment of the linear range and the test of a clinical sample are facilitated; the reagent is liquid, and has convenient use, low cost and good stability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an alpha 2-antifibrinolytic enzyme activity determination kit.
Background
Plasminogen is activated by PA (plasminogen activator), and after being converted into PL, the plasminogen not only plays a role in fibrinolysis and thrombolysis, but also participates in a series of physiological and pathological processes related to proteolysis in vivo. Plasmin is activated in blood and then is rapidly combined with alpha 2-antiplasmin to form plasmin-alpha 2-antiplasmin compound for inactivation, the decrease of fibrinolytic activity is one of important mechanisms causing thrombosis, alpha 2-antiplasmin (alpha 2-AP) is used for directly inhibiting the activity of plasmin, detecting the activity of alpha 2-antiplasmin in human body, being helpful for judging the fibrinolytic state in the body and being used for the auxiliary diagnosis of bleeding and thrombotic diseases; the activity of alpha 2-antiplasmin is generally determined clinically by a spectrophotometer method and an immunochemical method, although the method has certain operability, the repeatability of a detection result is poor, the anti-interference capability is poor, the result is easy to be influenced by various factors, and the real activity level of the alpha 2-antiplasmin in a patient body cannot be reflected.
With the clinical improvement of the requirement on the detection of the activity of the alpha 2-antiplasmin, the alpha 2-antiplasmin activity determination kit adapted to a full-automatic coagulation analyzer is firstly provided abroad, but the kit needs to be matched with the imported full-automatic coagulation analyzer and a matched kit for use, is expensive in pricing, and brings certain economic burden to hospitals and patients; secondly, most of the commercially available alpha 2-antifibrinolytic enzyme activity determination kits (chromogenic substrate method) are still in the form of freeze-dried powder, and are redissolved, inverted and uniformly mixed before use, and can be used after standing for a period of time, so that the operation of customers is not facilitated, and the detection efficiency is reduced; in addition, the matched complex solvent is not generally given in the box of the alpha 2-antifibrinolytic enzyme activity determination kit, a customer needs to prepare the complex solvent by himself, the quality of the used complex solvent cannot be controlled, and certain influence is brought to a test result.
Disclosure of Invention
The invention aims to solve the technical problem of providing an alpha 2-antiplasmin activity determination kit aiming at the defects of the prior art, which has the advantages of low price, full liquid, high reagent sensitivity, wide linear range, convenience and quickness.
The technical scheme adopted by the invention for solving the technical problems is as follows: an alpha 2-antifibrinolytic enzyme activity determination kit, comprising an R1 reagent, an R2 reagent and a diluting reagent;
the R1 reagent comprises recombinant bovine plasmin, a first buffer solution and a first auxiliary material, the concentration of the recombinant bovine plasmin is 5-7 IU/mL, and the pH value of the first buffer solution is 7.2-7.6;
the R2 reagent comprises a chromogenic substrate S-2403, a second buffer solution and a second auxiliary material, wherein the concentration of the chromogenic substrate S-2403 is 2-4 mmol/L, and the pH value of the second buffer solution is 7.2-7.6;
the diluting reagent comprises a third buffer solution and a third auxiliary material, and the pH value of the third buffer solution is 7.2-7.6.
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 and citric acid buffer solution.
Further, preferably, the first buffer solution, the second buffer solution and the third buffer solution are all Tris buffer solutions with the concentration of 100-150 mmol/L.
Further, preferably, the first auxiliary material comprises a first stabilizer, and the first stabilizer comprises one or more of sodium chloride, glutamic acid, sucrose, galactose, polyethylene glycol 2000, NP-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, glutamic acid, sucrose, polyethylene glycol 2000 and NP-40, wherein the mass percentages of the components in the R1 reagent are as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of sucrose, 1-3% of PEG-2000 and 0.5-1.5% of NP-40.
Further, preferably, the second auxiliary material comprises a second stabilizer, and the second stabilizer comprises at least one of sodium chloride, glutamic acid, sucrose, polyvinylpyrrolidone, NP-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride.
Further, it is preferred that the second stabilizer comprises the components: sodium chloride, glutamic acid, sucrose, PVP and NP-40, wherein the mass percentages of the components in the R2 reagent are as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of cane sugar, 1-3% of PVP and 0.5-1.5% of NP-40.
Further, preferably, the third auxiliary material further 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.
Further, preferably, the third stabilizer comprises sodium chloride, and the mass percentage of the sodium chloride 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, the R1 reagent, the R2 reagent and the diluting reagent preferably comprise 0.03-0.05% of Proclin-300 by mass percent.
Further, preferably, the R1 reagent and the R2 reagent each include a protease protective agent, and the protease protective agent is at least one of BSA, HAS, and Prionex.
Further, it is preferable that the protease protection agent is Prionix, and the mass percentages of Prionix in the R1 reagent and the R2 reagent are both 1 to 5%.
Further, preferably, the R1 reagent also comprises PAI-1, and the concentration of the PAI-1 is 1-2 IU/mL.
The invention has the beneficial effects that: the invention provides a 2 The kit reagent is in a liquid state, is convenient to use, has low cost, high sensitivity, wide linear range, strong anti-interference capability and good stability, is an original determination kit for detecting the alpha 2-antiplasmin activity in a full liquid state in China, can replace an imported alpha 2-AP activity determination kit, and fully meets the requirements of clinical examination; the cost of purchasing detection reagents in hospitals can be reduced, the detection cost of patients is reduced, and the burden is reduced; by using the recombinant bovine plasmin and the chromogenic substrate S-2403 as reagent raw materials, when the recombinant bovine plasmin and the chromogenic substrate S-2403 are used in a matched manner, the cutting efficiency is high during detection, the reaction signal is strong, the reaction speed is high, the reagent sensitivity is higher, the linear range is wider, the required reaction time is shorter, the discrimination of samples with different concentrations is increased, and the calibration operation, the establishment of the linear range and the clinical sample test are facilitated.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a plot of a linear regression of the correlation of the kit of example 1 of the invention with a reference kit;
FIG. 2 is a plot of a linear regression of the correlation of the kit of example 2 of the invention with a reference kit;
FIG. 3 is a plot of the linear regression of the correlation of the kit of example 3 of the invention with a reference kit.
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.
An alpha 2-antifibrinolytic enzyme activity determination kit, comprising an R1 reagent, an R2 reagent and a diluting reagent; the R1 reagent comprises recombinant bovine plasmin, a first buffer solution and a first auxiliary material, the concentration of the recombinant bovine plasmin is 5-7 IU/mL, and the pH value of the first buffer solution is 7.2-7.6; the R2 reagent comprises a chromogenic substrate S-2403, a second buffer solution and a second auxiliary material, wherein the concentration of the chromogenic substrate S-2403 is 2-4 mmol/L, and the pH value of the second buffer solution is 7.2-7.6; the diluting reagent comprises a third buffer solution and a third auxiliary material, and the pH value of the third buffer solution is 7.2-7.6.
The working principle of the kit is as follows: adding excessive recombinant bovine plasmin into a reaction system, wherein the recombinant bovine plasmin and alpha 2-AP (alpha 2-antiplasmin) in plasma form 1: 1, namely the recombinant bovine plasmin-alpha 2 antiplasmin complex, the residual recombinant bovine plasmin acts on a chromogenic substrate S-2403 to release p-nitroaniline (pNA), the depth of chromogenic reaction is positively correlated with the amount of the residual recombinant bovine plasmin, and is negatively correlated with the activity of the alpha 2 antiplasmin, thereby realizing the rapid detection of the activity of the alpha 2 antiplasmin.
According to the alpha 2-antiplasmin activity determination kit provided by the invention, the recombined bovine plasmin and the chromogenic substrate S-2403 are used as reagent raw materials, and when the recombined bovine plasmin and the chromogenic substrate S-2403 are used in a matching manner, the cutting efficiency is high, the reaction signal is strong, the reaction speed is high, the reagent sensitivity is higher, the linear range is wider, the required reaction time is shorter, the discrimination of samples with different concentrations is increased, and the calibration operation, the establishment of the linear range and the test of clinical samples are facilitated; 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, is a domestic pioneer full-liquid detection alpha 2-antiplasmin activity determination kit, can replace an imported alpha 2-AP activity determination kit, and fully meets the requirements of clinical examination; the cost of purchasing detection reagents in hospitals can be reduced, the detection cost of patients is reduced, and the burden is reduced.
The recombinant bovine plasmin is prepared by genetic engineering, the purity of the enzyme is higher, the unit activity is stronger, the use amount of the enzyme is lower, and the cost is saved; the chromogenic substrate S-2403 is a plasmin-specific high-efficiency substrate, the amino acid sequence of the substrate is specially adjusted and modified, and compared with other common chromogenic substrates, the plasmin-specific chromogenic substrate has higher sensitivity, higher cutting efficiency and quicker reaction; when the recombinant bovine plasmin and the chromogenic substrate S-2403 are used in a matching way, the cutting efficiency is high, the reaction signal is strong, the reaction speed is high, the reagent sensitivity is higher, the linear range is wider, the required reaction time is shorter, the discrimination of samples with different concentrations is increased, and the calibration operation, the establishment of the linear range and the clinical sample test are facilitated; in terms of reaction time, the R1 reagent containing the recombinant bovine plasmin and the sample to be detected are not incubated, the incubation time is only about 60s after the R2 reagent is added, the R1 reagent and the sample to be detected of the conventional kit are not incubated or the incubation time is between 60 and 180s, and the incubation time is between 120 and 240s after the R2 reagent of the conventional kit is added.
The first buffer solution, the second buffer solution and the third buffer solution are respectively at least one of Tris buffer solution, hepes buffer solution, MES buffer solution, MOPS buffer solution and citric acid buffer solution. The pH values of the first buffer solution, the second buffer solution and the third buffer solution are all 7.2-7.6, the R1 reagent, the R2 reagent and the diluting reagent can be effectively maintained within a preset range by adopting any one of the buffer solutions, namely, the solution is stabilized between the pH values of 7.2-7.6, and the first buffer solution, the second buffer solution and the third buffer solution do not cause adverse influence on the activity of alpha 2-antifibrinolytic enzyme to be measured in a sample to be detected; further, preferably, the first buffer solution, the second buffer solution and the third buffer solution are all Tris buffer solutions with the concentration of 100-150 mmol/L, under the buffer solutions, the recombinant bovine plasmin and the chromogenic substrate S-2403 can exert the maximum efficiency, the sensitivity of the reagent is improved, 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 first auxiliary material comprises a first stabilizer, and the first stabilizer comprises one or more of sodium chloride, glutamic acid, sucrose, galactose, polyethylene glycol 2000, NP-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride. The addition of the first 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, can also effectively protect the activity of the recombinant bovine plasmin in the reagent and improve the stability of the recombinant bovine plasmin; for example, the addition of sucrose, galactose, trehalose, glucose, gelatin and the like is beneficial to inhibiting the freeze denaturation of protein and can ensure that the freezing can not cause the precipitation and nonspecific aggregation of an R1 reagent, thereby ensuring the stability of the reagent in storage, transportation and the like, and gelatin macromolecules have a net structure and can generate a sieve pore effect, thereby performing space limitation on the protein, reducing the mutual collision among protein molecules and improving the stability of the protein to a certain extent; the addition of polyhydric alcohols such as polyethylene glycol 2000 and the like can improve the viscosity of the reagent, is beneficial to the uniform suspension of the recombinant bovine plasmin in the first buffer solution, is not easy to settle, plays a good role in stability, and can also be used as an antifreeze agent to effectively reduce the freezing point, thereby achieving a good freeze-thaw resistance effect; the addition of inorganic salts such as sodium chloride, potassium chloride and the like can be used for adjusting the osmotic pressure of the solution with the recombinant bovine plasmin, thereby improving the efficiency of the recombinant bovine plasmin in the determination of the alpha 2-antiplasmin; tween-20 and the like are added to play a role of a surfactant, so that the stability of the reagent is further improved; meanwhile, the addition of the first stabilizer can also play a certain role in protecting the protease so as to improve the stability of the kit.
In a particular embodiment, the first stabilizer comprises the components: sodium chloride, glutamic acid, sucrose, polyethylene glycol 2000 and NP-40, wherein the weight percentage of the components in the R1 reagent is as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of sucrose, 1-3% of PEG-2000 and 0.5-1.5% of NP-40; the reagent kit has high accuracy and good stability.
The second auxiliary material comprises a second stabilizer, and the second stabilizer comprises at least one of sodium chloride, glutamic acid, sucrose, polyvinylpyrrolidone (PVP), NP-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride. The addition of the second stabilizer can improve the stability of the R2 reagent, inhibit non-specific adsorption in a system on the premise of not influencing the sensitivity of the reaction system, and more importantly, improve the stability of the chromogenic substrate S-2403; for example, the addition of sucrose, trehalose, glucose, gelatin and the like is beneficial to inhibiting the freeze denaturation of protein and can ensure that the freezing can not cause the precipitation and non-specific aggregation of an R1 reagent, thereby ensuring the stability of the reagent in storage, transportation and the like, and gelatin macromolecules have a net structure and can generate a sieve pore effect, thereby performing space limitation on the protein, reducing the mutual collision among protein molecules and improving the stability of the protein to a certain extent; the addition of inorganic salts such as sodium chloride, potassium chloride and the like can be used for adjusting the osmotic pressure of the solution with the chromogenic substrate S-2403, so that the efficiency of the chromogenic substrate S-2403 in the determination of alpha 2-antiplasmin can be improved; tween-20 and the like are added to play a role of a surfactant, so that the stability of the reagent is further improved; meanwhile, the addition of the second stabilizer can also play a certain role in protecting the protease, so that the stability of the kit is further improved.
In a particular embodiment, the second stabilizer comprises the components: sodium chloride, glutamic acid, sucrose, PVP and NP-40, wherein the mass percentages of the components in the R2 reagent are as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of cane sugar, 1-3% of PVP and 0.5-1.5% of NP-40. The reagent kit has high accuracy and good stability.
The third auxiliary material also comprises a third stabilizing agent, and the third stabilizing agent 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 diluent is 0.5-1.5%; the reagent kit has high accuracy and good stability.
The R1 reagent, the R2 reagent and the diluting reagent respectively comprise a preservative, and the preservative is at least one of sodium benzoate, sodium azide, proclin-300, gentamicin and nitrite; the preservative can be 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. In a specific embodiment, the R1 reagent, the R2 reagent and the diluting reagent respectively comprise Proclin-300 with the mass percent of 0.03-0.05%, and the reagents have good antiseptic effect and are beneficial to improving the stability of the reagents.
The R1 reagent and the R2 reagent both comprise protease protective agents, the protease protective agents are at least one of BSA, HAS and Prionix, and the mass percentages of the protease protective agents in the R1 reagent and the R2 reagent are 1-5%. The protease protective agent is used for prolonging the stability of an R1 reagent and an R2 reagent in the kit, is beneficial to long-term use and storage of the kit, is beneficial to protecting the activity of enzyme and preventing thrombin denaturation, and plays a role in protecting recombinant bovine plasmin by improving the concentration of protein in a solution; prevent enzyme decomposition and nonspecific adsorption, and reduce the denaturation of some enzymes and adverse environmental factors such as heat, surface tension and chemical factors. Preferably, the protease protectant is Pronex.
The R1 reagent also includes PAI-1 (i.e., complete plasminogen activator inhibitor 1), PAI-1 at a concentration of 1-2 IU/mL. The influence of PLG in plasma can be reduced by adding a proper amount of PAI-1, the PLG in the plasma is prevented from interfering the determination of the alpha 2-antiplasmin activity, the PAI-1 reagent contains a certain concentration of PAI-1, the influence of Plasminogen (PLG) activation in a sample on a detection result is eliminated, and the accuracy of the determination result is further increased; the PAI-1 has the following action principle: PAI-1 inhibits tissue-type plasminogen activator (t-pa) and urokinase-type plasminogen activator (u-pa), thereby reducing production of active plasmin. The anti-interference capability of the kit can be enhanced, and the test accuracy can be improved.
The present invention is further explained below by means of specific embodiments.
Example 1
An alpha 2-antifibrinolytic enzyme activity determination kit, comprising an R1 reagent, an R2 reagent and a diluting reagent; 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
An alpha 2-antifibrinolytic enzyme activity determination kit, comprising an R1 reagent, an R2 reagent and a diluting reagent; 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
An alpha 2-antifibrinolytic enzyme activity determination kit, comprising an R1 reagent, an R2 reagent and a diluting reagent; 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 the comparative example 1, the recombinant bovine plasmin in the R1 reagent is replaced by the common bovine plasmin, the chromogenic substrate S-2403 in the R2 reagent is replaced by the chromogenic substrate S-2251, and other components and the using amounts are the same as those in the example 1.
Comparative example 2
Comparative example 2 contained no PAI-1, and the other components and amounts were the same as in example 1.
Comparative example 3
In comparative example 3, the protease inhibitor Prononex was not contained in the R1 reagent, the R2 reagent and the diluting reagent, and the other examples were the same as those of example 1.
The α 2-antiplasmin (α 2-AP) activity assay kits of examples 1-3 and comparative examples 1-3 were used for calibration, blank limit, accuracy, linear range, reproducibility, stability and clinical relevance tests.
1. Scaling and results thereof
Calibration curves were plotted using the single-point concentration and dilution calibration method using the calibrator (104.6%) on the fully automatic coagulation analyzer in examples 1-3 and comparative example 1. The calibration results are shown in tables 4-7. The calibration result should meet the requirements: linear regression equation R of standard curve 2 And the OD value signal-to-noise ratio of the calibration point of the C6/C5 calibrator is more than or equal to 0.98, and the OD value signal-to-noise ratio of the calibration point of the C6/C5 calibrator is more than or equal to 2.
TABLE 4 kit calibration results of example 1
TABLE 5 kit calibration results of example 2
TABLE 6 kit calibration results of example 3
TABLE 7 calibration results for the kit of comparative example 1
From the test results of tables 4 to 7, it was found that the standard curvilinear regression equation of examples 1 to 3 satisfies R 2 And the OD value signal-to-noise ratio of the calibration point of the C6/C5 calibrator is more than or equal to 0.98, and meets the requirement, thereby proving that the standard curve with better quality can be obtained in the examples 1-3. While comparative example 1 standard curvilinear regression equation R 2 Less than 0.98, the OD value signal-to-noise ratio of the calibration point of the C6/C5 calibration product is less than 2, the requirements are not met, the overall sensitivity of the reagent is poor, the quality of the obtained standard curve is poor, and furtherThe recombinant bovine plasmin and the chromogenic substrate S-2403 have good OD values when being used in a matching way, have high differentiation rate on samples with different concentrations, are easier to pull apart during calibration, have high cutting efficiency, strong reaction signals and high reaction speed, and ensure higher reagent sensitivity.
2. Margin test and results thereof
The blank samples were measured 20 times using the kits of examples 1 to 3, and the mean value (X), standard Deviation (SD), and blank limit (X +2 SD) were calculated according to the following equations (1) and (2), and the test results are shown in tables 8 to 10. The test result is required to be: the blank limit is less than or equal to 8 percent.
Formula (1):
formula (2):
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 example 1
TABLE 9 blank limit test results of example 2
TABLE 10 blank limit test results for example 3
From the test results of tables 8 to 10, it was found that the kits of examples 1 to 3 all meet the specified blank limit requirements.
3. Accuracy test and results thereof
Two alpha 2-antiplasmin (. Alpha.2-AP) accuracy control products were measured using examples 1 to 3 and comparative example 2, respectively, and the test was repeated 3 times for each of the accuracy control products, and the relative deviation was calculated according to the above equations (1) and (3), and the test results are shown in tables 11 to 14. The test result is required to be: the relative deviation should be within 15.00%.
Formula (3):
in the formula:
t-accuracy control item index value;
b-relative deviation.
TABLE 11 accuracy test results of example 1
TABLE 12 accuracy test results of example 2
TABLE 13 accuracy test results of example 3
TABLE 14 accuracy test results of comparative example 2
From the test results of tables 11 to 14, it was found that the relative deviation of examples 1 to 3 was small, demonstrating that examples 1 to 3 all have good accuracy. The relative deviation of comparative example 2 does not meet the requirement that they are within. + -. 15%, and it can be seen from the results of example 1 and comparative example 2 that the effect of PLG in plasma can be reduced and the interference of PLG in plasma with the measurement of. Alpha.2-antiplasmin activity can be prevented by adding an appropriate amount of PAI-1, the reagent of R1 in example 1 contains PAI-1 at a concentration which eliminates the effect of Plasminogen (PLG) activation in the sample on the measurement result, thereby increasing the accuracy of the measurement result, while comparative example 2 does not add PAI-1 and is interfered by PA in the sample, and the activated PLG forms extra PL, thereby resulting in the inaccurate measurement result of. Alpha.2-AP. Thus, comparative example 2 is less accurate than examples 1-3.
4. Linear range test and results thereof
Alpha 2-antiplasmin (alpha 2-AP) samples close to the upper limit of the linear range of the kit are respectively diluted into 5 samples with different concentrations, the kit of examples 1-3 and comparative example 1 is adopted to test each sample with different concentrations for 3 times, the theoretical concentration is (x), the mean value of the measured results is (y), 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 15-18. The test result is required to be: when the alpha 2-AP activity is in the linear range of 10-170%, the linear correlation coefficient r is more than or equal to 0.98.
TABLE 15 results of the linearity test of example 1
TABLE 16 results of the linearity test of example 2
TABLE 17 Linear test results of example 3
TABLE 18 Linear test results of comparative example 1
From the test results in tables 15-18, it was found that the linear correlation coefficient r of examples 1-3 was 0.99 or more, indicating that the kits of examples 1-3 all meet the above linear range requirements.WhileThe linear range r =0.95 of comparative example 1 is not satisfactory, and it can be seen from example 1 and comparative example 1 that the use of recombinant bovine plasmin and S-2403 (example 1) is more differentiated and has a wider linear range than the use of ordinary plasmin and substrate (comparative example 1).
5. Repeatability tests and results thereof
The alpha 2-antiplasmin (alpha 2-AP) low and high value quality controls were tested 10 times each using examples 1-3. The mean (X) and Standard Deviation (SD) of the mean values of the test results were calculated according to equations (1) and (2), and the Coefficient of Variation (CV) was calculated according to equation (4), and the test results are shown in tables 19 to 21. The test result is required to be: the Coefficient of Variation (CV) of the low-value quality control product is less than or equal to 10 percent; the Coefficient of Variation (CV) of the 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 19 results of the repeatability tests of example 1
TABLE 20 results of the repeatability tests of example 2
TABLE 21 results of the repeatability tests of example 3
From the test results of tables 19 to 21, it was found that examples 1 to 3 all met the above-mentioned reproducibility requirements and the results were good in reproducibility.
6. Stability testing and results thereof
1) The bottle opening stability is as follows: the R1 reagent, the R2 reagent and the diluting reagent of examples 1 to 3 were stored at 2 to 8 ℃ after decapping, and accuracy tests were performed on day 0, 14, 28, 30, and 32, and the test results are shown in Table 22. The test result is required to be: the R1 reagent, the R2 reagent and the diluting reagent are placed at 2-8 ℃ after being unpacked and can be stored stably for 30 days, namely, the relative deviation is within the range of +/-15%.
TABLE 22 open bottle stability test results of examples 1-3
2) From the test results in Table 22, it was found that examples 1-3 meet the above-mentioned requirements for decap stability, and that the R1 reagent, the R2 reagent and the diluting reagent can be stored stably at 2-8 ℃ for at least 32 days after decapping. Accelerated stability: the R1 reagent, the R2 reagent, and the diluting reagent 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, 12, 16, and 18, and the test results are shown in table 23. The test result is required to be: r1, R2 and the diluent can be stored for 16 days at 37 ℃ in an unopened state, namely, the relative deviation is within the range of +/-15%.
TABLE 23 accelerated stability test results for examples 1-3 and comparative example 3
From the test results in table 23, it was found that examples 1 to 3 meet the above requirement for accelerated stability, R1, R2 and the diluent can be stably stored at 37 ℃ for at least 18 days in the unopened state, and the relative deviation of comparative example 3 has not met the requirement of + -15% in the test on day 12, i.e., R1, R2 and the diluent cannot be stably stored at 37 ℃ for 12 days in the unopened state, and does not meet the requirement for accelerated stability, and from the results of examples 1 and comparative example 3, it is found that the addition of an appropriate amount of the enzyme protein protecting agent Prionex is advantageous for the long-term use and storage of the kit, is advantageous for protecting the activity of the enzyme, is advantageous for preventing the denaturation of thrombin, and provides the kit with excellent accelerated stability, and the kit of examples 1 to 3 has higher accelerated stability than the kit of comparative example 3.
7. Clinical relevance tests and results thereof
A group of clinical samples (40 examples) covering a linear range were simultaneously tested using a reference kit (manufacturer Siemens, lot No. 565098) purchased from a third party and 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 value of the kits of examples 1 to 3 as the y-axis, and the test results are shown in table 24 and fig. 1 to 3. The test result is required to be: the slope k of the linear regression equation is between 0.9 and 1.1 and the correlation coefficient R 2 ≥0.95。
TABLE 24 results of clinical relevance tests of examples 1-3
From the test results shown in Table 24, it was found that the results of testing clinical samples with the kits of examples 1 to 3 and the reference kit were subjected to linear regression analysis, and the slope k and the correlation R of the linear regression equation 2 The test kits of examples 1 to 3 are satisfactory, and have good correlation with the reference test kit.
The data and the figures show that the results obtained by using the kit of the invention to carry out the relevant 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. It will be apparent to those skilled in the art that various changes, modifications and substitutions can be made herein without departing from the scope of the invention as defined by the appended claims.
Claims (4)
1. An alpha 2-antifibrinolytic enzyme activity determination kit is characterized by comprising an R1 reagent, an R2 reagent and a diluting reagent;
the R1 reagent is composed of recombinant bovine plasmin, a first buffer solution, a protease protective agent, PAI-1, proclin-300 and a first auxiliary material, wherein the first auxiliary material is composed of sodium chloride, glutamic acid, sucrose, polyethylene glycol 2000 and NP-40, and the mass percentages of the components in the first auxiliary material in the R1 reagent are respectively as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of sucrose, 1-3% of polyethylene glycol 2000 and 0.5-1.5% of NP-40, wherein the concentration of the recombinant bovine plasmin is 5-7 IU/mL, the concentration of PAI-1 is 1-2 IU/mL, the mass percentage of Proclin-300 in the R1 reagent is 0.03-0.05%, the protease protective agent is at least one of BSA and Prionex, the mass percentage of the protease protective agent in the R1 reagent is 1-5%, and the pH of the first buffer solution is 7.2-7.6;
the R2 reagent consists of a chromogenic substrate S-2403, a second buffer solution, proclin-300, a protease protective agent and a second auxiliary material, wherein the second auxiliary material consists of sodium chloride, glutamic acid, sucrose, polyvinylpyrrolidone and NP-40, and the mass percentages of the components in the second auxiliary material in the R2 reagent are as follows: 0.5-1.5% of sodium chloride, 3-5% of glutamic acid, 3-5% of sucrose, 1-3% of polyvinylpyrrolidone and 0.5-1.5% of NP-40, wherein the concentration of the chromogenic substrate S-2403 is 2-4 mmol/L, the mass percentage of Proclin-300 in the R2 reagent is 0.03-0.05%, the protease protective agent is at least one of BSA and Prionix, the mass percentage of the protease protective agent in the R2 reagent is 1-5%, and the pH of the second buffer solution is 7.2-7.6;
the diluting reagent consists of a third buffer solution, proclin-300 and a third auxiliary material, wherein the third auxiliary material is sodium chloride, the mass percent of the sodium chloride in the diluting reagent is 0.5-1.5%, the mass percent of the Proclin-300 in the diluting reagent is 0.03-0.05%, and the pH value of the third buffer solution is 7.2-7.6;
the first buffer solution, the second buffer solution and the third buffer solution are all 100-150 mmol/L Tris buffer solution.
2. The kit of claim 1, wherein the first buffer solution of the R1 reagent is 100 mmol/L Tris buffer solution, the protease protection agent is BSA, the mass percentage of the BSA in the R1 reagent is 1%, the mass percentage of the Proclin-300 in the R1 reagent is 0.03%, the concentration of the recombinant bovine plasmin is 5IU/mL, the concentration of the PAI-1 is 1IU/mL, and the mass percentages of the components in the first auxiliary material in the R1 reagent are respectively: 0.5% sodium chloride, 3% glutamic acid, 3% sucrose, 1% polyethylene glycol 2000, 0.5% NP-40, wherein the pH of the R1 reagent is 7.2;
the second buffer solution of the R2 reagent is a 100 mmol/L Tris buffer solution, the protease protective agent is BSA, the mass percent of the BSA in the R2 reagent is 1%, the mass percent of the Proclin-300 in the R2 reagent is 0.03%, the concentration of the chromogenic substrate S-2403 is 2 mmol/L, and the mass percent of each component in the second auxiliary material in the R2 reagent is respectively: 0.5% sodium chloride, 3% glutamic acid, 3% sucrose, 1% polyvinylpyrrolidone, 0.5% NP-40, wherein the pH of the R2 reagent is 7.2;
the third buffer solution of the diluting reagent is 100 mmol/L Tris buffer solution, the mass percent of the Proclin-300 in the diluting reagent is 0.03%, the mass percent of the sodium chloride in the diluting reagent is 0.5%, and the pH value of the diluting reagent is 7.2.
3. The kit of claim 1, wherein the first buffer solution of the R1 reagent is 125 mmol/L Tris buffer solution, the protease protection agent is BSA, the mass percentage of the BSA in the R1 reagent is 3%, the mass percentage of the Proclin-300 in the R1 reagent is 0.04%, the concentration of the recombinant bovine plasmin is 6IU/mL, the concentration of the PAI-1 is 1.5IU/mL, and the mass percentages of the components in the first auxiliary material in the R1 reagent are respectively: 1% sodium chloride, 4% glutamic acid, 4% sucrose, 2% polyethylene glycol 2000 and 1% np-40, said R1 reagent having a pH of 7.4;
the second buffer solution of the R2 reagent is 125 mmol/L Tris buffer solution, the protease protective agent is BSA, the mass percent of the BSA in the R2 reagent is 3%, the mass percent of the Proclin-300 in the R2 reagent is 0.04%, the concentration of the chromogenic substrate S-2403 is 3mmol/L, and the mass percent of each component in the second auxiliary material in the R2 reagent is respectively: 1% sodium chloride, 4% glutamic acid, 4% sucrose, 2% polyvinylpyrrolidone, 1% NP-40, wherein the R2 reagent has a pH of 7.4;
the third buffer solution of the diluting reagent is 125 mmol/L Tris buffer solution, the mass percent of the Proclin-300 in the diluting reagent is 0.04%, the mass percent of the sodium chloride in the diluting reagent is 1%, and the pH value of the diluting reagent is 7.4.
4. The kit according to claim 1, wherein the first buffer solution of the R1 reagent is 150mmol/L Tris buffer solution, the protease protection agent is Prionex, the mass percentage of the Prionex in the R1 reagent is 5%, the mass percentage of the Proclin-300 in the R1 reagent is 0.05%, the concentration of the recombinant bovine plasmin is 7IU/mL, the concentration of the PAI-1 is 2IU/mL, and the mass percentages of the components in the first auxiliary material in the R1 reagent are respectively: 1.5% sodium chloride, 5% glutamic acid, 5% sucrose, 3% polyethylene glycol 2000, 1.5% np-40, said R1 reagent having a pH of 7.6;
the second buffer solution of the R2 reagent is 150mmol/L Tris buffer solution, the protease protective agent is Prionix, the mass percent of Prionix in the R2 reagent is 5%, the mass percent of Proclin-300 in the R2 reagent is 0.05%, the concentration of the chromogenic substrate S-2403 is 4 mmol/L, and the mass percent of each component in the second auxiliary material in the R2 reagent is respectively as follows: 1.5% sodium chloride, 5% glutamic acid, 5% sucrose, 3% polyvinylpyrrolidone, 1.5% NP-40, wherein the pH of the R2 reagent is 7.6;
the third buffer solution of the diluting reagent is 150mmol/L Tris buffer solution, the mass percent of the Proclin-300 in the diluting reagent is 0.05 percent, the mass percent of the sodium chloride in the diluting reagent is 1.5 percent, and the pH value of the diluting reagent is 7.6.
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| CN108982850A (en) * | 2018-06-28 | 2018-12-11 | 广州市伊川生物科技有限公司 | It is a kind of for detecting the kit of Pepsinogen II |
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| AT398004B (en) * | 1992-04-14 | 1994-08-25 | Berbi Gmbh | METHOD FOR DETERMINING PLASMINE-ALPHA2-ANTIPLASMINE COMPLEXES USING A COMPLEX-SPECIFIC MONOCLONAL ANTIBODY |
| EP0950095A2 (en) * | 1996-11-12 | 1999-10-20 | The Scripps Research Institute | TISSUE TYPE PLASMINOGEN ACTIVATOR (t-PA) VARIANTS: COMPOSITIONS AND METHODS OF USE |
| US6485904B1 (en) * | 1997-06-23 | 2002-11-26 | Pfizer, Inc. | DNA encoding a plasminogen activating protein |
| JP4577992B2 (en) * | 1998-09-29 | 2010-11-10 | リューベン・リサーチ・アンド・デベロップメント・ベー・ゼット・ウェー | Use of a compound that reduces α2 antiplasmin in vivo for the preparation of a composition for the treatment of ischemic stroke |
| EP1062953A1 (en) * | 1999-06-22 | 2000-12-27 | Leuven Research & Development vzw | Use of compounds that reduce alpha2-antiplasmin in vivo for the preparation of a composition for the treatment of ischemic stroke |
| KR20140015289A (en) * | 2011-01-05 | 2014-02-06 | 쓰롬보제닉스 엔.브이. | Plasminogen and plasmin variants |
| CN107177611B (en) * | 2017-05-23 | 2020-03-20 | 江苏康禾生物制药有限公司 | DNA molecule for coding tissue type plasminogen activator and recombinant cell strain thereof |
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| US20210330762A1 (en) * | 2020-04-23 | 2021-10-28 | Shenzhen Bay Laboratory | Medical application of mutant plasminogen and plasmin polypeptide therapeutics |
| CN114058675B (en) * | 2020-08-04 | 2022-11-15 | 深圳市帝迈生物技术有限公司 | Stabilizer, thrombin time testing reagent, preparation method thereof and kit |
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| CN107727587A (en) * | 2017-09-22 | 2018-02-23 | 宁波瑞源生物科技有限公司 | A kind of Antithrombin III assay kit and its detection method |
| CN108982850A (en) * | 2018-06-28 | 2018-12-11 | 广州市伊川生物科技有限公司 | It is a kind of for detecting the kit of Pepsinogen II |
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