CN114814245A - Protein C activity determination kit based on chromogenic substrate method - Google Patents
Protein C activity determination kit based on chromogenic substrate method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a protein C activity determination kit based on a chromogenic substrate method, wherein the protein C is a vitamin K-dependent plasma serine protease zymogen and comprises an R1 reagent, an R2 reagent and a diluting reagent; the R1 reagent comprises a protein C activator, a first buffer solution and a first auxiliary material; the R2 reagent comprises a chromogenic substrate Pca-5297, a second buffer solution and a second auxiliary material; 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; according to the invention, the protein C activator and the chromogenic substrate Pca-5297 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 sample discrimination is increased, and the establishment of the linear range and the clinical sample test 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 a protein C activity determination kit based on a chromogenic substrate method.
Background
Protein C (PC for short) is a vitamin K-dependent plasma serine protease zymogen, synthesized primarily in the liver, which is converted to activated protein C (APC for short) by thrombin or thrombin-thrombomodulin complex. APC and its cofactor protein S (PS for short) form a complex, have activity of inactivating Va and VIIIa and increasing fibrinolysis, and thus have anticoagulation effect. When PC is deficient, factors Va and VIIIa are inactivated less and the fibrinolytic capacity of blood circulation is reduced, so that excessive fibrin formation is promoted to cause thrombosis, and therefore, the determination of PC plays an important role in the prevention, monitoring and treatment of diseases.
At present, methods for quantitative detection of protein C can be divided into three categories: immunization, APTT coagulation and chromogenic substrate. Although the antigen-antibody reaction in the immunological method can accurately detect the content of the PC antigen, the screening of the specific antibody requires highly purified PC, and the condition is very strict; secondly, the whole detection process is long in time, and the requirements of emergency patients and clinical timely diagnosis cannot be met. The APTT coagulation method is characterized in that after snake venom is used for activating PC, the PC is added into blood plasma to be detected, wherein the content of the PC corresponds to the prolonged proportion of the blood plasma coagulation time, the method can specifically detect the content of protein C, but the method has more influencing factors on the result, and the experience of inspectors often directly influences the accuracy and the repeatability of the detection result. The thrombin-thrombin regulatory protein is used as an activator in the early chromogenic substrate method, but because a PC inhibitor and an interfering substance exist in plasma, the activity of PC in the plasma cannot be directly detected, and the PC needs to be separated from the plasma, so that a large amount of manpower and material resources are wasted, and the detection speed is slow.
Most of PC activity determination kits (chromogenic substrate methods) sold in China are mainly imported kits, are expensive, have long picking period, are mostly in the form of freeze-dried powder, are complex in manufacturing process and high in cost, the bottle difference of the reagents is enlarged due to freeze-drying, the freeze-dried powder reagents need to be redissolved before use, and can be used after standing for a period of time, so that the operation of customers is not facilitated; in the PC activity determination kit, the re-solvent is not additionally provided in the kit, the quality of the re-solvent selected by a client cannot be controlled, and certain influence is brought to a test result; the reagent has poor sensitivity, narrow linear range and poor specificity, so that the repeatability of a detection result is poor, the anti-interference capability is poor, the result is easily influenced by various factors, and the real protein C activity level in a human body cannot be reflected.
Disclosure of Invention
The invention aims to solve the technical problem of providing a protein C activity determination kit based on a chromogenic substrate method, which is low in price, full liquid, high in reagent sensitivity, high in specificity and wide in linear range.
The technical scheme adopted by the invention for solving the technical problems is as follows: a protein C activity determination kit based on a chromogenic substrate method, wherein the protein C is a vitamin K-dependent plasma serine protease zymogen and comprises an R1 reagent, an R2 reagent and a diluting reagent;
the R1 reagent comprises a protein C activator, a first buffer solution and a first auxiliary material, wherein the pH value of the first buffer solution is 7.2-7.6;
the R2 reagent comprises a chromogenic substrate Pca-5297, a second buffer solution and a second auxiliary material, wherein 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, the concentration of the protein C activator is preferably 0.1-0.5 IU/mL.
Further, preferably, the protein C activator is a snake venom extract.
Further, the concentration of the chromogenic substrate Pca-5297 is preferably 0.5-2 mg/ml.
Further, the first buffer solution, the second buffer solution and the third buffer solution are preferably at least one of Tris buffer solution, MES buffer solution, MOPS buffer solution, citric acid buffer solution and PBS 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 50-100 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, mannitol, sucrose, polyethylene glycol 2000 and tween-20, wherein the mass percentages of the components in the R1 reagent are as follows: 1.0-3.0% of sodium chloride, 3-5% of mannitol, 1-3% of sucrose, 0.5-1.5% of polyethylene glycol 2000 and 0.1-0.5% of tween-20.
Further, preferably, the second auxiliary material comprises a second stabilizer, and the second stabilizer comprises at least one of sodium chloride, galactose, 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: mannitol, galactose, polyvinylpyrrolidone and tween-20, wherein the mass percentages of the components in the R2 reagent are as follows: 3-5% of mannitol, 1-3% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-0.5% of tween-20.
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, it is preferable that the preservatives of the R1 reagent, the R2 reagent and the diluting reagent are Proclin-300, and the mass percentage of the Proclin-300 in the R1 reagent, the R2 reagent and the diluting reagent is 0.03-0.05%.
Further, it is preferable that each of the R1 reagent and the dilution reagent includes a protease protective agent, and the protease protective agent is at least one of BSA, HSA, and Prionex.
Further, the protease protection agent is preferably Prionix, and the mass percentages of the Prionix in the R1 reagent and the dilution reagent are both 0.03-0.05%.
The invention has the beneficial effects that: according to the protein C activity determination kit based on the chromogenic substrate method, a protein C activator and a chromogenic substrate Pca-5297 are used as reagent raw materials, when the chromogenic substrate Pca-5297 specific to activated protein C is used together with the protein C activator, the cutting efficiency of the activated protein C for cutting the chromogenic substrate Pca-5297 is higher, a reaction signal is stronger, the reagent sensitivity is higher, the linear range is wider, meanwhile, the combination of other non-specific signals can be eliminated by the chromogenic substrate Pca-5297, so that the specificity of the kit is stronger, 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; 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 initiated full-liquid detection kit for detecting the activity of the protein C, can replace an imported protein C activity detection kit, fully meets the requirement of clinical examination, can reduce the cost for purchasing detection reagents in hospitals, and simultaneously reduces the detection cost of patients and the burden.
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.
A protein C activity determination kit based on a chromogenic substrate method, wherein the protein C is a vitamin K-dependent plasma serine protease zymogen and comprises an R1 reagent, an R2 reagent and a diluting reagent; the R1 reagent comprises a protein C activator, a first buffer solution and a first auxiliary material, wherein the pH value of the first buffer solution is 7.2-7.6; the R2 reagent comprises a chromogenic substrate Pca-5297, a second buffer solution and a second auxiliary material, 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 a protein C activator into the plasma to be detected, activating the protein C into Activated Protein C (APC), specifically cutting the chromogenic substrate Pca-5297 by the APC to enable the chromogenic substrate Pca-5297 to release chromogenic group pNA (pNA is nitroaniline, and the release amount of pNA is measured at 405 nm), enabling the chromogenic degree of a reaction system to be in parallel relation with the content of the protein C in the detected plasma, and finally calculating the PC activity in the plasma to be detected according to a calibration curve.
According to the protein C activity determination kit based on the chromogenic substrate method, a protein C activator and a chromogenic substrate Pca-5297 are used as reagent raw materials, when the chromogenic substrate Pca-5297 specific to activated protein C is matched with the protein C activator for use, the activated protein C has higher cutting efficiency for cutting the chromogenic substrate Pca-5297, stronger reaction signals, higher reagent sensitivity and wider linear range, meanwhile, the chromogenic substrate Pca-5297 can exclude the combination of other non-specific signals, so that the specificity of the kit is stronger, 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; in terms of reaction time, the R1 reagent containing the protein C activator is added into the sample to be tested for incubation for 60s, the R2 reagent containing the chromogenic substrate Pca-5297 is added, the incubation time is about 60-120s, the R1 reagent and the sample to be tested of the existing kit are added for incubation for 240-300s, and the incubation time after the R2 reagent of the existing kit is added is between 120-240 s. 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 originated full-liquid detection kit for detecting the activity of the protein C, can replace an imported protein C activity detection kit, fully meets the requirement of clinical examination, can reduce the cost for purchasing detection reagents in hospitals, and simultaneously reduces the detection cost of patients and the burden.
Further, preferably, the protein C activator is a snake venom extract, the snake venom extract can specifically activate the protein C and exclude interference of other substances in plasma to the protein C, and the concentration of the protein C activator is preferably 0.1-0.5 IU/mL, at which the protein C activator has higher activation efficiency to the protein C, the protein C is rapidly activated to be activated to the protein C. The chromogenic substrate Pca-5297 is a specific high-efficiency substrate of the activated protein C, the amino acid sequence of the substrate is specially adjusted and modified, and compared with other common chromogenic substrates, the chromogenic substrate has higher sensitivity to the activated protein C, higher cutting efficiency and quicker reaction; preferably, the concentration of the chromogenic substrate Pca-5297 is 0.5-2 mg/ml, and at this concentration, the chromogenic substrate Pca-5297 has higher sensitivity to activated protein C and higher cleavage efficiency.
The first buffer solution, the second buffer solution and the third buffer solution are respectively at least one of Tris buffer solution, MES buffer solution, MOPS buffer solution, citric acid buffer solution and PBS 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 pH values of the R1 reagent, the R2 reagent and the diluting reagent are stabilized between 7.2-7.6, and the first buffer solution, the second buffer solution and the third buffer solution do not cause adverse effect on the activity of the measured protein C 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 50-100mmol/L, under the buffer solutions, the protein C activator and the chromogenic substrate Pca-5297 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, wherein the first stabilizer comprises one or more of sodium chloride, glutamic acid, sucrose, galactose, polyethylene glycol 2000, NP-40, gelatin, Tween-20 (Tween-20), trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride. The stability of the R1 reagent can be improved by adding the first stabilizing agent, the nonspecific adsorption in a system can be inhibited on the premise of not influencing the sensitivity of the reaction system, more importantly, the activity of protein C in a sample to be detected can be effectively protected, and the stability of the R1 reagent can be improved; if cane sugar, galactose, trehalose, glucose, gelatin and the like are added, on one hand, the freeze denaturation of protein can be inhibited, and the condition that R1 reagent is precipitated and non-specific aggregation can not be caused by freezing can be ensured, so that the stability of reagent storage, transportation and the like can be ensured, on the other hand, gelatin macromolecules have a net structure and can generate a sieve pore effect, the space limitation can be performed on the protein, the mutual collision among protein molecules is reduced, and the stability of the protein is improved to a certain extent; the addition of polyhydric alcohols such as polyethylene glycol 2000 can improve the viscosity of the reagent, and is beneficial to the uniform suspension of the protein C activator in the first buffer solution, and the protein C activator is not easy to settle, so that the protein C activator has a good stability effect, and can also be used as an antifreeze agent to effectively reduce the freezing point, so that a good freeze-thaw resistance effect is achieved; the addition of inorganic salts such as sodium chloride, potassium chloride and the like can be used for adjusting the osmotic pressure of the R1 reagent, so that the efficiency of activating the protein C into activated protein when the protein C activator is used for measuring the activity of the protein C 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 first stabilizer can also play a certain role in protecting the protease, so that the stability of the kit is improved.
In a particular embodiment, the first stabilizer comprises the components: sodium chloride, mannitol, sucrose, PEG-2000 (polyethylene glycol 2000), tween-20, and the mass percentage of the above components in the R1 reagent is: 1.0-3.0% of sodium chloride, 3-5% of mannitol, 1-3% of sucrose, 0.5-1.5% of PEG-2000 and 0.1-0.5% of Tween-20. Under the above proportion, the reagent stability of the kit is facilitated, and the accuracy is high when the activity of the protein C is detected.
The second auxiliary material comprises a second stabilizer, wherein the second stabilizer comprises at least one of sodium chloride, galactose, glutamic acid, sucrose, polyvinylpyrrolidone, NP-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride, and can be one of the components or a combination of a plurality of components; the addition of the second stabilizer can improve the stability of the R2 reagent, can inhibit nonspecific adsorption in a system on the premise of not influencing the sensitivity of a reaction system, and more importantly, enables the chromogenic substrate Pca-5297 to be uniformly dispersed in the second buffer solution, can also effectively protect the activity of protein C in a sample to be detected, and improves the stability of the R2 reagent; if cane sugar, galactose, trehalose, glucose, gelatin and the like are added, on one hand, the freeze denaturation of protein can be inhibited, and the condition that R2 reagent is precipitated and non-specific aggregation can not be caused by freezing can be ensured, so that the stability of reagent storage, transportation and the like can be ensured, on the other hand, gelatin macromolecules have a net structure and can generate a sieve pore effect, the space limitation can be performed on the protein, the mutual collision among protein molecules is reduced, and the stability of the protein is improved 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 an R2 reagent with a chromogenic substrate Pca-5297 and improving the efficiency of the chromogenic substrate Pca-5297 for being cut by activated protein C when the activity of the protein C is measured, thereby improving the detection efficiency of the activity of the protein C; tween-20 and the like are added to play a role of a surfactant, so that the stability of the R2 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 reagent kit is improved.
In a specific embodiment, the second stabilizer comprises the components: mannitol, galactose, polyvinylpyrrolidone and tween-20, wherein the mass percentages of the components in the R2 reagent are as follows: 3-5% of mannitol, 1-3% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-0.5% of tween-20. Under the above proportion, the reagent stability of the kit is facilitated, and the accuracy is high when the activity of the protein C is detected.
The third auxiliary material also comprises a third stabilizing agent, wherein the third stabilizing agent comprises one or more of sodium chloride, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride. The stability of the diluting reagent can be improved by adding the third stabilizing agent, the nonspecific adsorption in a system can be inhibited on the premise of not influencing the sensitivity of a reaction system, more importantly, the sample to be detected can be well diluted, the osmotic pressure of the sample to be detected can be adjusted by adding the sodium chloride, so that the activity of the protein C in the sample to be detected is not easily damaged, and the activity of the protein C in the sample to be detected can be effectively detected.
In a specific embodiment, the third stabilizer comprises sodium chloride, and the mass percentage of the sodium chloride in the diluting agent is 0.5-1.5%. Under the above proportion, the reagent stability of the kit is facilitated, and the accuracy of measuring the protein C activity of a sample to be measured is improved.
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 preservatives of the R1 reagent, the R2 reagent and the diluting reagent are Proclin-300, the mass percentages of the Proclin-300 in the R1 reagent, the R2 reagent and the diluting reagent are 0.03-0.05%, and the kit has a good preservative effect and is beneficial to improving the stability of the reagents.
The R1 reagent and the diluting reagent both comprise protease protective agent, and the protease protective agent is at least one of BSA, HSA and Prionex. The addition of the protease protective agent is beneficial to prolonging the stability of an R1 reagent and a diluting reagent in the kit, is beneficial to long-term use and storage of the kit, is beneficial to protecting the activity of the protein C and preventing the protein C from denaturation, and plays a role in protecting the protein C by improving the concentration of the protein in the solution; prevent enzyme decomposition and nonspecific adsorption, and reduce enzyme denaturation, and adverse environmental factors such as heat, surface tension and chemical factors.
In a specific embodiment, the protease protective agent is Prionix, and the mass percentages of the Prionix in the R1 reagent and the diluting reagent are both 0.03-0.05%. The stability of the reagent is improved in the proportion.
The present invention is further explained below by means of specific embodiments.
Example 1
A protein C activity determination kit based on a chromogenic substrate method comprises 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
A protein C activity determination kit based on a chromogenic substrate method comprises 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
A protein C activity determination kit based on a chromogenic substrate method comprises 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 comparative example 1, the Protein C activator (snake venom extract) in the reagent R1 was replaced with thrombin-thrombin regulatory Protein, the chromogenic substrate Pca-5297 in the reagent R2 was replaced with chromogenic substrate S-2366, and the other components and amounts were the same as in example 1.
Comparative example 2
In comparative example 2, Tris buffer in R1, R2 and diluting agent was replaced with Hepes buffer, and other components and amounts were the same as in example 1.
Comparative example 3
Comparative example 3 wherein the R1 reagent contained Mg 2+ (magnesium chloride), other components and amounts were the same as in example 1.
Comparative example 4
In comparative example 4, neither the R1 reagent nor the diluent contained the protease inhibitor Prononex, and the other examples were the same as in example 1.
The protein C activity assay kits of examples 1-3 and comparative examples 1-4 were used for calibration, margin, accuracy, linear range, reproducibility, stability, and clinical relevance testing.
1. Scaling and results thereof
The protein C activity assay kits of examples 1-3 and comparative example 2 were used to complete the calibration of the protein C project in conjunction with a protein C calibrator on a fully automated coagulation analyzer. The calibrator was a single-point calibrator, and the instrument automatically concentrated/diluted to 5 concentration levels of C0, C1, C2, C3, C4, and C5 (the concentrations of C0 to C5 increase in sequence), and the calibration results are shown in tables 4 to 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 of the protein C Activity assay kit of example 1
TABLE 5 calibration results of the protein C Activity assay kit of example 2
TABLE 6 calibration results of the protein C Activity assay kit of example 3
TABLE 7 calibration results of the protein C Activity assay kit of comparative example 2
The test results in tables 4-7 show that r of the curvilinear regression equations in examples 1-3 is not less than 0.980, and meets the requirements, and the results prove that examples 1-3 can obtain better calibration curves. The r of the standard curvilinear regression equation of the comparative example 2 is less than 0.980, the standard curvilinear regression equation is not satisfactory, the OD values of adjacent calibrators are not obviously different, and the sensitivity of the reagent and the drawing of a calibration curve are influenced. Further shows that when a Protein C activator (snake venom extract) and a chromogenic substrate Pca-5297 are matched and used in a Tris buffer solution, the kit has a good OD value, high sample discrimination for different concentrations, easy pulling during calibration, high cutting efficiency, strong reaction signal and high reaction speed, and ensures that the reagent sensitivity is higher.
2. Margin test and results thereof
A blank sample was measured 20 times using the protein C measurement kit of examples 1 to 3, and the average value (2) was calculated according to the following formulas (1) and (1)
) 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 protein C is less than or equal to 6 percent.
Formula (1):
equation (2):
；
in the formula:
n is the number of tests;
i-serial number of test;
b-relative deviation;
SD-standard deviation.
TABLE 8 blank Limit test results for the protein C Activity assay kit of example 1
TABLE 9 blank Limit test results for the protein C Activity assay kit of example 2
TABLE 10 blank limit test results for the protein C Activity assay kit of example 3
From the test results in tables 8 to 10, it was found that examples 1 to 3 all meet the specified margin requirements.
3. Accuracy test and results thereof
The protein C accuracy control products were measured using the protein C activity measurement kits of examples 1 to 3 and comparative example 3, and the test was repeated 3 times, and the relative deviation was calculated according to the above formulas (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 + -10.00%.
Formula (3):
；
in the formula:
t-accuracy control item index value;
b-relative deviation.
TABLE 11 accuracy test results of the protein C Activity assay kit of example 1
TABLE 12 accuracy test results of the protein C Activity assay kit of example 2
TABLE 13 accuracy test results of the protein C Activity assay kit of example 3
TABLE 14 accuracy test results of the protein C activity assay kit of comparative example 3
The test results in tables 11-14 show that the relative deviation of the examples 1-3 is relatively small, and the examples 1-3 are proved to have good accuracy. The relative deviation of comparative example 3 does not meet the requirements of being all within the range of ± 10.0%. From the results of example 1 and comparative example 3, Mg 2+ Does inhibit activation of protein C, so that the test result does not truly reflect the activity of protein C in the sample to be tested, and therefore, the method of comparative example 3The accuracy was inferior to those of examples 1 to 3.
4. Linear range test and results thereof
Respectively diluting high-value samples close to the upper limit of the linear range of the kit into 5 samples with different concentrations, testing the samples with each concentration for 3 times, calculating a linear regression equation by taking the theoretical concentration as (xi) and the mean value of the actual measurement results as (yi), and calculating the correlation coefficient (r) of linear regression, wherein the test results are shown in tables 15-18. The test result is required to be: when the linear range is 10-140%, the linear correlation coefficient r is more than or equal to 0.980
TABLE 15 results of the Linear Range test of the protein C Activity assay kit of example 1
TABLE 16 results of the Linear Range test of the protein C Activity assay kit of example 2
TABLE 17 results of the Linear Range test of the protein C Activity assay kit of example 3
TABLE 18 results of the Linear Range test of the protein C Activity assay kit of comparative example 1
The test results in tables 15-18 show that the linear correlation coefficient r is not less than 0.99, the linear interval of the kit can reach 10% -140%, and the kit of examples 1-3 meets the requirements of the linear range and has wide linear range. While the linear range r =0.985 of comparative example 1, the linear interval of the kit is 20% -120%, which is not satisfactory. As can be seen from example 1 and comparative example 1, the protein C activator (snake venom extract) and chromogenic substrate Pca-5297 used in combination have a higher discrimination and a wider linear range than other protein C activators and other chromogenic substrates.
5. Repeatability tests and results thereof
The protein C activity assay kit of examples 1-3 was used to test protein C low-value quality control substances and protein C high-value quality control substances 10 times each. Calculating the average value of the test results according to the formulas (1) and (2) ((
) And Standard Deviation (SD), calculating Coefficient of Variation (CV) according to formula (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 high-value quality control product and the low-value quality control product of the protein C is less than or equal to 10 percent.
Formula (4):
in the formula: CV is the coefficient of variation.
TABLE 19 results of the reproducibility test of the protein C activity assay kit of example 1
TABLE 20 results of repeated tests of the protein C Activity assay kit of example 2
TABLE 21 results of the reproducibility test of the protein C Activity assay kit of example 3
From the test results in tables 19 to 21, it was found that examples 1 to 3 all meet the above-mentioned reproducibility requirements.
6. Stability testing and results thereof
1) The bottle opening stability is as follows: the R1 reagent, the R2 reagent and the diluting reagent in the protein C activity assay kit of examples 1 to 3 were stored at 2 to 8 ℃ after decapping, and accuracy tests were performed on days 0, 10, 20, 30, 35, and 40, and the test results are shown in tables 22 to 24. The test result is required to be: the R1 reagent, the R2 reagent and the diluting reagent in the protein C activity determination kit are bottled and stored at 2-8 ℃ for 40 days, namely, the relative deviation is within the range of +/-10%.
TABLE 22 open-bottle stability test results of the protein C Activity assay kit of example 1
TABLE 23 open-bottle stability test results of the protein C Activity assay kit of example 2
TABLE 24 open-bottle stability test results of the protein C Activity assay kit of example 3
As shown in the test results in tables 22 to 24, examples 1 to 3 satisfy the above requirements for the bottle-opening stability, and the R1 reagent, the R2 reagent and the diluting reagent can be stored stably for at least 40 days at 2 to 8 ℃ in the bottle-opened state. Accelerated stability: the R1 reagent, the R2 reagent, and the diluent reagent in the protein C activity assay kit of examples 1 to 3 and comparative example 4 were stored at 37 ℃ in an unopened state, and accuracy tests were performed on days 0, 8, 12, and 16, and the test results are shown in tables 25 to 28. The test result is required to be: the R1 reagent, R2 reagent and the diluting reagent in the protein C activity determination kit can be stored at 37 ℃ for 16 days in an unopened state, namely, the relative deviation is within the range of +/-10%.
TABLE 25 accelerated stability test results of the protein C Activity assay kit of example 1
TABLE 26 accelerated stability test results of the protein C Activity assay kit of example 2
TABLE 27 accelerated stability test results of the protein C Activity assay kit of example 3
TABLE 28 accelerated stability test results of the protein C Activity assay kit of comparative example 4
As shown in the test results in tables 25 to 28, examples 1 to 3 satisfy the requirement of accelerated stability, and the R1 reagent, the R2 reagent and the diluting reagent can be stored stably at 37 ℃ for at least 16 days in an unopened state, the relative deviation of comparative example 4 has not met the requirements of being all within + -10% at the time of detection on day 16, that is, the R1 reagent, the R2 reagent and the diluent were not stored stably at 37 ℃ for 16 days in an unopened state, and did not meet the requirement of accelerated stability, as is clear from the results of example 1 and comparative example 4 in which no protein protectant Prionix was added, the long-term use and storage of the kit are facilitated by adding a proper amount of the enzyme protein protective agent Prionix, the activity of the enzyme is protected, the denaturation of the protein C is prevented, so that the kits had excellent accelerated stability, and the kits of examples 1 to 3 had higher accelerated stability than that of comparative example 4.
7. Clinical relevance tests and results thereof
A group of clinical samples (40 cases) covering a linear range were simultaneously tested using a reference kit (STAGO protein C activity assay kit (chromogenic substrate method), REF 00671) and the protein C activity assay kit of examples 1-3, and a linear regression analysis was performed using the actual measurement value of the reference kit as the x-axis and the actual measurement value of the kit of examples 1-3 as the y-axis, and the test results are shown in tables 29-31 and FIGS. 1-3. 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 greater than or equal to 0.975.
TABLE 29 results of the clinical relevance test of the protein C Activity assay kit of example 1
TABLE 30 results of the test of clinical relevance of the protein C Activity assay kit of example 2
TABLE 31 results of the clinical relevance test of the protein C Activity assay kit of example 3
From the test results of tables 29 to 31 and fig. 1 to 3, it is found that the results of the test of the clinical samples by the kits of examples 1 to 3 and the reference kit are subjected to linear regression analysis, and the slope k and the correlation r of the linear regression equation both meet the requirements, so that the kits of examples 1 to 3 and the reference kit are proved to 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. 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 (16)
1. A protein C activity determination kit based on a chromogenic substrate method, wherein the protein C is a vitamin K-dependent plasma serine protease zymogen, and is characterized by comprising an R1 reagent, an R2 reagent and a diluting reagent;
the R1 reagent comprises a protein C activator, a first buffer solution and a first auxiliary material, wherein the pH value of the first buffer solution is 7.2-7.6;
the R2 reagent comprises a chromogenic substrate Pca-5297, a second buffer solution and a second auxiliary material, wherein 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.
2. The kit of claim 1, wherein the concentration of the protein C activator is 0.1 to 0.5 IU/mL.
3. The kit of claim 2, wherein the protein C activator is a snake venom extract.
4. The kit according to claim 1, characterized in that the concentration of the chromogenic substrate Pca-5297 is 0.5-2 mg/ml.
5. The kit according to claim 1, wherein the first buffer, the second buffer and the third buffer are at least one of Tris buffer, MES buffer, MOPS buffer, citrate buffer and PBS buffer.
6. The kit according to claim 5, wherein the first buffer, the second buffer and the third buffer are all Tris buffers with the concentration of 50-100 mmol/L.
7. The kit of claim 1, wherein the first excipient comprises a first stabilizer comprising one or more of sodium chloride, glutamic acid, sucrose, galactose, polyethylene glycol 2000, NP-40, gelatin, tween-20, trehalose, glucose, β -cyclodextrin, mannitol, and potassium chloride.
8. The kit of claim 7, wherein the first stabilizer comprises the components: sodium chloride, mannitol, sucrose, polyethylene glycol 2000 and tween-20, wherein the mass percentages of the components in the R1 reagent are as follows: 1.0-3.0% of sodium chloride, 3-5% of mannitol, 1-3% of sucrose, 0.5-1.5% of polyethylene glycol 2000 and 0.1-0.5% of tween-20.
9. The kit of claim 1, wherein the second excipient comprises a second stabilizer comprising at least one of sodium chloride, galactose, glutamic acid, sucrose, polyvinylpyrrolidone, NP-40, gelatin, tween-20, trehalose, glucose, β -cyclodextrin, mannitol, and potassium chloride.
10. The kit of claim 9, wherein the second stabilizer comprises the components: mannitol, galactose, polyvinylpyrrolidone and tween-20, wherein the mass percentages of the components in the R2 reagent are as follows: 3-5% of mannitol, 1-3% of galactose, 0.5-1.5% of polyvinylpyrrolidone and 0.1-0.5% of tween-20.
11. The kit according to claim 1, wherein the third excipient further 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.
12. The kit according to claim 11, wherein the third stabilizer comprises sodium chloride, and the mass percentage of the sodium chloride in the diluent is 0.5-1.5%.
13. The kit of claim 1, wherein the R1 reagent, R2 reagent, and the diluting reagent each comprise a preservative that is at least one of sodium benzoate, sodium azide, Proclin-300, gentamicin, and nitrite.
14. The kit according to claim 13, wherein the preservatives of the R1 reagent, the R2 reagent and the diluting reagent are Proclin-300, and the mass percentage of the Proclin-300 in the R1 reagent, the R2 reagent and the diluting reagent is 0.03-0.05%.
15. The kit of claim 1, wherein the R1 reagent and the diluent reagent each comprise a protease protectant that is at least one of BSA, HSA, and Prionex.
16. The kit according to claim 15, wherein the protease protection agent is Prionix, and the mass percentages of the Prionix in the R1 reagent and the dilution reagent are 0.03-0.05%.
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| CN115932239A (en) * | 2022-12-22 | 2023-04-07 | 广州市进德生物科技有限公司 | Oxidized low-density lipoprotein protective agent, detection kit for determining oxidized low-density lipoprotein and application of detection kit |
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