CN110954380A - Matrix for biochemical calibrator and biochemical calibrator - Google Patents
Matrix for biochemical calibrator and biochemical calibrator Download PDFInfo
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- CN110954380A CN110954380A CN201911126149.3A CN201911126149A CN110954380A CN 110954380 A CN110954380 A CN 110954380A CN 201911126149 A CN201911126149 A CN 201911126149A CN 110954380 A CN110954380 A CN 110954380A
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Abstract
The invention is applicable to the technical field of biochemistry, and particularly relates to a matrix for biochemical standard substances and a biochemical standard substance, wherein each 1 liter of the matrix for biochemical standard substances comprises: 10-100 mmol of pH regulator, 2-50 g of sugar protective agent, 2-100 g of polymer protective agent, 5-50 g of protein protective agent, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt. According to the matrix for the biochemical standard provided by the embodiment of the invention, when the analysis component is added into the matrix for the biochemical standard, each component in the matrix for the biochemical standard can have a good protection effect on the analysis component, especially, the structural variation caused by dehydration of the analysis component in the freeze-drying process can be avoided, and the effect of the analysis component can be effectively retained.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a matrix for a biochemical calibrator and the biochemical calibrator.
Background
In clinical diagnosis, in order to diagnose a sample conveniently, a calibration product is usually needed, and the calibration product can contain one or even dozens of analysis components, and the quality of the calibration product is directly related to whether a detection result is accurate and reliable. The currently commonly used calibrator has two forms of liquid and freeze-dried powder, the liquid calibrator is convenient to use but has poor stability, and particularly, the biochemical calibrator containing enzymes is easy to inactivate in solution storage and has short shelf life. Therefore, biochemical calibrators are also typically stored in a lyophilized form.
The existing freeze-dried powder is usually prepared by dissolving analysis components in a serum matrix, and then freeze-drying, because partial activity loss exists in the freeze-drying process, part of the freeze-dried powder needs to be re-dissolved firstly, the content of the analysis components in the freeze-dried powder is measured to be used as a calibration value, when the freeze-dried powder needs to be used, the rest freeze-dried powder is added into water in proportion to be re-dissolved, and the previously measured calibration value is used as a standard value to perform subsequent calculation. However, the existing calibrator using serum as the matrix of the calibrator has two main problems, one is that the activity loss of the same batch of freeze-dried products is different in the freeze-drying process, the difference between bottles of the same batch of freeze-dried products is large, that is, the content of the analysis component measured by the redissolved partial freeze-dried powder is used as the standard value, so that a large error exists, and the subsequent detection result is affected, and the other is that the activity of the freeze-dried powder is gradually reduced in the freeze-drying preservation process, so that the activity of the freeze-dried powder is different from the previously measured calibration value, and the subsequent detection result is also affected.
Therefore, in the prior art, the calibrator with serum as a matrix has the technical problems that the activity loss difference is large in the freeze-drying process, and the freeze-dried product is unstable in the freeze-drying storage process to influence the accurate result of the subsequent detection.
Disclosure of Invention
The embodiment of the invention aims to provide a matrix for a biochemical calibrator, and aims to solve the technical problems that the activity loss difference of the existing calibrator taking serum as the matrix is large in the freeze-drying process, and the freeze-dried product is unstable in the freeze-drying storage process to influence the accurate result of the subsequent detection.
The embodiment of the invention is realized by that, the matrix for the biochemical calibrator comprises the following components in 1 liter:
10-100 mmol of pH regulator, 2-50 g of sugar protective agent, 2-100 g of polymer protective agent, 5-50 g of protein protective agent, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt.
The other purpose of the embodiment of the invention is to provide a biochemical calibrator, which is prepared by adding analytical components into the matrix for the biochemical calibrator, and performing freeze drying after bagging, wherein the biochemical calibrator is freeze-dried powder.
The embodiment of the invention provides a substrate for a biochemical calibrator, which comprises 10-100 mmol of pH regulator, 2-50 g of sugar protectant, 2-100 g of polymer protectant, 5-50 g of protein protectant, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt per 1L of substrate for the biochemical calibrator. According to the matrix for the biochemical calibrator provided by the embodiment of the invention, when analytical components are added into the matrix for the biochemical calibrator, the carbohydrate protective agent and the polymer protective agent can increase the viscosity of a solution and prevent the ice crystal from growing too fast in a freezing process, so that the spatial structure of an enzyme molecule is maintained, the enzyme activity loss is reduced, the protein protective agent can improve the solid content in a system, the surfactant can reduce the interfacial tension between ice and water in the freezing process and prevent the enzyme molecule from being adsorbed on an ice-water interface to damage the spatial structure, the pH regulator ensures the pH of the matrix and avoids the enzyme from being inactivated due to the extreme pH environment, the change of the pH in the freeze-drying process is inhibited by the addition of amino acid, the activity loss of the enzyme due to the change of the pH is further avoided, the alcohol organic solvent can improve the vapor pressure of the system, the sublimation is facilitated, and the collapse of a surface freeze-dried layer in the freeze-drying process can be avoided, the stability of the freeze-dried powder is improved, active centers of the analysis components are magnesium salts and zinc salts, a certain amount of metal ions are added to further stabilize the activity of the analysis components, the substances have good protection effects on various different analysis components, the loss of the activity of the analysis components in the freeze-drying process is reduced through the protection effect of the substances on the analysis components, and the average activity recovery rate of the prepared biochemical calibration freeze-dried powder after redissolution is higher than 75%.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a matrix for biochemical calibrator, wherein each 1 liter of the matrix for biochemical calibrator comprises:
10-100 mmol of pH regulator, 2-50 g of sugar protective agent, 2-100 g of polymer protective agent, 5-50 g of protein protective agent, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt.
In the examples of the present invention, since a common analytical component is usually an enzyme, the following explanation will be mainly made with an enzyme as an analytical component.
In the embodiment of the invention, a certain amount of pH regulator is added into the biochemical calibrator matrix, so that the pH of the solution can be controlled to be 6-8, and the enzyme is prevented from being denatured and inactivated under an extreme pH condition. In a preferred embodiment of the present invention, the pH adjusting agent is included in an amount of 30 to 50 mmol/1 l of the biochemical calibrator matrix, and further, the pH adjusting agent is included in an amount of 40 mmol/1 l of the biochemical calibrator matrix.
As a preferred embodiment of the present invention, the pH regulator is selected from hydrogen phosphate and dihydrogen phosphate. Compared with other pH regulators, the hydrogen phosphate and the dihydrogen phosphate form a phosphate buffer solution, which not only helps to maintain the pH value of the solution, but also helps to maintain the stability of the solution because the osmotic pressure of the solution is similar to that of the physiological environment of a human body.
In the embodiment of the invention, the carbohydrate protective agent and the polymer protective agent can increase the viscosity of the solution, prevent ice crystals from growing too fast in the freezing process, and can tightly wrap enzyme molecules after drying, and in addition, hydroxyl contained in the carbohydrate can replace a surface hydration layer when the enzyme dries and loses water, and form a hydrogen bond with the enzyme, namely, the enzyme structure can be stabilized, the spatial structure of the enzyme molecule can be effectively maintained, and the activity loss of the enzyme in the freeze-drying process is reduced.
In the embodiment of the invention, the protein protective agent can increase the solid content in the system, and can be used as an excipient to participate in the freeze drying of the enzyme, and the surfactant can reduce the interfacial tension between ice and water in the freezing process, so that enzyme molecules are prevented from being adsorbed on an ice-water interface to damage the space structure of the enzyme molecules. Also, the present invention is not particularly limited to specific proteinaceous protective agents, which may be human serum albumin, bovine serum albumin, ovalbumin, etc., and surfactants, which may be betaine, triton X-100, sodium lauryl sulfate, etc. In a preferred embodiment of the present invention, the matrix for biochemical calibrator comprises 20-40 g of protein protectant and 2-4 g of surfactant per 1l of matrix for biochemical calibrator, and further comprises 30 g of protein protectant and 3 g of surfactant per 1l of matrix for biochemical calibrator.
In the embodiment of the invention, because the amino acid has both an acidic functional group and a basic functional group, the addition of the amino acid can inhibit the change of the pH of the solution in the freezing process, and avoid the loss of the activity of the enzyme caused by the change of the pH in the freezing process. In a preferred embodiment of the present invention, the biochemical calibrator includes 10 to 20 mmol of amino acids per 1 liter of the biochemical calibrator, and further includes 15 mmol of amino acids per 1 liter of the biochemical calibrator.
As a preferred embodiment of the invention, the amino acid is glycine, the structure of glycine is simplest compared with other amino acids, and the molecular forms of glycine in aqueous solutions with different acid and alkali are different, so that the adaptability is stronger, and the protection performance on enzyme is better.
In embodiments of the invention, antioxidants and protease inhibitors are effective in reducing the loss of activity during storage due to oxidation and hydrolysis of the enzyme. Likewise, antioxidants and protease inhibitors will generally be used in serum matrices, and this is not specifically illustrated by the present invention. In a preferred embodiment of the present invention, the biochemical calibrator matrix includes 20 to 40 mmol of the antioxidant and 0.1 to 0.5 mmol of the protease inhibitor per 1 liter of the biochemical calibrator matrix, and further includes 30 mmol of the antioxidant and 0.3 mmol of the protease inhibitor per 1 liter of the biochemical calibrator matrix.
In the embodiment of the invention, the freeze-drying process utilizes water sublimation under the condition of low temperature and reduced pressure to dehydrate the material at low temperature, if the sublimation process absorbs heat to reduce the temperature of the material to slow down the sublimation rate, the drying time is finally overlong, and a surface freeze-drying layer collapses to cause incomplete drying, thereby affecting the stability of the enzyme. In a preferred embodiment of the present invention, 1 liter of the biochemical calibrator matrix contains 10 to 20 g of the alcohol organic solvent, and further 1 liter of the biochemical calibrator matrix contains 15 g of the alcohol organic solvent.
In a preferred embodiment of the present invention, t-butanol is selected as the alcohol organic solvent, and (1) t-butanol has a higher vapor pressure and is miscible with water at any ratio as compared with other alcohol organic solvents. (2) When a small amount of tert-butyl alcohol is mixed with water, the crystallization state of the water can be changed, needle crystals are formed in the freeze-drying process, tubular passages are formed after ice crystals are sublimated, the sublimation rate is obviously improved, and freeze-dried finished products are loose and porous and are more favorable for redissolution. (3) The tert-butyl alcohol can be fully volatilized in the freeze-drying process, almost has no residue, and does not influence the detection value of the calibrator.
In the present example, the preservative was added primarily to increase shelf life of the standard, and the sodium chloride was added primarily to form an isotonic solution. The mechanism of action of the above substances is not described in detail in the present invention. As a preferred embodiment of the present invention, the matrix for biochemical calibrator contains 0.3 g preservative and 9 g sodium chloride per 1 l.
In the embodiment of the invention, the active center of the enzyme usually contains metal ions such as magnesium ions and zinc ions, and certain amount of magnesium ions and zinc ions are added, so that the activity of the enzyme in the freeze-drying process can be improved to a certain extent. In a preferred embodiment of the present invention, 1 to 2 mmol of the magnesium salt and/or the zinc salt is contained in 1 liter of the matrix for biochemical calibrator, and further 1.5 mmol of the magnesium salt and/or the zinc salt is contained in 1 liter of the matrix for biochemical calibrator.
The embodiment of the invention provides a substrate for a biochemical calibrator, which comprises 10-100 mmol of pH regulator, 2-50 g of sugar protectant, 2-100 g of polymer protectant, 5-50 g of protein protectant, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt per 1L of substrate for the biochemical calibrator. According to the matrix for the biochemical calibrator provided by the embodiment of the invention, when analytical components are added into the matrix for the biochemical calibrator, the carbohydrate protective agent and the polymer protective agent can increase the viscosity of a solution and prevent the ice crystal from growing too fast in a freezing process, so that the spatial structure of an enzyme molecule is maintained, the enzyme activity loss is reduced, the protein protective agent can improve the solid content in a system, the surfactant can reduce the interfacial tension between ice and water in the freezing process and prevent the enzyme molecule from being adsorbed on an ice-water interface to damage the spatial structure, the pH regulator ensures the pH of the matrix and avoids the enzyme from being inactivated due to the extreme pH environment, the change of the pH in the freeze-drying process is inhibited by the addition of amino acid, the activity loss of the enzyme due to the change of the pH is further avoided, the alcohol organic solvent can improve the vapor pressure of the system, the sublimation is facilitated, and the collapse of a surface freeze-dried layer in the freeze-drying process can be avoided, the stability of the freeze-dried powder is improved, active centers of the analysis components are magnesium salts and zinc salts, a certain amount of metal ions are added to further stabilize the activity of the analysis components, the substances have good protection effects on various different analysis components, the loss of the activity of the analysis components in the freeze-drying process is reduced through the protection effect of the substances on the analysis components, and the average activity recovery rate of the prepared biochemical calibration freeze-dried powder after redissolution is higher than 75%.
The embodiment of the invention also provides a biochemical calibrator, which is prepared by adding analytical components into the matrix for the biochemical calibrator, and performing freeze drying after bag separation, wherein the biochemical calibrator is freeze-dried powder.
As a preferred embodiment of the present invention, the analytical component comprises one or more of alkaline phosphatase, α -amylase, alanine aminotransferase, pancreatic amylase, aspartate aminotransferase, mitochondrial isozymes of aspartate aminotransferase, creatine kinase MB isozymes, cholinesterase, γ -glutamyl transferase, glutamate dehydrogenase, α -hydroxybutyrate dehydrogenase, lactate dehydrogenase isozymes, and lipases.
In another preferred embodiment of the present invention, when the addition is required, the amount of alkaline phosphatase added is 100 to 300U, the amount of α -amylase added is 100 to 200U, the amount of alanine aminotransferase added is 100 to 200U, the amount of pancreatic amylase added is 100 to 200U, the amount of aspartate aminotransferase mitochondrial isozyme added is 20 to 100U, the amount of creatine kinase added is 200 to 500U, the amount of creatine kinase MB isozyme added is 50 to 150U, the amount of cholinesterase added is 3000 to 7000U, the amount of γ -glutamyl transferase added is 50 to 150U, the amount of glutamate dehydrogenase added is 10 to 80U, the amount of α -hydroxybutyrate dehydrogenase added is 200 to 400U, the amount of lactate dehydrogenase isozyme added is 200 to 400U, and the amount of lipase added is 20 to 100U, U is a standard activity unit, that is an amount of substrate capable of converting 1. mu. mol of enzyme under specific international conditions.
To further illustrate the benefits of the present invention over the prior art, the following examples and related experimental data are provided to explain the present invention, it should be noted that a great deal of experimental trial and error has been performed during the course of the specific experiments, and only some of the examples with typical results have been selected from the following examples and comparative examples.
Example 1:
(1) preparing a biochemical calibrator matrix:
adding 10 millimole of sodium hydrogen phosphate and 10 millimole of sodium dihydrogen phosphate into 500mL of distilled water to prepare a buffer solution;
to the buffer was added 2 g of sucrose, 2 g of β -dextran, 2 g of human serum albumin, 0.5 g of betaine, 5 g of glycine, 0.02 mmol of sodium D-isoascorbate, 0.05 mmol of phenylmethylsulfonyl fluoride, 0.05 mmol of serine protease inhibitor, 5 g of t-butanol, 0.2 g of sodium azide, 8.5 g of sodium chloride and 0.1 mmol of magnesium chloride and 0.1 mmol of zinc chloride in that order;
and (5) fixing the buffer solution to 1L to obtain the matrix for the biochemical calibrator.
(2) Preparation of biochemical calibrator (lyophilized powder):
adding 100U of alkaline phosphatase, 100U of α -amylase, 100U of alanine aminotransferase, 100U of pancreatic amylase, 100U of aspartate aminotransferase, 20U of aspartate aminotransferase mitochondrial isozyme, 200U of creatine kinase, 50U of creatine kinase MB isozyme, 3000U of cholinesterase, 50U of gamma-glutamyltransferase, 10U of glutamate dehydrogenase, 200U of α -hydroxybutyrate dehydrogenase, 200U of lactate dehydrogenase isozyme, and 20U of lipase to the substrate for biochemical calibrator to obtain biochemical calibrator solution;
and (3) subpackaging, weighing and freeze-drying the prepared biochemical calibrator solution to prepare a biochemical calibrator (freeze-dried powder), simultaneously extracting five parts of the freeze-dried powder for redissolution, and determining the active content of each analysis component as initial enzyme activity (namely calibration value) by using biochemical reagents and calibrators corresponding to each analysis component.
And (3) preserving the residual biochemical calibrator for 7 days at 37 ℃, redissolving, determining the activity content of each analysis component as the preserved enzyme activity by using the biochemical reagent and calibrator corresponding to each analysis component, calculating the enzyme activity recovery rate (the ratio of the preserved enzyme activity to the initial enzyme activity), and reacting the stability of the freeze-dried powder in the preservation process by the enzyme activity recovery rate, wherein the specific detection result is shown in the following table 1. In the examples and comparative examples which follow, the methods for determining the initial enzyme activity and the enzyme activity after preservation are the same as those described above unless otherwise specified.
Example 2:
(1) preparing a biochemical standard substance matrix:
adding 100 mmol of sodium hydrogen phosphate and 100 mmol of sodium dihydrogen phosphate into 500mL of distilled water to prepare a buffer solution;
to the buffer were added in this order 50 g sucrose, 100 g β -dextran, 50 g human serum albumin, 5 g betaine, 20 g glycine, 50 mmol sodium D-erythorbate, 2 mmol phenylmethylsulfonyl fluoride, 2 mmol serine protease inhibitor, 30 g t-butanol, 0.5 g sodium azide, 9.5 g sodium chloride and 5 mmol magnesium chloride and 5 mmol zinc chloride;
and (5) fixing the buffer solution to 1L to obtain the matrix for the biochemical calibrator.
(2) Preparation of biochemical calibrator (lyophilized powder):
adding 100U of alkaline phosphatase, 100U of α -amylase, 100U of alanine aminotransferase, 100U of pancreatic amylase, 100U of aspartate aminotransferase, 20U of aspartate aminotransferase mitochondrial isozyme, 200U of creatine kinase, 50U of creatine kinase MB isozyme, 3000U of cholinesterase, 50U of gamma-glutamyltransferase, 10U of glutamate dehydrogenase, 200U of α -hydroxybutyrate dehydrogenase, 200U of lactate dehydrogenase isozyme, and 20U of lipase to the substrate for biochemical calibrator to obtain biochemical calibrator solution;
the initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the results of the detection are shown in table 1 below.
Example 3:
(1) preparing a biochemical calibrator matrix:
adding 30 millimole of potassium hydrogen phosphate and 30 millimole of potassium dihydrogen phosphate into 500mL of distilled water to prepare a buffer solution;
to the buffer were added, in order, 10 g of lactose, 10 g of polyvinylpyrrolidone, 20 g of bovine serum albumin, 2 g of triton X-100, 10 g of mmol of glycine, 20 mmol of vitamin E, 0.1 mmol of phenylmethylsulfonyl fluoride, 10 g of t-butanol, 0.2 g of sodium azide, 8.5 g of sodium chloride and 1 mmol of magnesium chloride;
and (5) fixing the buffer solution to 1L to obtain the matrix for the biochemical calibrator.
(2) Preparation of biochemical calibrator (lyophilized powder):
adding 300U of alkaline phosphatase, 200U of α -amylase, 200U of alanine aminotransferase, 200U of pancreatic amylase, 200U of aspartate aminotransferase, 100U of aspartate aminotransferase mitochondrial isozyme, 500U of creatine kinase, 150U of creatine kinase MB isozyme, 7000U of cholinesterase, 150U of gamma-glutamyltransferase, 80U of glutamate dehydrogenase, 400U of α -hydroxybutyrate dehydrogenase, 400U of lactate dehydrogenase isozyme, and 100U of lipase to the substrate for biochemical calibrator to obtain biochemical calibrator solution;
the initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the results of the detection are shown in Table 2 below.
Example 4:
(1) preparing a biochemical calibrator matrix:
adding 50 millimole of potassium hydrogen phosphate and 50 millimole of potassium dihydrogen phosphate into 500mL of distilled water to prepare a buffer solution;
adding 30 g of maltose, 50 g of polyethylene glycol, 20 g of ovalbumin, 4 g of sodium dodecyl sulfate, 20 g of millimole glycine, 40 millimole lecithin, 0.5 millimole serine protease inhibitor, 20 g of tert-butyl alcohol, 0.5 g of sodium azide, 9.5 g of sodium chloride and 2 millimole magnesium chloride to the buffer solution in sequence;
and (5) fixing the buffer solution to 1L to obtain the matrix for the biochemical calibrator.
(2) Preparation of biochemical calibrator (lyophilized powder):
adding 300U of alkaline phosphatase, 200U of α -amylase, 200U of alanine aminotransferase, 200U of pancreatic amylase, 200U of aspartate aminotransferase, 100U of aspartate aminotransferase mitochondrial isozyme, 500U of creatine kinase, 150U of creatine kinase MB isozyme, 7000U of cholinesterase, 150U of gamma-glutamyltransferase, 80U of glutamate dehydrogenase, 400U of α -hydroxybutyrate dehydrogenase, 400U of lactate dehydrogenase isozyme, and 100U of lipase to the substrate for biochemical calibrator to obtain biochemical calibrator solution;
the initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the results of the detection are shown in Table 2 below.
Example 5:
(1) preparing a biochemical calibrator matrix:
adding 40 mmol of sodium hydrogen phosphate and 40 mmol of sodium dihydrogen phosphate into 500mL of distilled water to prepare a buffer solution;
to the buffer were added in this order 20 g of sucrose, 30 g of β -dextran, 30 g of human serum albumin, 3 g of betaine, 15 g of glycine, 30 mmol of sodium D-isoascorbate, 0.3 mmol of phenylmethylsulfonyl fluoride, 0.3 mmol of serine protease inhibitor, 15 g of t-butanol, 0.3 g of sodium azide, 9 g of sodium chloride, 1.5 mmol of magnesium chloride and 1.5 mmol of zinc chloride;
and (5) fixing the buffer solution to 1L to obtain the matrix for the biochemical calibrator.
(2) Preparation of biochemical calibrator (lyophilized powder):
adding 200U of alkaline phosphatase, 150U of α -amylase, 150U of alanine aminotransferase, 150U of pancreatic amylase, 150U of aspartate aminotransferase, 60U of aspartate aminotransferase mitochondrial isozyme, 350U of creatine kinase, 100U of creatine kinase MB isozyme, 5000U of cholinesterase, 100U of gamma-glutamyltransferase, 50U of glutamate dehydrogenase, 300U of α -hydroxybutyrate dehydrogenase, 300U of lactate dehydrogenase isozyme and 60U of lipase to the substrate for biochemical calibrator to obtain biochemical calibrator solution;
the initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the detection results are shown in table 3 below.
Example 6: (investigating the differences between the hydrogen/dihydrogen phosphate salts and the optional use of other pH regulators)
The same as other steps of example 5, except that the "buffer preparation by adding 40 mmol of sodium hydrogen phosphate and 40 mmol of sodium dihydrogen phosphate to 500mL of distilled water" step was specifically "buffer preparation by adding 40 mmol of citric acid and 40 mmol of sodium citrate to 500mL of distilled water".
The initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the detection results are shown in table 3 below.
Example 7: (examining differences between Glycine and the choice of other amino acids)
The same procedure as in example 5 was followed except that 15 mmole of glycine added was replaced by 15 mmole of glutamic acid.
The initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the results are shown in Table 4 below.
Example 8: (examine the differences between tert-butanol and other organic solvents of alcohols)
The same procedure as in example 5 was followed except that 15 g of t-butanol was added as 15 g of glycerol.
The initial enzyme activity and the preserved enzyme activity were measured by the same detection method as in example 1, and the results are shown in Table 4 below.
Comparative example 1:
(1) preparing serum matrix of biochemical calibrator
Collecting human blood materials, and detecting and removing unqualified blood samples; separating serum, and filtering to obtain serum matrix of biochemical calibrator; taking 1L serum matrix for use
(2) Preparation of Biochemical calibrator to the serum substrate of the biochemical calibrator was added 200U of alkaline phosphatase, 150U of α -amylase, 150U of alanine aminotransferase, 150U of pancreatic amylase, 150U of aspartate aminotransferase, 60U of aspartate aminotransferase mitochondrial isozymes, 350U of creatine kinase, 100U of creatine kinase MB isozyme, 5000U of cholinesterase, 100U of γ -glutamyltransferase, 50U of glutamate dehydrogenase, 300U of α -hydroxybutyrate dehydrogenase, 300U of lactate dehydrogenase isozyme, 60U of lipase to obtain a biochemical calibrator solution;
the initial enzyme activity and the preserved enzyme activity were measured by the same assay method as in example 1, and the assay results are shown in Table 5 below.
Comparative example 2: (investigation of the Effect of addition of alcohol organic solvent on Biochemical calibrators)
The same procedure as in example 5 was followed except that t-butanol was not added.
The initial enzyme activity and the preserved enzyme activity were measured by the same assay method as in example 1, and the assay results are shown in Table 5 below.
Comparative example 3: (examination of the Effect of addition of magnesium salt and Zinc salt on the Biochemical standards)
The same procedure as in example 5 was followed except that zinc chloride and magnesium chloride were not added.
The initial enzyme activity and the preserved enzyme activity were measured by the same assay method as in example 1, and the assay results are shown in Table 6 below.
The data in tables 1 to 6 are specifically as follows:
table 1:
table 2:
table 3:
table 4:
table 5:
table 6:
the experimental data of the embodiment 1-5 in table 1, table 2 and table 3 show that the matrix for the calibrator provided by the invention has high protective performance and strong stability on the analysis components, and the recovery rates of the enzyme activity after reconstitution are all higher than 75%, and the contents in table 3, table 5 and table 6 show that the enzyme activity of the existing lyophilized powder using serum as the matrix is about 60-70% after reconstitution, while the substitute for the serum matrix provided by the invention has strong protective performance on the enzyme in a proper proportion, and can still keep more than 90% of the enzyme activity after reconstitution after being preserved for 7 days at 37 ℃. In addition, the enzyme activity loss during the freeze-drying process is also reduced from about 20% to about 10%. In addition, it can be seen from the above table 3 and table 4 that, in selecting the pH adjuster, the amino acid, and the alcohol organic solvent, the effect of selecting the hydrogen phosphate/dihydrogen phosphate as the pH adjuster is better, and the effect is improved to a certain extent by selecting glycine as the amino acid and tert-butyl alcohol as the alcohol organic solvent compared with other amino acids and alcohol organic solvents.
In addition, because the serum components are complex and can not keep a uniform and stable state like a buffer solution, the invention also solves the technical problems that the uniformity after freeze-drying is poor and the difference among the same batch of calibrator bottles is large when the serum is used as a matrix in the prior art. The experiment of example 5 and comparative example 1 was repeated 5 times, two batches of 5 biochemical calibrators were prepared, and the CV value of each enzyme activity after reconstitution of 5 biochemical calibrators in each batch was calculated (the coefficient of variation, i.e. the ratio of standard deviation to average, it is obvious that the larger the coefficient of variation, the larger the difference between bottles, the better the calculated result), the inter-bottle CV value was reduced from 8% -13% to 0.2% -5% compared to the same batch of biochemical calibrators using serum as the matrix, i.e. the biochemical standards prepared in each batch were more uniform.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A biochemical calibrator substrate comprising, for every 1 liter of said biochemical calibrator substrate:
10-100 mmol of pH regulator, 2-50 g of sugar protective agent, 2-100 g of polymer protective agent, 5-50 g of protein protective agent, 0.5-5 g of surfactant, 5-20 mmol of amino acid, 0.02-50 mmol of antioxidant, 0.05-2 mmol of protease inhibitor, 5-30 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 0.1-5 mmol of magnesium salt and/or zinc salt.
2. The biochemical calibrator substrate according to claim 1, wherein said biochemical calibrator substrate comprises, for every 1 liter:
30-50 mmol of pH regulator, 10-30 g of sugar protective agent, 10-50 g of polymer protective agent, 20-40 g of protein protective agent, 2-4 g of surfactant, 10-20 mmol of amino acid, 20-40 mmol of antioxidant, 0.1-0.5 mmol of protease inhibitor, 10-20 g of alcohol organic solvent, 0.2-0.5 g of preservative, 8.5-9.5 g of sodium chloride and 1-2 mmol of magnesium salt and/or zinc salt.
3. The biochemical calibrator substrate according to claim 1, wherein said biochemical calibrator substrate comprises, for every 1 liter:
40 millimoles of pH regulator, 20 grams of sugar protective agent, 30 grams of polymer protective agent, 30 grams of protein protective agent, 3 grams of surfactant, 15 millimoles of amino acid, 30 millimoles of antioxidant, 0.3 millimoles of protease inhibitor, 15 grams of alcohol organic solvent, 0.3 gram of preservative, 9 grams of sodium chloride and 1.5 millimoles of magnesium and/or zinc salt.
4. A biochemical calibrator substrate according to any one of claims 1 to 3, wherein the pH adjustor is selected from the group consisting of hydrogen phosphate and dihydrogen phosphate.
5. A biochemical calibrator substrate according to any one of claims 1 to 3, wherein said amino acid is glycine.
6. A substrate for biochemical calibrators according to any one of claims 1 to 3, wherein the protease inhibitor is selected from phenylmethylsulfonyl fluoride and/or a serine protease inhibitor.
7. A substrate for biochemical calibrators according to any one of claims 1 to 3, wherein the alcohol-based organic solvent is tert-butanol.
8. A biochemical calibrator prepared by adding an analytical component to the matrix for biochemical calibrators according to any one of claims 1 to 8, bagging the analytical component, and freeze-drying the packaged analytical component, wherein the biochemical calibrators are freeze-dried powders.
9. A biochemical calibrator according to claim 8, wherein said analytical component comprises one or more of alkaline phosphatase, α -amylase, alanine aminotransferase, pancreatic amylase, aspartate aminotransferase mitochondrial isozymes, creatine kinase MB isozymes, cholinesterase, gamma-glutamyltransferase, glutamate dehydrogenase, α -hydroxybutyrate dehydrogenase, lactate dehydrogenase isozymes, and lipases.
10. A biochemical calibrator according to claim 9, wherein the amount of alkaline phosphatase to be added is 100 to 300U, the amount of α -amylase to be added is 100 to 200U, the amount of alanine aminotransferase to be added is 100 to 200U, the amount of pancreatic amylase to be added is 100 to 200U, the amount of aspartate aminotransferase to be added is 20 to 100U, the amount of creatine kinase to be added is 200 to 500U, the amount of creatine kinase MB isozyme to be added is 50 to 150U, the amount of cholinesterase to be added is 3000 to 7000U, the amount of γ -glutamyltransferase to be added is 50 to 150U, the amount of glutamate dehydrogenase to be added is 10 to 80U, the amount of α -hydroxybutyrate dehydrogenase to be added is 200 to 400U, the amount of lactate dehydrogenase to be added is 200 to 400U, the amount of γ -glutamyl transferase isozyme to be added is 200 to 150U, and the amount of lipase to be added is 100U, based on 1 liter of a substrate for biochemical calibrator.
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