CN113005176A - Stabilizer, prothrombin time detection reagent, preparation method and kit thereof - Google Patents

Stabilizer, prothrombin time detection reagent, preparation method and kit thereof Download PDF

Info

Publication number
CN113005176A
CN113005176A CN201911330746.8A CN201911330746A CN113005176A CN 113005176 A CN113005176 A CN 113005176A CN 201911330746 A CN201911330746 A CN 201911330746A CN 113005176 A CN113005176 A CN 113005176A
Authority
CN
China
Prior art keywords
calcium lactate
glycine
prothrombin time
reagent
time detection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201911330746.8A
Other languages
Chinese (zh)
Inventor
李云霞
何圣侠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Dymind Biotechnology Co Ltd
Original Assignee
Shenzhen Dymind Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Dymind Biotechnology Co Ltd filed Critical Shenzhen Dymind Biotechnology Co Ltd
Priority to CN201911330746.8A priority Critical patent/CN113005176A/en
Publication of CN113005176A publication Critical patent/CN113005176A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Neurosurgery (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention relates to a stabilizer, a prothrombin time detection reagent and a preparation method thereof, wherein the stabilizer comprises calcium lactate and glycine, the ratio of the calcium lactate to the glycine is 10 mmol-100 mmol: 5g to 50 g. The stabilizer comprises calcium lactate and glycine, wherein calcium lactate can supplement calcium ion and can be used as buffer and cosolvent, and glycine is used for stabilizing protein space structure of thromboplastin. Compared with the traditional prothrombin time detection reagent adopting calcium chloride, when the stabilizing agent is applied to the prothrombin time detection reagent, the calcium lactate is combined with the glycine to avoid precipitates in the prothrombin time detection reagent and improve the stability of the reagent, so that the accuracy and the stability of a detection result are ensured.

Description

Stabilizer, prothrombin time detection reagent, preparation method and kit thereof
Technical Field
The invention relates to the technical field of detection, in particular to a stabilizer, a prothrombin time detection reagent containing the stabilizer, a preparation method of the prothrombin time detection reagent, and a kit containing the prothrombin time detection reagent.
Background
The prothrombin time determination is a more ideal and commonly used sieving test of an extrinsic coagulation path, a coagulation method is used for determining the Prothrombin Time (PT), rabbit brain tissue factors, namely thromboplastin, in fresh rabbit brain powder is dissolved into a buffer solution through constant-temperature water bath, calcium ion thromboplastin with specific concentration is added into plasma to be detected after anticoagulation and centrifugation by sodium citrate, a compound is formed by the thromboplastin in the plasma again containing calcium ions and the thromboplastin VII and the calcium ions to activate the thromboplastin X to become Xa, the Xa, factor VII and the calcium ions convert the prothrombin into thrombin, the thrombin converts the fibrinogen into fibrin so as to perform cross-linking coagulation, and the time required by the coagulation determination is the prothrombin time.
In the prior art, calcium chloride is often added into a prothrombin time detection reagent to provide calcium ions when a technology for extracting thromboplastin from animal tissues is used, but in the specific implementation process, the PT reagent prepared by calcium chloride is unstable in detection result, and active substance tissue factors in a solution are easy to separate out to cause excessive precipitates in the reagent, so that the activity of the PT reagent is reduced, and the detection result of a sample is influenced.
Disclosure of Invention
In view of the above, there is a need for a stabilizer which can improve the stability of a prothrombin time detecting reagent when applied to the prothrombin time detecting reagent.
In addition, it is necessary to provide a prothrombin time detecting reagent comprising the above stabilizer and a method for preparing the same.
A stabilizer for a prothrombin time detection reagent comprises calcium lactate and glycine, wherein the ratio of the calcium lactate to the glycine is 10 mmol-100 mmol: 5g to 50 g.
A prothrombin time detection reagent comprises a thromboplastin extracting solution, a buffer solution, a preservative and the stabilizer;
wherein the concentration of the calcium lactate is 10 mmol/L-100 mmol/L, and the concentration of the glycine is 5 g/L-50 g/L.
A preparation method of a prothrombin time detection reagent is used for preparing the prothrombin time detection reagent and comprises the following steps:
mixing the rabbit brain powder and the buffer solution, then carrying out water bath at a certain temperature, centrifuging after the water bath is finished, and taking supernatant fluid, namely the thromboplastin extracting solution;
dissolving calcium lactate in the buffer solution to obtain a calcium lactate mother solution; and
and adding the calcium lactate mother liquor into the thromboplastin extracting solution, uniformly mixing, adding glycine, uniformly mixing again, and adding a preservative to obtain the prothrombin time detection reagent, wherein the final concentration of the calcium lactate is 10-100 mmol/L, and the final concentration of the glycine is 5-50 g/L.
A kit, comprising the prothrombin time detecting reagent.
The stabilizer comprises calcium lactate and glycine, wherein calcium lactate can supplement calcium ion and can be used as buffer and cosolvent, and glycine is used for stabilizing protein space structure of thromboplastin. Compared with the traditional prothrombin time detection reagent adopting calcium chloride, when the stabilizing agent is applied to the prothrombin time detection reagent, calcium lactate with good biocompatibility is added into the prothrombin time detection reagent, so that calcium ions can be supplemented, and the stabilizing agent can also be used as a buffering agent and a cosolvent. Therefore, the calcium lactate and the glycine are combined for use, so that the active substances in the prothrombin time detection reagent have good stabilizing and protecting effects, precipitates in the prothrombin time detection reagent can be avoided, the stability of the reagent is improved, and the accuracy and the stability of a detection result are ensured.
In addition, in the prothrombin time measuring reagent comprising the stabilizer, glycine with the concentration of 5 g/L-50 g/L can inhibit the crystallization of the buffer solution, thereby avoiding the change of the pH value of the reagent and preventing the protein from being denatured.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Wherein:
FIG. 1 is a flowchart of a method for preparing a prothrombin time detecting reagent according to an embodiment.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description taken in conjunction with the accompanying drawings. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The application discloses an embodiment of a stabilizing agent for a prothrombin time detection reagent.
The stabilizer comprises calcium lactate and glycine, wherein the proportion of the calcium lactate to the glycine is 10 mmol-100 mmol: 5g to 50 g.
In particular, the ratio of calcium lactate to glycine may be 50 mmol: 5g, 50 mmol: 15g, 50 mmol: 25g, 50 mmol: 35g, 50 mmol: 45g or 50 mmol: 50 g.
The stabilizer comprises calcium lactate and glycine, wherein calcium lactate can supplement calcium ion and can be used as buffer and cosolvent, and glycine is used for stabilizing protein space structure of thromboplastin. Compared with the traditional prothrombin time detection reagent adopting calcium chloride, when the stabilizing agent is applied to the prothrombin time detection reagent, calcium lactate with good biocompatibility is added into the prothrombin time detection reagent, so that calcium ions can be supplemented, and the stabilizing agent can also be used as a buffering agent and a cosolvent. Therefore, the calcium lactate and the glycine are combined for use, so that the active substances in the prothrombin time detection reagent have good stabilizing and protecting effects, precipitates in the prothrombin time detection reagent can be avoided, the stability of the reagent is improved, and the accuracy and the stability of a detection result are ensured.
In this embodiment, the stabilizer further comprises mannitol, wherein the ratio of mannitol to glycine is 1g to 10 g: 5g to 50 g.
Specifically, the ratio of mannitol to glycine may be 5 g: 5g, 5 g: 10g, 5 g: 15g, 5 g: 20g, 5 g: 25g, 5 g: 30g, 5 g: 35g, 5 g: 40g, 5 g: 45g or 5 g: 50 g.
Mannitol is used to stabilize the protein space structure of thromboplastin. Mannitol has a large number of hydroxyl groups, can replace water molecules in a solvent to be combined with polar residues of protein molecules, and plays a role in protecting a high molecular structure.
In addition, mannitol stabilizes the enzyme protein, prevents aggregation of cell membrane protein molecules, and protects the enzyme protein structure from being damaged.
In this embodiment, the stabilizer further comprises bovine serum albumin, wherein the ratio of bovine serum albumin to glycine is 1g to 10 g: 5g to 50 g.
Specifically, the ratio of bovine serum albumin to glycine may be 5 g: 5g, 5 g: 10g, 5 g: 15g, 5 g: 20g, 5 g: 25g, 5 g: 30g, 5 g: 35g, 5 g: 40g, 5 g: 45g or 5 g: 50 g.
Bovine serum albumin is used to maintain the structural stability of thromboplastin.
In this embodiment, the stabilizer further comprises a surfactant and a saccharide, and the ratio of the surfactant, the saccharide and the glycine is 0.5g to 5 g: 1 g-10 g: 5g to 50 g.
Specifically, the ratio of surfactant, saccharide and glycine may be 2.5 g: 5 g: 5g, 2.5 g: 5 g: 15g, 2.5 g: 5 g: 25g, 2.5 g: 5 g: 35g, 2.5 g: 5 g: 45g or 2.5 g: 5 g: 50 g.
In the embodiment, the surfactant is polyethylene glycol with the molecular weight of 6000-20000, and the saccharide is at least one selected from sucrose and maltose.
The molecular weight of the polyethylene glycol is 6000-20000, so that the thromboplastin can be uniformly dispersed.
Sucrose and maltose are used to maintain the spatial structure of thromboplastin.
Polyethylene glycol and saccharides have a large amount of hydroxyl groups, and can replace water molecules in a solvent to be combined with polar residues of protein molecules, so that the effect of protecting a high molecular structure is achieved.
The application also provides a prothrombin time detection reagent of an embodiment, which comprises a thromboplastin extracting solution, a buffer solution, a preservative and the stabilizer.
In the prothrombin time detection reagent, the concentration of calcium lactate is 10 mmol/L-100 mmol/L, and the concentration of glycine is 5 g/L-50 g/L. The selection of an appropriate concentration allows the activity of prothrombin to be optimized.
Specifically, the concentration of calcium lactate in the reagent for measuring prothrombin time may be 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L or 100mmol/L, and the concentration of glycine may be 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50 g/L.
The buffer solution is mainly used for maintaining the pH of the prothrombin time detection reagent, and has stable property and does not interfere the reaction.
In the embodiment, the buffer solution is Tris-HCl buffer solution with the concentration of 20 mmol/L-100 mmol/L, pH of 6.5-8. In other embodiments, other buffer types may be selected.
In the present embodiment, the preservative is ProClin150, ProClin200, ProClin300, or ProClin5000, and the concentration of the preservative is 0.1g/L to 0.5 g/L.
The prothrombin time detection reagent comprises calcium lactate and glycine, wherein the calcium lactate can supplement calcium ions and can also be used as a buffering agent and a cosolvent, and the glycine is used for stabilizing the protein space structure of thromboplastin. Compared with the traditional prothrombin time detection reagent adopting calcium chloride, calcium lactate with better biocompatibility is added into the prothrombin time detection reagent, so that calcium ions can be supplemented, and the calcium lactate can also be used as a buffering agent and a cosolvent.
In addition, glycine at a concentration of 5g/L to 50g/L inhibits crystallization of the buffer, thereby avoiding changes in the pH of the reagent and preventing protein denaturation. Therefore, the calcium lactate and the glycine are combined for use, so that the active substances in the prothrombin time detection reagent have good stabilizing and protecting effects, precipitates in the prothrombin time detection reagent can be avoided, the stability of the reagent is improved, and the accuracy and the stability of a detection result are ensured.
In the embodiment, the prothrombin time detection reagent further comprises mannitol, and the concentration of the mannitol is 1 g/L-10 g/L.
Specifically, the concentration of mannitol may be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, or 10 g/L.
Mannitol is used to stabilize the protein space structure of thromboplastin. Mannitol has a large number of hydroxyl groups, can replace water molecules in a solvent to be combined with polar residues of protein molecules, and plays a role in protecting a high molecular structure.
Mannitol stabilizes the enzyme protein and prevents aggregation of cell membrane protein molecules, thereby protecting the enzyme protein structure from being damaged.
In the present embodiment, the reagent for measuring prothrombin time further comprises Bovine Serum Albumin (BSA), and the concentration of the BSA is 1g/L to 10 g/L.
Specifically, the concentration of bovine serum albumin may be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, or 10 g/L.
Bovine serum albumin is used to maintain the structural stability of thromboplastin. In the PT reagent, BSA dissociates mainly into BSA-Electrostatic repulsion between surface groups stretches molecular chains to present an expanded conformation, the characteristic viscosity number is almost the highest, and interaction between protein and surrounding water molecules or polyhydroxy stabilizers is facilitated.
In the present embodiment, the reagent for measuring prothrombin time further comprises a surfactant and a saccharide, wherein the concentration of the surfactant is 0.5g/L to 5g/L, and the concentration of the saccharide is 1g/L to 10 g/L.
Specifically, the concentration of the surfactant may be 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L or 5g/L, and the concentration of the saccharide may be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10 g/L.
In the embodiment, the surfactant is polyethylene glycol with the molecular weight of 6000-20000, and the saccharide is at least one selected from sucrose and maltose.
The molecular weight of the polyethylene glycol is 6000-20000, so that the thromboplastin can be uniformly dispersed.
Sucrose and maltose are used to maintain the spatial structure of thromboplastin.
Polyethylene glycol and saccharides have a large amount of hydroxyl groups, and can replace water molecules in a solvent to be combined with polar residues of protein molecules, so that the effect of protecting a high molecular structure is achieved.
With reference to fig. 1, the present application also discloses a method for preparing a prothrombin time detecting reagent according to an embodiment, which is used for preparing the prothrombin time detecting reagent, and includes the following steps:
s10, mixing the rabbit brain powder and the buffer solution, carrying out water bath at a certain temperature, centrifuging after the water bath is finished, and taking the supernatant, namely the thromboplastin extracting solution.
In S10, mixing the rabbit brain powder and the buffer solution, and carrying out water bath at a certain temperature: mixing the rabbit brain powder and the buffer solution, then bathing for 30-60 min at 35-40 ℃, and stirring or shaking up once every 3-10 min so as to fully extract.
In this embodiment, the temperature of the water bath of S10 was 37 ℃ and the time of the water bath of S10 was 40 min.
In the present embodiment, the rotation speed of the centrifugation is 6000rpm, and the time of the centrifugation is 10 min.
In the embodiment, the addition amount of the rabbit brain powder is 20 g/L. In actual operation, the appropriate addition amount of the rabbit brain powder can be selected according to actual requirements.
S20, dissolving calcium lactate in the buffer solution to obtain a calcium lactate mother solution.
In S20, calcium lactate is dissolved in buffer solution under the condition of water bath at 45-60 ℃.
In this embodiment, the calcium lactate is dissolved in the buffer under water bath conditions at 50 ℃.
The mother liquor of calcium lactate may be in a relatively high concentration, e.g. 1 mol/L.
And S30, adding the calcium lactate mother liquor obtained in the step S20 into the thromboplastin extracting solution obtained in the step S10, uniformly mixing, adding glycine, uniformly mixing again, and adding a preservative to obtain the prothrombin time detection reagent.
In the prothrombin time detection reagent, the final concentration of calcium lactate is 10 mmol/L-100 mmol/L, and the final concentration of glycine is 5 g/L-50 g/L.
In this embodiment, the final concentration of calcium lactate may be 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L or 100mmol/L, and the final concentration of glycine may be 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50 g/L.
Specifically, when at least one of mannitol, bovine serum albumin, a surfactant and a saccharide is required to be added, mannitol, bovine serum albumin, a surfactant and a saccharide are added simultaneously with glycine.
In the present embodiment, the preservative is ProClin150, ProClin200, ProClin300, or ProClin5000, and the concentration of the preservative is 0.1g/L to 0.5 g/L.
The application also discloses a kit of an embodiment, which comprises the prothrombin time detection reagent.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1
Dissolving 50g of rabbit brain powder into 250mL of Tris-HCl buffer solution with pH of 7.2 and concentration of 50mM, carrying out water bath at 37 ℃ for 40min, shaking up every 5min, centrifuging at 6000rpm/min for 10min after the water bath is finished, and taking the supernatant, namely the prothrombin stock solution.
Dissolving calcium lactate in 25mL Tris-HCl buffer solution with pH of 7.2 and concentration of 50mM in a water bath at 50 deg.C to obtain 1M calcium lactate mother liquor.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding glycine to enable the final concentration of the glycine to be 10g/L, uniformly stirring, adding Proclin300 to enable the final concentration of the Proclin300 to be 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Example 2
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of calcium lactate of 10mM, uniformly mixing, adding glycine to enable the final concentration of glycine to be 50g/L, uniformly stirring, adding Proclin300 to enable the final concentration of Proclin300 to be 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Example 3
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of calcium lactate of 100mM, uniformly mixing, adding glycine to enable the final concentration of glycine to be 5g/L, uniformly stirring, adding Proclin300 to enable the final concentration of Proclin300 to be 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Example 4
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding glycine, mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of glycine is 10g/L, the final concentration of mannitol is 5g/L, the final concentration of bovine serum albumin is 5g/L, the final concentration of polyethylene glycol is 2.5g/L and the final concentration of sucrose is 5g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Example 5
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding glycine, mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of glycine is 10g/L, the final concentration of mannitol is 1g/L, the final concentration of bovine serum albumin is 1g/L, the final concentration of polyethylene glycol is 0.5g/L and the final concentration of sucrose is 1g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Example 6
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding glycine, mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of glycine is 10g/L, the final concentration of mannitol is 10g/L, the final concentration of bovine serum albumin is 10g/L, the final concentration of polyethylene glycol is 5g/L and the final concentration of sucrose is 10g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Comparative example 1
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding serine, mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of serine is 10g/L, the final concentration of mannitol is 10g/L, the final concentration of bovine serum albumin is 10g/L, the final concentration of polyethylene glycol is 5g/L and the final concentration of sucrose is 10g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Comparative example 2
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding glutamic acid, mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of glutamic acid is 10g/L, the final concentration of mannitol is 10g/L, the final concentration of bovine serum albumin is 10g/L, the final concentration of polyethylene glycol is 5g/L and the final concentration of sucrose is 10g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Blank example
The thromboplastin stock solution and the calcium lactate mother liquor obtained in example 1 were used.
Adding a small amount of calcium lactate mother liquor into a small amount of thromboplastin stock solution according to the final concentration of 20mM of calcium lactate, uniformly mixing, adding mannitol, bovine serum albumin, polyethylene glycol and sucrose to ensure that the final concentration of the mannitol is 10g/L, the final concentration of the bovine serum albumin is 10g/L, the final concentration of the polyethylene glycol is 5g/L and the final concentration of the sucrose is 10g/L, uniformly stirring, adding Proclin300 to ensure that the final concentration of the Proclin300 is 0.2g/L, and uniformly stirring to obtain the PT reagent. Wherein the total amount of the prepared PT reagent is 5 mL.
Test example
The PT reagents prepared in examples 1-6, comparative examples 1-2 and the blank were observed to produce translucent emulsions for the PT reagents prepared in examples 1-6, comparative examples 1-2 and the blank.
The PT reagents prepared in examples 1 and 4-6, comparative examples 1-2 and blank example were measured on an Emo Express full-automatic hemagglutination instrument of Stago using Stago-original-packed normal quality control plasma and abnormal quality control plasma, and plasma coagulation time was recorded. Wherein, the PT measurement normal value range is as follows: 10.5 to 14.5; ISI value: 1.0 + -0.05.
ISI international sensitivity index: ISI represents the slope of the standard tissue thromboplastin versus calibration curve for each batch of tissue thromboplastin. The standard tissue thromboplastin ISI is 1.0. The lower the ISI value of the reagent, the closer to 1.0, the more sensitive.
In a specific test, in order to ensure the accuracy of a test result, taking example 1 as an example, 5 times of preparation of the PT reagent are repeated according to the reaction conditions of example 1 to obtain 5 parts of the PT reagent, 10 times of repeated measurement are performed on each part of the PT reagent, and a final average value is taken as a measured value.
The PT reagents prepared in examples 1 and 4 to 6, comparative examples 1 to 2 and the blank example were measured for precision, and the results of the measurements are shown in table 1 below. Wherein S is the standard deviation, X is the average value, and the error CV is S/X.
Table 1: precision measurement values of PT reagents prepared in examples 1 and 4-6, comparative examples 1-2 and blank example
Mean value X Standard deviation S Coefficient of variation CV
Example 1 13.30 0.26 1.96％
Example 4 12.15 0.10 0.82％
Example 5 12.54 0.08 0.64％
Example 6 12.63 0.09 0.71％
Comparative example 1 13.82 0.29 2.07％
Comparative example 2 13.78 0.31 2.22％
Blank example 14.46 0.29 1.99％
It can be seen from table 1 that the test precision of PT prepared in examples 1 and 4-6 is significantly better than that of comparative example and blank example.
The PT reagents prepared in example 1, example 4, comparative examples 1-2 and the blank example were subjected to stability testing. The stability test includes stability after unsealing, stability under simulated conditions, accelerated destruction test without unsealing, and long-term stability test.
The PT reagents prepared in examples 1 and 4, comparative examples 1 to 2 and the blank example were subjected to a stability test after unsealing at a temperature of 4 c, and the test results are shown in table 2 below.
Table 2: stability test after unsealing of PT reagent prepared in examples 1 and 4, comparative example 1 and blank example
Figure BDA0002329489420000111
Figure BDA0002329489420000121
With reference to table 2, the results of the stability after unsealing tests of examples 1 and 4 show that the measured value of normal quality control plasma is within the normal range within 30 days, the measured value of abnormal quality control plasma is within the given range, and both of them have a coefficient of variation of less than 5%.
The PT reagents prepared in examples 1 and 4, comparative examples 1 to 2 and the blank example were subjected to a high temperature transportation condition test at a temperature of 38 c, and the test results are shown in table 3 below.
Table 3: high temperature transport condition test of PT reagents prepared in examples 1 and 4, comparative example 1 and blank example
Figure BDA0002329489420000122
With reference to table 3, the test results of the high temperature transportation condition tests of examples 1 and 4 show that under the high temperature transportation condition, the normal quality control and the abnormal quality control are both within the given range within 10 days, and the deviation is less than 5%.
The PT reagents prepared in examples 1 and 4, comparative examples 1 to 2 and the blank example were subjected to a refrigerated transport condition test at a temperature of-5 c, and the test results are shown in table 4 below.
Table 4: refrigerated transport condition testing of PT reagents prepared in examples 1 and 4, comparative example 1 and blank
Figure BDA0002329489420000131
With reference to table 4, the results of the freeze transportation condition test experiments of examples 1 and 4 show that both the normal quality control and the abnormal quality control are within the given range within 30 days under the freeze transportation condition, and the deviation is less than 5%.
The PT reagents prepared in examples 1 and 4, comparative examples 1 to 2 and the blank example were subjected to an accelerated 37 c destruction test at a temperature of 37 c, and the test results are shown in table 5 below.
Table 5: accelerated 37 ℃ Damage test of PT reagents prepared in examples 1 and 4, comparative example 1 and blank
Figure BDA0002329489420000132
Figure BDA0002329489420000141
With reference to table 5, the 37 ℃ accelerated release test results of examples 1 and 4 show that the plasma detection values of normal quality control and abnormal quality control are substantially stable within the given range within 21 days.
As can be seen from the above tables 1 to 5, the PT reagent prepared in example 4 is significantly superior to comparative example 1 and the blank in precision and stability. The precision and stability of the test of the PT reagent prepared in example 1 were slightly better than those of comparative example 1, and were significantly better than those of the blank example.
That is, in example 1, the prepared PT reagent still has high stability and precision in the stability test, even when only calcium lactate and glycine are added, and conventional stabilizers such as mannitol, bovine serum albumin, polyethylene glycol, and sucrose are not added.
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 claims. 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.

Claims (11)

1. A stabilizer used for a prothrombin time detection reagent is characterized by comprising calcium lactate and glycine, wherein the ratio of the calcium lactate to the glycine is 10 mmol-100 mmol: 5g to 50 g.
2. The stabilizer according to claim 1, further comprising mannitol in a ratio of 1 to 10 g: 5g to 50 g.
3. The stabilizer according to claim 2, further comprising bovine serum albumin, wherein the ratio of bovine serum albumin to the glycine is 1g to 10 g: 5g to 50 g.
4. The stabilizer according to any one of claims 1 to 3, further comprising a surfactant and a saccharide, wherein the ratio of the surfactant, the saccharide and the glycine is 0.5g to 5 g: 1 g-10 g: 5g to 50 g.
5. The stabilizer according to claim 4, wherein the surfactant is polyethylene glycol having a molecular weight of 6000 to 20000, and the saccharide is at least one selected from sucrose and maltose.
6. A prothrombin time detection reagent comprising a thromboplastin extract, a buffer, a preservative and the stabilizer of any one of claims 1 to 5;
wherein the concentration of the calcium lactate is 10 mmol/L-100 mmol/L, and the concentration of the glycine is 5 g/L-50 g/L.
7. The prothrombin time detecting reagent according to claim 6, wherein the buffer is Tris-HCl buffer with a concentration of 20mmol/L to 100mmol/L, pH of 6.5 to 8;
the preservative is ProClin150, ProClin200, ProClin300 or ProClin5000, and the concentration of the preservative is 0.1 g/L-0.5 g/L.
8. A method for producing a prothrombin time detecting reagent according to claim 6 or 7, comprising the steps of:
mixing the rabbit brain powder and the buffer solution, then carrying out water bath at a certain temperature, centrifuging after the water bath is finished, and taking supernatant fluid, namely the thromboplastin extracting solution;
dissolving calcium lactate in the buffer solution to obtain a calcium lactate mother solution; and
and adding the calcium lactate mother liquor into the thromboplastin extracting solution, uniformly mixing, adding glycine, uniformly mixing again, and adding a preservative to obtain the prothrombin time detection reagent, wherein the final concentration of the calcium lactate is 10-100 mmol/L, and the final concentration of the glycine is 5-50 g/L.
9. The method for preparing a prothrombin time detecting reagent according to claim 8, wherein the operation of mixing the rabbit brain powder and the buffer solution and then performing water bath at a certain temperature comprises: mixing the rabbit brain powder and the buffer solution, then bathing for 30-60 min at 35-40 ℃, and stirring or shaking up once every 3-10 min.
10. The method for preparing a reagent for detecting prothrombin time according to claim 8, wherein the dissolving of calcium lactate in the buffer is performed by: dissolving the calcium lactate in the buffer solution under the water bath condition of 45-60 ℃.
11. A kit comprising the prothrombin time detecting reagent according to claim 6 or 7.
CN201911330746.8A 2019-12-20 2019-12-20 Stabilizer, prothrombin time detection reagent, preparation method and kit thereof Pending CN113005176A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911330746.8A CN113005176A (en) 2019-12-20 2019-12-20 Stabilizer, prothrombin time detection reagent, preparation method and kit thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911330746.8A CN113005176A (en) 2019-12-20 2019-12-20 Stabilizer, prothrombin time detection reagent, preparation method and kit thereof

Publications (1)

Publication Number Publication Date
CN113005176A true CN113005176A (en) 2021-06-22

Family

ID=76382140

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911330746.8A Pending CN113005176A (en) 2019-12-20 2019-12-20 Stabilizer, prothrombin time detection reagent, preparation method and kit thereof

Country Status (1)

Country Link
CN (1) CN113005176A (en)

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3522148A (en) * 1965-08-13 1970-07-28 Dade Reagents Inc Stabilized thromboplastin preparation
US5358853A (en) * 1992-08-03 1994-10-25 Akzo Av Liquid thromboplastin reagent
WO2001042222A1 (en) * 1999-12-08 2001-06-14 Pharmacia Corporation Polymorphic crystalline forms of celecoxib
US20030044872A1 (en) * 1999-12-24 2003-03-06 Masahiro Okuda Means of stabilizing compositions and reagents
WO2004063036A1 (en) * 2003-01-09 2004-07-29 Baxter Healthcare S.A. Safety containers for biologically active substances and method for producing said container
CN1952169A (en) * 2005-10-18 2007-04-25 上海太阳生物技术有限公司 In-vitro detection diagnosis kit for clinical examination of prothrombin time (PT)
CN101183109A (en) * 2006-11-14 2008-05-21 希森美康株式会社 Reagent for measuring clotting time and method for manufacturing the reagent
US20100015684A1 (en) * 2001-10-10 2010-01-21 Neose Technologies, Inc. Factor vii: remodeling and glycoconjugation of factor vii
CN103180439A (en) * 2010-07-09 2013-06-26 比奥根艾迪克依蒙菲利亚公司 Chimeric clotting factors
CN104046606A (en) * 2013-03-15 2014-09-17 希森美康株式会社 Method for producing recombinant prothrombin, vector dna, and reagent kit
CN106350499A (en) * 2016-09-30 2017-01-25 四川迈克生物科技股份有限公司 Stabilizer of thrombin solution
WO2017023992A1 (en) * 2015-08-05 2017-02-09 Bristol-Myers Squibb Company Novel substituted glycine derived fxia inhibitors
US20170283853A1 (en) * 2016-03-30 2017-10-05 Sysmex Corporation Reagent for prothrombin time measurement, method for production thereof, and method for measurement of prothrombin time
WO2019222329A1 (en) * 2018-05-15 2019-11-21 Voyager Therapeutics, Inc. Compositions and methods for delivery of aav

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3522148A (en) * 1965-08-13 1970-07-28 Dade Reagents Inc Stabilized thromboplastin preparation
US5358853A (en) * 1992-08-03 1994-10-25 Akzo Av Liquid thromboplastin reagent
WO2001042222A1 (en) * 1999-12-08 2001-06-14 Pharmacia Corporation Polymorphic crystalline forms of celecoxib
US20030044872A1 (en) * 1999-12-24 2003-03-06 Masahiro Okuda Means of stabilizing compositions and reagents
US20100015684A1 (en) * 2001-10-10 2010-01-21 Neose Technologies, Inc. Factor vii: remodeling and glycoconjugation of factor vii
WO2004063036A1 (en) * 2003-01-09 2004-07-29 Baxter Healthcare S.A. Safety containers for biologically active substances and method for producing said container
CN1952169A (en) * 2005-10-18 2007-04-25 上海太阳生物技术有限公司 In-vitro detection diagnosis kit for clinical examination of prothrombin time (PT)
CN101183109A (en) * 2006-11-14 2008-05-21 希森美康株式会社 Reagent for measuring clotting time and method for manufacturing the reagent
CN103180439A (en) * 2010-07-09 2013-06-26 比奥根艾迪克依蒙菲利亚公司 Chimeric clotting factors
CN104046606A (en) * 2013-03-15 2014-09-17 希森美康株式会社 Method for producing recombinant prothrombin, vector dna, and reagent kit
WO2017023992A1 (en) * 2015-08-05 2017-02-09 Bristol-Myers Squibb Company Novel substituted glycine derived fxia inhibitors
US20170283853A1 (en) * 2016-03-30 2017-10-05 Sysmex Corporation Reagent for prothrombin time measurement, method for production thereof, and method for measurement of prothrombin time
CN106350499A (en) * 2016-09-30 2017-01-25 四川迈克生物科技股份有限公司 Stabilizer of thrombin solution
WO2019222329A1 (en) * 2018-05-15 2019-11-21 Voyager Therapeutics, Inc. Compositions and methods for delivery of aav

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NULL: ""凝血酶原时间测定试剂（凝固法）"", 《国家药品监督管理局官网》 *
陈玉祥编著: "《分子药剂学》", 30 January 2010 *

Similar Documents

Publication Publication Date Title
EP2047280B1 (en) Standard/reference/control for blood coagulation testing
US5254350A (en) Method of preparing a thromboplastin extract
US3955925A (en) Preparation of optically clear serum
US20060246052A1 (en) Means for stabilization of thrombin and compositions
WO2021063392A1 (en) Thrombin solution, kit, stabilization method for thrombin, detection reagent, measurement method of thrombin time, and use of defoamer
EP1092157A1 (en) Coagulation controls for pt and aptt assays
CN110887970B (en) Extraction buffer solution, rabbit brain extraction solution, PT detection reagent and PT detection kit
CN114544983A (en) Preparation method of normal value quality control product for blood coagulation and platelet function analyzer
CN104822387A (en) Thrombin solution and methods of use thereof
US20050136499A1 (en) Control plasma for thrombin activity tests
CN113005176A (en) Stabilizer, prothrombin time detection reagent, preparation method and kit thereof
CN106350499B (en) The stabilizer of thrombin solution
CN115517243B (en) Plasma preservation solution, coagulation quality control product and application thereof
CN112557665B (en) Validation reagent for lupus anticoagulant and preparation method thereof
JP5425062B2 (en) Method for measuring glycoalbumin and the like using a control sample containing D-mannitol
CN115704749A (en) Kit for preparing glycosylated hemoglobin quality control product and preparation method thereof
CN114350743B (en) Aryl sulfatase calibrator and application thereof
CN112433057B (en) Screening reagent for lupus anticoagulant and preparation method thereof
CN117247989A (en) Liquid thrombin time determination reagent and preparation method thereof
CN117949661A (en) Protein S activity determination kit
CN110133304B (en) Composition, reagent containing the composition and application thereof
CN117568445B (en) Preparation method and application of TAT (TAT) and PIC (PIC) composite quality control product
EP1229048A1 (en) Thromboplastin reagent and method for manufacturing the same
EP3926343A1 (en) Hemostatic enzyme and carboxymethyl chitosan-containing composition for blood coagulation test, and use thereof
CN116410258A (en) Factor XI deficiency plasma protective agent and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20210622

RJ01 Rejection of invention patent application after publication