CN109207462B - Abnormal prothrombin, preparation method thereof, detection agent containing abnormal prothrombin, kit and application - Google Patents
Abnormal prothrombin, preparation method thereof, detection agent containing abnormal prothrombin, kit and application Download PDFInfo
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- CN109207462B CN109207462B CN201810867852.9A CN201810867852A CN109207462B CN 109207462 B CN109207462 B CN 109207462B CN 201810867852 A CN201810867852 A CN 201810867852A CN 109207462 B CN109207462 B CN 109207462B
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- 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)
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- 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
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- 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)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
Abstract
The invention discloses an abnormal prothrombin, a preparation method thereof, a detection agent containing the abnormal prothrombin and a kit. The preparation method comprises the following steps: the decarboxylation treatment is carried out on the prothrombin by a chemical catalysis method. The detection agent or kit comprises the abnormal prothrombin. The preparation method directly adopts a chemical catalysis method to carry out decarboxylation treatment, so that the gamma-glutamic acid in at least part of glutamic acid regions of the prothrombin is converted into de-gamma-glutamic acid. The preparation method can be used for obtaining the abnormal prothrombin with high yield, high titer and high stability, and the detection agent or the kit can ensure the detection accuracy of the abnormal prothrombin, and is convenient and rapid.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to an abnormal prothrombin, a preparation method thereof, a detection agent containing the abnormal prothrombin, a kit containing the abnormal prothrombin and application of the abnormal prothrombin and the kit.
Background
The primary liver cancer is cancer and swelling of liver cells or intrahepatic bile duct cells, is one of the most common malignant tumors of the digestive system, and seriously threatens the life and health of people. The incidence of the disease of the male is higher than that of the female, about more than sixty thousand patients with new-onset liver cancer all the year round worldwide, the fifth place of malignant tumor is, and the east Asia and the Pacific region are liver cancer high incidence regions. The number of primary liver cancer in China is more than half of the number of primary liver cancer all over the world, and is one of the common malignant tumors in China. The mortality rate of the primary liver cancer is high, and the mortality rate of the primary liver cancer is the third place in the mortality rate of malignant tumors.
Normally, the prothrombin precursor in the liver is post-translationally carboxylated by gamma-glutamyl-carboxylase (gamma-glutamyl-carboxylase GLA) to form prothrombin, which is then secreted into the blood. Thus, prothrombin is a serum coagulation factor synthesized in the liver and is also a precursor to thrombin. There are 10 glutamic acid residues in the glutamic acid region of prothrombin precursors, and when sufficient vitamin K is present, these glutamic acid residues will all be converted to gamma-carboxyglutamic acid to become prothrombin. When vitamin K is deficient or a vitamin K antagonist is present, the glutamic acid is not fully carboxylated and is called Des-gamma-carboxyprothrombin (DCP). Because the carboxyl group of gamma-carboxyglutamic acid is a functional domain that binds calcium, its deficiency deprives the basis for calcium binding, DCP is free of prothrombin function and is therefore also referred to as aberrant prothrombin.
Thus, prothrombin is active as a prothrombin by carboxylating 10 glutamic acid residues in the structure at positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 32 to gamma-carboxyglutamic acid in the presence of vitamin K-dependent gamma-glutamyl carboxylase and epoxide reductase (VKOR). Abnormal prothrombin in contrast to prothrombin, the structural feature of abnormal prothrombin is that one or more glutamic acid residues in the glutamic acid domain (Gla domain) cannot be carboxylated to gamma-carboxyglutamic acid, thereby losing the clotting function.
In the case of hepatocellular carcinoma patients, the modification of the above-mentioned gamma-glutamyl carboxylase contained in the prothrombin precursor in the liver to form prothrombin by post-translational carboxylation is hindered, and abnormal prothrombin is produced in the liver. Abnormal prothrombin may appear in the blood when vitamin K is deficient in hepatocytes, or when treated with anticoagulant drugs (warfarin). Therefore, the abnormal prothrombin is used as a tumor marker for the auxiliary diagnosis of the primary liver cancer HCC.
Compared with the common tumor marker AFP of the liver cancer, the liver cancer abnormal prothrombin has high specificity (the negative accurate judgment rate of patients without liver cancer) when the intensity of the abnormal prothrombin is discussed, and the liver cancer abnormal prothrombin positive rate can reach 93-95 percent even when the patients with liver cirrhosis and chronic hepatitis are used as a control group. Studies of the structure of abnormal prothrombin revealed that 2 of these domains are similar to Hepatocyte Growth Factor (HGF), and that both domains are thought to promote mitosis in mature hepatocytes. The expression of abnormal prothrombin in serum and tissues is closely related to the degree of deterioration of liver cancer and the degree of invasion of blood vessels. Meanwhile, researches show that the abnormal prothrombin can promote the proliferation and the transfer of human umbilical vein endothelial cell strains, influence the proliferation and the transfer of human liver cancer cells and cell signal conduction paths, and enhance the activity of liver cancer cell matrix metalloproteinase. Therefore, the examination of abnormal prothrombin is helpful for timely discovering and treating liver cancer.
In the existing diagnostic reagent products for detecting abnormal prothrombin, an antigen calibrator and a quality control product containing the abnormal prothrombin are needed. The conventional antigen calibrator and quality control product has the following conventional methods for the source and preparation of abnormal prothrombin:
(1) abnormal prothrombin extracted from plasma of liver cancer patient: the extraction process has high requirement, low yield and high cost, is difficult to stably store in a liquid environment, and may need a freeze-drying storage process, and when the freeze-dried product is used as a product calibrator or a quality control product, certain inconvenience is caused to customers;
(2) the abnormal prothrombin polypeptide is prepared by the expression of a genetic engineering method: due to uncertainty and randomness of the number of decarboxylation sites of the GLA segment of the abnormal thrombin, the recombinant antigen cannot be accurately expressed and prepared, the expression difficulty is high, meanwhile, the recombinant antigen is possibly subjected to an expression mechanism of host bacteria and lacks necessary modification, a secondary tertiary structure is definitely not available or almost impossible to be consistent with natural protein, and the recombinant abnormal prothrombin antigen which can be comparable with the natural antigen is possibly difficult to screen;
(3) preparation of abnormal prothrombin by chemical synthesis: abnormal prothrombin is prepared by chemical synthesis such as Fmoc method (fluorene methoxycarbonyl method), tBoc method (tert-butylcarbonyl method) etc. but chemical synthesis cannot achieve the coiling or folding of the spatial structure of the protein, so that secondary and tertiary structures are lacking; the abnormal prothrombin titer obtained by preparation cannot meet the requirement.
(4) Thermochemical decarboxylation procedure to produce abnormal prothrombin: although the thermochemical decarboxylation method is currently accepted by most researchers or manufacturers, the thermal decarboxylation preparation process requires high-temperature (greater than 110 ℃) treatment for a long time (more than 10 hours), and the high temperature inevitably damages secondary and tertiary structures of proteins, thereby directly influencing the biological activity or the antigenic potency of the proteins.
Therefore, the existing methods for obtaining the abnormal prothrombin have high difficulty and high cost, and simultaneously, due to the limitation of each method, the titer activity of the prepared abnormal prothrombin is not ideal, so that the yield is not high. Meanwhile, secondly, in order to stably store the abnormal prothrombin prepared by the prior art method, the abnormal prothrombin is generally frozen at-20 ℃ or-80 ℃ to form freeze-dried powder at present. However, the abnormal prothrombin lyophilized powder can denature the protein in repeated freeze/thaw cycles, leading to the formation of aggregates with reduced activity, and thus to reduced biological activity of the abnormal prothrombin.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an abnormal prothrombin and a preparation method thereof, so as to solve the technical problems of high difficulty, high cost, low titer activity and poor stability of the conventional method for obtaining the abnormal prothrombin.
The invention also aims to provide a detection reagent and a kit for detecting abnormal prothrombin, so as to solve the problems of low biological activity and poor storage stability of the abnormal prothrombin in the conventional detection reagent and kit for abnormal prothrombin.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a method for producing abnormal prothrombin. The preparation method of the abnormal prothrombin comprises the following steps:
performing decarboxylation on prothrombin by adopting a chemical catalysis method to convert gamma-glutamic acid in at least part of glutamic acid region of the prothrombin into de-gamma-glutamic acid.
Preferably, the method for preparing the abnormal prothrombin comprises the following steps:
mixing and dissolving the prothrombin and a catalyst to prepare a mixed solution;
drying the mixed solution in a supporting environment to obtain mixed powder;
and carrying out thermal decarboxylation catalytic reaction treatment on the mixture powder in a hydrogen environment.
Based on the preparation method, the invention also provides the abnormal prothrombin prepared by the preparation method.
In yet another aspect of the invention, a detection agent is provided. The detection agent comprises the abnormal prothrombin.
In yet another aspect of the invention, a kit for detecting abnormal prothrombin is provided. The kit comprises the abnormal prothrombin or the detection agent.
In still another aspect of the invention, a method for using the detection agent or the kit is provided. The detection agent or the kit is applied to the clinical detection of the abnormal prothrombin.
Compared with the prior art, the preparation method of the abnormal prothrombin directly carries out decarboxylation on the prothrombin by a chemical catalysis method, and carries out decarboxylation reaction on carboxyl of a prothrombin glutamic acid region, so that gamma-glutamic acid is converted into de-gamma-glutamic acid, thereby ensuring that the formed abnormal prothrombin structure is a natural abnormal prothrombin structure and endowing the abnormal prothrombin with high biological activity. In addition, the method has easily controlled process conditions, and the prepared abnormal prothrombin has high and stable activity, high cost, high yield and low cost. Thereby effectively overcoming the defects of the prior chemical synthesis method, the prior genetic engineering method and the method for extracting the abnormal prothrombin from the blood plasma of the liver cancer patient.
The detection agent of the present invention is a solution having a predetermined concentration formed by using the abnormal prothrombin of the present invention. Because the abnormal thrombin has high titer and good stability, the abnormal thrombin in the detection agent has high biological activity, good stability and low cost.
The kit for detecting the abnormal prothrombin comprises the abnormal prothrombin or the detection agent, so the kit has high abnormal prothrombin titer and good stability, and the kit can accurately, conveniently and quickly detect the abnormal prothrombin in a sample.
Drawings
FIG. 1 is a flow chart of a method for producing abnormal prothrombin according to an embodiment of the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Interpretation of the nomenclature:
abnormal prothrombin: des-gamma-carboxypropylhrombin (DCP) is a Protein (Protein Induced by Vitamin K Absence or Antagonist-II) Induced by Vitamin K, and is abbreviated as PIVKA-II.
In one aspect, the embodiments of the present invention provide a method for preparing abnormal prothrombin. The preparation method of the abnormal prothrombin comprises the following steps:
performing decarboxylation on prothrombin by adopting a chemical catalysis method to convert gamma-glutamic acid in at least part of glutamic acid region of the prothrombin into de-gamma-glutamic acid.
In this way, the preparation method of the abnormal prothrombin directly carries out decarboxylation treatment on prothrombin by a chemical catalysis method, so that at least part of carboxyl groups in a glutamic acid region of the prothrombin are subjected to decarboxylation reaction, namely, gamma-glutamic acid is converted into de-gamma-glutamic acid, and thus the target product abnormal prothrombin is obtained.
In one embodiment, the decarboxylation is performed by performing a chemical catalysis method on prothrombin to obtain a decarboxylation process as shown in fig. 1, which includes the following steps:
step S01: mixing and dissolving the prothrombin and a catalyst to prepare a mixed solution;
step S02: drying the mixed solution in a supporting environment to obtain mixed powder;
step S03: and carrying out chemical catalytic decarboxylation treatment on the mixture powder in a hydrogen environment.
In step S01, after the catalyst and the prothrombin are mixed, the catalyst and the prothrombin can be uniformly dispersed and fully contacted, so that the catalyst can fully play a role in the subsequent thermal decarboxylation catalytic reaction process to prepare the abnormal prothrombin with stable performance. In one embodiment, the catalyst comprises catalyst I and catalyst II; the catalyst I is a transition metal compound for providing transition metal ions, the catalyst II is an oxide, and in a further embodiment, the molar concentration ratio of the catalyst I to the catalyst II is 1 (1-3). Therefore, the composite catalyst can effectively catalyze and decarboxylate prothrombin in a hydrogen environment through the synergistic interaction between the transition metal catalyst and the oxide catalyst, so that the temperature required by the decarboxylation process is obviously reduced, the decarboxylation efficiency is improved, and the decarboxylation reaction time is effectively shortened. As the decarboxylation conditions such as decarboxylation temperature, time and the like become mild, the structure of more than two dimensions of the prothrombin protein in the decarboxylation treatment process is effectively protected from being damaged, so that the high yield and high cost of the abnormal thrombin are effectively ensured, and the production cost is reduced. Therefore, the preparation method of the abnormal prothrombin can simultaneously consider the advantages of high yield and high cost of the abnormal thrombin and low production cost, thereby effectively overcoming the defects of the existing chemical synthesis method (the valence is low due to the damage to the secondary and tertiary structures), the genetic engineering method (the main difficulty is high, the valence is low, the cost is high), the thermochemical decarboxylation method (the main existence is low in biological activity or antigen valence due to long-time high-temperature treatment, and the cost is high).
In a specific embodiment, the transition metal compound comprises at least one of transition metal nitrate, transition metal chloride, transition metal sulfate, and transition metal acetate; wherein the transition metal ions comprise at least one of palladium ions, platinum ions, rhodium ions, silver ions, ferrous ions and copper ions; the oxide comprises at least one of aluminum oxide, silicon dioxide and gallium sesquioxide. The specific catalyst I and the specific catalyst II are selected, so that the synergistic catalytic action of the catalyst I and the catalyst II can be improved, the decarboxylation temperature is further obviously reduced, the decarboxylation time is shortened, the stability of an abnormal prothrombin structure, particularly a secondary structure and a tertiary structure, in the catalytic decarboxylation process is improved, and the titer activity of the generated abnormal prothrombin is improved.
In another embodiment, the prothrombin and the catalyst are mixed according to the mass ratio of 1 (5-30), so that the prothrombin can be subjected to decarboxylation in the chemical catalytic decarboxylation reaction treatment in the subsequent step. In addition, the mixing of the two can be a conventional mixing manner, such as mechanical stirring and the like. After the mixing treatment, the mixture is subjected to a dissolving treatment, and the solvent for dissolving may be a conventional solvent capable of dissolving and securing the biological activity of the thrombin, such as water, a buffer solution, etc. The concentration of the prepared mixed solution can be flexibly adjusted and controlled according to the requirement, and the concentration can be any concentration which can ensure that the mixed solution and the solution can be uniformly dispersed.
In step S02, the mixed solution is dried to remove the solvent in the mixed solution, so as to obtain a mixture of the prothrombin and the catalyst. The support environment refers to the environment in which the mixture is dried, in particular to the gas-phase support environment. The support environment is a gas phase support environment to ensure that the prothrombin is not affected by external factors to ensure the bioactivity, therefore, the gas phase support environment can be vacuum or inert atmosphere such as nitrogen and argon. In one embodiment, the drying process is performed in a vacuum at 45-60 ℃.
In another embodiment, the method of drying the mixed solution in the supporting environment in step S02 is to combine the mixed solution with a solid support and then perform a vacuum drying process. The solid support is adopted to bind and adsorb the prothrombin and the catalyst in the mixed solution, so that the prothrombin and the catalyst can be uniformly dispersed, and the drying treatment efficiency of the mixed solution can be accelerated. In one embodiment, the solid support comprises at least one of a pure metal, an alloy, and a resin. Wherein the resin can be conductive or non-conductive, low melting point or high melting point, liquid phase or solid phase, block or powder, and can be used or processed for use. Such as: aluminum-based silica gel, dextran series (nitrocellulose aluminum film), polystyrene, polychloroprene, polyethylene, polymethyl methacrylate, and the like. In specific embodiments, the mass ratio of the prothrombin to the solid support is controlled to be: 1, (1000-3000), wherein the vacuum drying temperature is 45-60 ℃.
In step S03, the powder of the mixture is subjected to a chemical catalytic decarboxylation treatment in a hydrogen atmosphere, and the prothrombin in the mixture undergoes a decarboxylation reaction under the catalytic action of the catalyst, such as the mixed catalyst containing the catalyst i and the catalyst ii. In one embodiment, the temperature of the chemical catalytic decarboxylation is 80-100 ℃ and the time is 2-4 h.
When the mixture powder is formed by combining and adsorbing the solid phase support, after the chemical catalytic decarboxylation reaction treatment, the method further comprises the following steps of:
and dissolving the mixture powder subjected to the chemical catalytic decarboxylation, collecting supernatant, and dissolving the abnormal prothrombin generated by the chemical catalytic decarboxylation in the supernatant.
Therefore, the above-mentioned method for preparing the abnormal prothrombin directly performs decarboxylation on the chemical catalysis method of the prothrombin, thereby effectively protecting the prothrombin from being damaged, improving the effect of the chemical catalysis decarboxylation treatment and endowing the prepared abnormal prothrombin with high biological activity. In addition, the abnormal prothrombin is prepared by adopting prothrombin as a raw material, and the source of the prothrombin is rich and easy to obtain, so that the cost of the abnormal prothrombin is effectively reduced. Secondly, the preparation method has easily controlled process conditions, such as low temperature which is lower than 100 ℃, short reaction time which is 2-4 hours, and the prepared abnormal prothrombin has high and stable activity, namely high titer, high stability, high yield, low temperature and relatively short time, and effectively reduces the cost. Therefore, the above-mentioned method for preparing abnormal prothrombin can effectively overcome the defects of the existing chemical synthesis method (low main titer), the genetic engineering method (mainly difficult, low titer and high cost), the thermochemical decarboxylation method (mainly long-time high-temperature treatment causes low biological activity or antigen titer and high cost) and the extraction method for obtaining abnormal prothrombin from the plasma of liver cancer patients (mainly inconvenient to use and low in amount).
The method for preparing the abnormal prothrombin is based on the method for preparing the abnormal prothrombin. The embodiment of the invention also provides abnormal prothrombin. The abnormal prothrombin is prepared by the abnormal prothrombin preparation method. Thus, the abnormal prothrombin-abnormal prothrombin is structurally stable and highly active.
In a further aspect, embodiments of the present invention provide a detection agent, in particular a detection agent for detecting abnormal prothrombin. The detection agent comprises the abnormal prothrombin described above. The detection agent adopts the solution with stable concentration formed by the abnormal prothrombin, and the abnormal prothrombin has the advantages of high abnormal prothrombin titer and good stability, so the detection agent can be stored for a long time, and the content of the abnormal prothrombin can keep high activity and stability under the normal-temperature liquid state. Because the detection agent has the advantages, in one embodiment, the detection agent can be used as a calibrator and/or a quality control product for detecting abnormal prothrombin, so that the accuracy of the calibrator and the quality control product is improved.
In yet another aspect, embodiments of the invention provide a kit for detecting abnormal prothrombin. The kit for detecting abnormal prothrombin comprises the following reagents: abnormal prothrombin according to the embodiments of the invention described above or a detection agent according to the embodiments of the invention described above. The specification, such as volume, of the detection agent can be flexibly set according to the specification of the kit, wherein the concentration of the abnormal prothrombin contained in the detection agent can also be flexibly adjusted according to the detection requirement. In one embodiment, the calibrator for detecting abnormal prothrombin contained in the kit is provided with a low-concentration calibrator and a high-concentration calibrator, and in a specific embodiment, the concentration of the low-concentration calibrator may be, but is not limited to, 200mAU/mL, and the concentration of the high-concentration calibrator may be, but is not limited to, 10000 mAU/mL. In one embodiment, the quality control material contained in the kit comprises a quality control material 1 and a quality control material 2, and in a specific embodiment, the concentration of the quality control material 1 can be, but is not limited to, 50mAU/mL, and the concentration of the quality control material 2 can be, but is not limited to, 5000 mAU/mL.
Of course, the kit may further comprise other necessary reagents for detecting abnormal prothrombin, such as reagents related to chemiluminescence immunoassay, specifically, an anti-abnormal prothrombin antibody-coated magnetosphere reagent, a labeled reagent for labeling the anti-abnormal prothrombin antibody, and the like. The labelling agent may be an ABEI labelling agent.
Therefore, the kit for detecting abnormal prothrombin has high titer and good stability because the kit contains the abnormal prothrombin or the detection agent (such as a calibrator or/and a quality control material), so that the kit is ensured to detect the abnormal prothrombin accurately, conveniently and quickly.
In addition, the detection of the abnormal prothrombin content in a sample using the kit for detecting abnormal prothrombin may be performed according to a conventional chemiluminescence immunoassay.
Now, the method for producing abnormal prothrombin, the preservation solution for abnormal prothrombin, and the use thereof according to the embodiment of the present invention will be described in further detail with reference to specific examples.
1. Method for producing abnormal Prothrombin
Example 11 example 19
This example 11-19 provides a method for the preparation of abnormal prothrombin, respectively. The preparation method of the abnormal prothrombin comprises the following steps:
(1) and (3) dialysis: placing commercial 1mg of human plasma prothrombin in a dialysis bag, dialyzing in 1L of 0.1M sodium bicarbonate solution with pH8.0 at 4 deg.C for 2 h;
(2) mixing and dissolving human prothrombin with a catalyst I and a catalyst II according to the proportion in the following table 1 to prepare a mixed solution;
(3) combining the mixed solution in the step (2) with a solid phase support according to the proportion in the following table 1, and drying in vacuum to obtain mixture powder;
(4) performing thermocatalytic decarboxylation treatment on the mixture powder obtained in the step (3) in a hydrogen environment in the following table 1 to convert gamma-glutamic acid in at least part of glutamic acid regions of the prothrombin into de-gamma-glutamic acid, so as to generate abnormal prothrombin;
(5) and (3) dissolving the reaction product in the step (4) in 50mM Tris-HCl buffer solution, and taking a supernatant after ultrasonic mixing, wherein the supernatant is the abnormal prothrombin.
Comparative examples 11 to 14
The abnormal prothrombin provided in comparative examples 11 to 14 was obtained according to the conventional method shown in Table 1 below.
TABLE 1
2. Detection reagents for detecting abnormal Prothrombin and kit embodiments
Examples 21 to 29 and comparative examples 21 to 24
This example 21-29 and comparative example 21-comparative example 24 provide a kit for detecting abnormal prothrombin, respectively, comprising the following components:
(1) anti-aberrant prothrombin antibody coated magnetic microspheres, wherein the concentration of magnetic microspheres is: 1mg/mL, the concentration of the anti-abnormal prothrombin antibody is 10 mug/mL; the magnetic microsphere coated by the anti-abnormal prothrombin antibody is prepared by the following method:
a certain amount of magnetic spheres (Estapor series carboxylated microspheres M1-180/20 manufactured by Merck) were taken, 10mg of DCC was added per mg of magnetic spheres, and then a DMF solution was added to make the concentration of the magnetic spheres 20mg/ml, and the mixture was placed in a 38 ℃ water bath and shaken or mechanically stirred for 2 hours. Removing supernatant from the activated nano magnetic ball solution, and then adding carbonate buffer solution with pH of 9.5 to make the concentration of the carbonate buffer solution reach 20mg/ml (measured by nano magnetic balls); then, an anti-abnormal prothrombin antibody was added in an amount of 10. mu.g per mg of the magnetic sphere, and the mixture was left to react at room temperature for 2 hours with shaking. And (3) carrying out magnetic separation, and washing the solid for three times by using washing liquor (0.5% BSA solution) to obtain the magnetic ball coated with the abnormal prothrombin resisting antibody, namely the detection agent. The thus-obtained detection reagent was added to a diluent (carbonate buffer, phosphate buffer, borate buffer or Tris buffer) to a concentration of 1mg/ml for use.
(2) An ABEI-labeled anti-aberrant prothrombin antibody, wherein ABEI concentration: 250ng/mL, concentration of anti-aberrant prothrombin antibody: 5000 ng/mL; the ABEI labeled anti-abnormal prothrombin antibody is prepared according to the following method:
1mg of the anti-abnormal prothrombin antibody was adjusted to 1ml in volume with 0.1mol/L carbonate buffer (pH9.5), and then placed in a dialysis bag and dialyzed against pH9.5 carbonate buffer for 1 hour. The dialyzed anti-abnormal prothrombin antibody was removed, 50. mu.g of ABEI-hemisuccinamic acid-NHS was added and shaken at room temperature for 1.5 hours.
The G-25 gel column was mounted, and after being washed clean with purified water, the column was equilibrated with PBS buffer at pH 7.4. And after the equilibrium elution of the G-25 gel column is finished, purifying the ABEI-marked anti-abnormal prothrombin antibody by passing through the column, and then collecting a protein solution with a peak value. The collected protein solution was diluted to 0.15. mu.g/ml by adding an equal volume of the protective solution containing 5% BSA.
(3) The calibrators in examples 21-29 shown in table 2:
a low-concentration calibrator comprising an abnormal prothrombin antigen dissolved in a stock solution at a concentration of: 200 mAU/mL;
a high-concentration calibrator comprising an abnormal prothrombin antigen dissolved in a storage solution at a concentration of: 10000 mAU/mL;
(4) quality controls in examples 21 to 29 shown in Table 2:
the quality control product 1 contains an abnormal prothrombin antigen, wherein the abnormal prothrombin antigen is dissolved in a preservation solution, and the concentration is as follows: 50 mAU/mL;
and the quality control product 2 contains an abnormal prothrombin antigen, and the abnormal prothrombin antigen is dissolved in the preservation solution, and the concentration is as follows: 5000 mAU/mL;
(5) the preservation diluent of the low-concentration calibrator, the high-concentration calibrator, the quality control product 1 and the quality control product 2 is 50mM Tris-HCl buffer solution.
TABLE 2
Experimental methods, data and results analysis
(1) And (3) yield determination: the abnormal prothrombin product prepared by the method from 1mg of the fed prothrombin is taken, the supernatant is taken for mass concentration measurement and titer concentration measurement, and the yield is calculated as follows:
yield (titer concentration/mass concentration) 100%,
wherein, the titer concentration is the concentration detected by a Fujirebio abnormal prothrombin determination kit, the unit is mAU/mL, meanwhile, 1mAU/mL is 1ng/mL, the mass concentration is the protein mass concentration determined by a Qbit fluorescence quantitative method, and the unit is mg/mL.
The calculation results are shown in Table 3.
The specific experimental steps for determining the protein concentration of abnormal prothrombin by using the Qbit fluorescence quantitative method are as follows:
and (3) uniformly mixing 10 mu L of Qbit dye solution with 1990 mu L of Qbit buffer solution to obtain Qbit working solution, mixing 10 mu L of standard substance (test sample) and 190 mu L of Qbit working solution in a PCR-05-C tube, reacting at room temperature for 15min, placing the PCR-05-C tube in a Qbit fluorescence quantitative instrument for reading, and converting to obtain the protein concentration of the abnormal prothrombin.
(2) And (3) measuring the titer: the titer concentrations of the abnormal prothrombin in examples 11 to 19 and comparative examples 11 to 14 were measured by the abnormal prothrombin assay kit, and the calculation results are shown in Table 3.
The measurement method is as follows:
1) the first step of reaction: adding 50 mu L of the abnormal thrombin stock solution prepared in the examples 11-19 and the comparative examples 1-4, 100 mu L of buffer solution and 20 mu L of magnetic sphere system solution, uniformly mixing, incubating for 10min at 37 ℃, and cleaning for 3 times in a magnetic environment;
2) the second step of reaction: add 200. mu.L of the luminescent marker system solution and mix well. Incubating at 37 ℃ for 10min to form a luminescent immune complex which comprises the anti-abnormal prothrombin antibody coated by the magnetic microspheres, the abnormal prothrombin to be detected, the anti-abnormal prothrombin antibody and the ABEI;
4) and (3) detection: the luminescent immune complex is precipitated by applying a magnetic field, the supernatant is removed and washed with an automatic perfusion system buffer, a chemiluminescent activator is added and the relative light intensity (RLU) emitted is detected. And converting to obtain the titer of the abnormal thrombin.
(3) And (3) stability evaluation:
the test kit comprises: an abnormal prothrombin assay kit (chemiluminescence immunoassay) prepared according to the following method;
and (3) testing conditions are as follows: a test group (oscillating at constant temperature of 180r/min at 37 ℃) and a control group (oscillating at constant temperature of 180r/min at 2-8 ℃);
testing a sample: a low-concentration calibrator, a high-concentration calibrator, a quality control product 1 and a quality control product 2;
and (3) testing time: continuously tracking for 7 days;
other descriptions: the test samples under two groups of test conditions are continuously tracked for 7 days of RLU, the change condition of the RLU is observed, and the phenomenon that whether the abnormal prothrombin in a calibration product and a quality control product is subjected to protein degradation or denaturation or not in the transportation process of a product is simulated, so that the titer of an antigen is reduced, and the performance and the stability of the reagent are influenced is simulated.
The test method comprises the following steps: and calculating and counting the average relative deviation of the RLUs of the product calibration product and the quality control product which are continuously tracked for 7 days by the test samples under the two groups of test conditions under the examples 21-29 and the comparative examples 21-24.
The results are shown in Table 4.
TABLE 3
TABLE 4
As can be seen from the data in tables 3 and 4, the yield, titer and stability of abnormal prothrombin were slightly better than those of 1:3 in example 21 and example 29, when the ratio of prothrombin to solid support was 1mg:2 g.
Comparing example 21 with example 28, it can be seen that the ratio of prothrombin to solid support and the reaction temperature and time synergistically affect the yield, titer and stability of the abnormal prothrombin produced;
compared with examples 29 and 28, the yield, titer and stability of 3h at 90 ℃ are slightly better than those of 100 ℃ for 2 h;
compared with example 27, the yield, titer and stability of prothrombin and catalyst are better than 1mg to 30mg when the ratio of prothrombin to catalyst is 1mg to 15 mg;
compared with example 26, the yield, titer and stability of the abnormal prothrombin prepared by the synergistic effect of prothrombin, catalyst ratio and reaction temperature and time can be obtained;
compared with examples 27 and 26, the yield, titer and stability of 3h at 90 ℃ are slightly better than those of 80 ℃ for 4 h;
pd can be obtained in example 21 as compared with example 252+: the yield, the titer and the stability of the oxide which is 1:2 are better than 1: 3;
example 21 compares with example 23 and shows that the catalyst is formed from Pd2+、SiO2、Al2O3The effect of the combination of the three is better than that of the two;
compared with example 22, it can be seen that the kind and ratio of the catalyst synergistically affect the yield, titer and stability of the produced abnormal prothrombin;
compared with example 24, it can be shown that the transition metal ion species in catalyst I also affects the yield, titer and stability of the produced abnormal prothrombin;
from the comparative analysis, the catalyst was formed from Pd2+、SiO2、Al2O3The combination effect of the three is superior to that of Pd2+And SiO2Or Pd2+And Al2O3，Pd2+And oxide SiO2(or Al)2O3) The molar concentration ratio of (1) - (3), optimally 1: 2; the mass ratio of the prothrombin to the catalyst is 1mg (5-30) mg, and the optimal mass ratio is 1mg to 15 mg; the mass ratio of prothrombin to solid support is: 1mg (1-3) g, optimally 1mg:2 g; introduction of H2The reaction temperature is 80-100 ℃, the reaction time is 2-4h, the optimal reaction time is 90 ℃, and the reaction time is 3 h; the vacuum rotary drying temperature is as follows: 45-60 ℃.
As can be seen from the data in tables 3 and 4, the abnormal prothrombin provided by the embodiment of the invention has high yield, high titer and stability, and the standard substance or quality control substance containing the abnormal prothrombin can be stably maintained in a liquid state for a long time and has good biological activity.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for preparing abnormal prothrombin comprises the following steps:
subjecting prothrombin to a chemocatalytic decarboxylation process to convert gamma-glutamic acid of at least a portion of the glutamic acid region of the prothrombin to des-gamma-glutamic acid;
the decarboxylation treatment method comprises the following steps:
mixing and dissolving the prothrombin and a catalyst to prepare a mixed solution;
drying the mixed solution in a supporting environment to obtain mixed powder;
performing chemical catalytic decarboxylation treatment on the mixture powder in a hydrogen environment;
the catalyst comprises a catalyst I and a catalyst II; wherein, the catalyst I is a transition metal compound for providing transition metal ions, and the catalyst II is an oxide;
the mass ratio of the prothrombin to the catalyst is 1 (5-30).
2. The method of claim 1, wherein: the transition metal compound comprises at least one of transition metal nitrate, transition metal chloride, transition metal sulfate and transition metal acetate; and/or
The transition metal ions comprise at least one of palladium ions, platinum ions, rhodium ions, silver ions, ferrous ions and copper ions; and/or
The oxide comprises at least one of aluminum oxide, silicon dioxide and gallium sesquioxide.
3. The production method according to claim 1 or 2, characterized in that: the molar concentration ratio of the catalyst I to the catalyst II is 1 (1-3).
4. The production method according to any one of claims 1 to 2, characterized in that: the mixed solution is dried in a supporting environment by combining the mixed solution with a solid support and then drying the combined solution in vacuum.
5. The method of claim 4, wherein: the solid support comprises at least one of pure metal, alloy and resin; and/or
The mass ratio of the prothrombin to the solid support is as follows: 1, (1000-3000); and/or
The vacuum drying temperature is 45-60 ℃.
6. The method of claim 4, wherein: and after the vacuum drying treatment, dissolving the mixture powder subjected to the chemical catalytic decarboxylation reaction treatment, and collecting supernatant.
7. The production method according to any one of claims 1 to 2, 5 and 6, wherein: the temperature of the chemical catalytic decarboxylation reaction treatment is 80-100 ℃, and the time is 2-4 hours.
8. A preparation method of a detection agent is characterized by comprising the following steps: comprising the steps of the method for producing abnormal prothrombin according to any one of claims 1 to 7.
9. The method for producing a detection agent according to claim 8, characterized in that: the prepared detection agent is used as a calibrator or/and a quality control product for detecting abnormal prothrombin.
10. A preparation method of a kit for detecting abnormal prothrombin is characterized by comprising the following steps: the kit comprises the steps of the method for preparing the detection agent according to any one of claims 9 to 9.
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