CN114112587A - Preparation method of glycated albumin calibrator or quality control product - Google Patents
Preparation method of glycated albumin calibrator or quality control product Download PDFInfo
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- CN114112587A CN114112587A CN202111415865.0A CN202111415865A CN114112587A CN 114112587 A CN114112587 A CN 114112587A CN 202111415865 A CN202111415865 A CN 202111415865A CN 114112587 A CN114112587 A CN 114112587A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N2001/2893—Preparing calibration standards
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Abstract
The invention belongs to the technical field of in-vitro diagnostic reagents, and provides a preparation method of a glycated albumin calibrator or a quality control product, aiming at overcoming the defects of complicated production process, high cost and easy change of measured value of a liquid form product of the glycated albumin calibrator or the quality control product, which comprises the following steps: human recombinant albumin or serum is taken as a raw material to carry out glycosylation; adding glycosylation inhibitor or terminator, wherein the inhibitor or terminator is one or more selected from metal chelating agent, thioctic acid or carnosine, the concentration of the metal chelating agent is 0.1-3g/L, the concentration of the thioctic acid is 0.1-1g/L, and the concentration of the carnosine is 0.05-1 g/L; adding unfrozen liquid, wherein the volume ratio of the unfrozen liquid to the glycosylation reaction liquid is 1:3-1:1, and the unfrozen liquid is 1, 2-propylene glycol and/or 1, 3-propylene glycol; storing the prepared glycated albumin calibrator or quality control product at a temperature below-20 deg.C. The method has the advantages of simple production process, low cost, good stability of the reagent and convenient use.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to a preparation method of a glycated albumin calibrator or quality control material.
Background
Glucose in the human body undergoes a non-enzymatic glycosylation reaction with the N-terminal of serum proteins, 90% of which are bound to the 189 nd lysine in the serum protein chain to form a high molecular ketoamine structure, collectively referred to as Glycated serum proteins, and more than 90% of which are Glycated serum Albumin (GA), so that the total level of Glycated serum Albumin can be reflected. Glycated albumin is an index that reflects the average blood glucose level over the past 2-3 weeks, and is shorter than the "gold standard" glycated hemoglobin in the blood glucose test. Therefore, GA has an advantage over glycated hemoglobin in confirming therapeutic effects and adjusting clinical drug doses.
However, the preparation process of the traditional glycated albumin calibrator generally comprises the steps of glycosylation, filtration or dialysis to remove unreacted glucose, so as to avoid the change of a measured value caused by further glycosylation, and the preparation process of the glycated albumin calibrator after dialysis is in a form of freeze-dried powder, so that the preparation process is complicated and is not beneficial to mass production. For example, patent CN105092336A uses serum as raw material, and the finished product is obtained by centrifugation, glycosylation, dialysis purification, and freeze drying; CN1103076213A adopts human recombinant albumin as a raw material, and the finished product is obtained by glycosylation, dialysis and purification, and then freeze drying; CN104764638A, also using human recombinant albumin as raw material to carry out glycosylation, adopting tangential flow filtration technology in the purification process, and before the above patent is made into finished products, all the above patents need to go through purification steps in order to remove unreacted glucose and small molecule glycosylated polypeptide, and avoid the continuous change of reagent measured value in glycosylation reaction. And finally, the raw materials are all prepared into freeze-dried powder, so that the process flow is relatively complex. In view of the fact that the glycosylation process of the calibrator and the quality control is influenced by a plurality of interference factors, and the measured value of the liquid form product is easy to change, no report related to the liquid glycated albumin calibrator and the quality control is found at present.
Disclosure of Invention
The invention aims to overcome the defects that the process is complicated in the production process of the glycated albumin calibrator and the glycated albumin quality control product in the form of freeze-dried powder and the measured value of a liquid product is easy to change, and provides a preparation method of the glycated albumin calibrator or the glycated albumin quality control product.
The invention is realized by the following technical scheme:
a preparation method of a glycated albumin calibrator or quality control product comprises the following steps:
(1) human recombinant albumin or serum is taken as a raw material to carry out glycosylation, and the glycosylation degree is determined according to the concentration of a calibrator or a quality control product;
(2) adding glycosylation reaction inhibitor or terminator to maintain the glycosylated protein at a certain concentration, wherein the inhibitor or terminator is one or more selected from metal chelating agent, lipoic acid or carnosine, the final concentration of the metal chelating agent is 0.1-3g/L, the final concentration of the lipoic acid is 0.1-1g/L, and the final concentration of the carnosine is 0.05-1 g/L;
(3) adding unfrozen liquid, wherein the volume ratio of the unfrozen liquid to the glycosylation reaction liquid is 1:3-1:1, and the unfrozen liquid is 1, 2-propylene glycol or 1, 3-propylene glycol or the combination thereof;
(4) storing the prepared glycated albumin calibrator or quality control product at a temperature below-20 deg.C.
Further, in the step (2), the final concentration of the metal chelating agent is 0.5g/L, the final concentration of zinc sulfate is 0.35g/L, and the final concentration of carnosine is 0.75 g/L.
Further, the metal chelating agent in the step (2) is nitrilotriacetic acid.
Further, the method also comprises a step (4) of storing the glycated albumin calibrator or quality control product at a temperature ranging from-20 ℃ to-30 ℃.
Further, the storage time of the step (4) is 12 months at most.
Has the advantages that:
(1) the progress of glycosylation reaction is inhibited by adding an inhibitor or a terminator, and the progress of glycosylation reaction is further inhibited by low-temperature storage, so that the stability of the reagent is ensured;
(2) dialysis or filtration is not needed, so that the complex steps of preparing freeze-dried powder are avoided, the production process is simpler, the cost is low, and the method is more suitable for mass production;
(3) in the redissolution process of the freeze-dried powder, because the volume of the added redissolution is possibly deviated, the water content of each batch of freeze-dried powder is also possibly different, and dust is possibly blown out in the uncovering process, the possibility of error is brought, and the reagent is a liquid reagent, so that the problem is avoided;
(4) the liquid reagent is more convenient to take and use.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The specific degree of glycosylation reaction solution is determined according to the required calibration and quality control concentration value, wherein the early glycosylation process is not in the invention content, the invention aims to solve the problem of avoiding the purification process by adding glycosylation reaction inhibitor or terminator, and simplifying the production process by making the glycosylation reaction inhibitor or terminator into a liquid form, and the feasibility of the simplified production process provided by the invention is illustrated only by specific examples. Thus any other form of inhibition or termination of the glycosylation reaction may be inspired by the present invention and is also within the scope of the invention.
Example 1:
and (2) placing the solution at 37 ℃, carrying out glycosylation for 36h, mixing uniformly, testing by using a glycated albumin detection kit, wherein the concentration of glycated albumin in the solution is 7.31 +/-0.54 g/L, adding a metal chelating agent nitrilotriacetic acid (nitrilotriacetic acid) of 0.5g/L into the solution, mixing uniformly, and mixing with 1, 3-propanediol according to a volume ratio of 1:1, mixing, fully mixing again, then subpackaging in 1mL vials, storing at-20 ℃, respectively taking out one vial at different time periods, testing the content of the glycated albumin, and measuring the value shown in the following table:
table 1 example 1 stability of glycated albumin reagent
| Time | 7 days | 1 month | 3 months old | 6 months old | 12 months old |
| Glycated Albumin concentration (g/L) | 7.32 | 7.29 | 7.25 | 7.33 | 7.31 |
The glycated albumin calibrator or quality control product prepared by the embodiment has a uniform state and stable measured value (which does not exceed the target value range) within 12 months of storage at-20 ℃, and is suitable for mass production.
Example 2
And (2) placing the solution at 37 ℃, carrying out glycosylation reaction for 72h, mixing uniformly, testing by using a glycated albumin detection kit, wherein the concentration of glycated albumin in the solution is 21.43 +/-1.61 g/L, adding alpha-lipoic acid into the solution, and mixing uniformly, and then mixing the solution with 1, 3-propanediol according to a volume ratio of 1: 2, mixing, fully mixing again, then subpackaging in 1mL vials, storing at-20 ℃, respectively taking out one vial at different time periods, testing the content of the glycated albumin, and measuring the value shown in the following table:
table 2 example 2 stability of glycated albumin reagent
| Time | 7 days | 1 month | 3 months old | 6 months old | 12 months old |
| Glycated Albumin concentration (g/L) | 21.41 | 21.38 | 21.46 | 21.45 | 21.52 |
The glycated albumin calibrator or quality control product prepared by the method has uniform state and stable measured value (not exceeding the target value range) within 12 months of storage at-20 ℃, and is suitable for mass production.
Example 3:
and (2) placing the solution at 37 ℃, carrying out glycosylation reaction for 36h, mixing uniformly, testing by using a glycated albumin detection kit, wherein the concentration of glycated albumin in the solution is 7.79 +/-0.57 g/L, adding carnosine into the solution, and mixing uniformly, and then mixing the solution with 1, 3-propanediol according to a volume ratio of 1:1, mixing, fully mixing again, then subpackaging in 1mL vials, storing at-20 ℃, respectively taking out one vial at different time periods, testing the content of the glycated albumin, and measuring the value shown in the following table:
table 3 example 3 stability of glycated albumin reagent
| Time | 7 days | 1 month | 3 months old | 6 months old | 12 months old |
| Glycated Albumin concentration (g/L) | 7.83 | 7.79 | 7.75 | 7.85 | 7.83 |
The glycated albumin calibrator or quality control product prepared by the method has uniform state and stable measured value (not exceeding the target value range) within 12 months of storage at-20 ℃, and is suitable for mass production.
Example 4:
placing the solution at 37 ℃, carrying out glycosylation reaction for 48h, mixing uniformly, testing by using a glycated albumin detection kit, wherein the concentration of glycated albumin in the solution is 11.67 +/-0.87 g/L, and adding a metal chelating agent nitrilotriacetic acid into the solution to a final concentration of 0.25 g/L; carnosine with a final concentration of 0.35g/L is fully and uniformly mixed, and then mixed with 1,3 propylene glycol according to a volume ratio of 1:1, mixing, fully mixing again, then subpackaging in 1mL vials, storing at-20 ℃, respectively taking out one vial at different time periods, testing the content of the glycated albumin, and measuring the value shown in the following table:
table 4 example 4 stability of glycated albumin reagent
| Time | 7 days | 1 month | 3 months old | 6 months old | 12 months old |
| Glycated Albumin concentration (g/L) | 11.72 | 11.69 | 11.75 | 11.73 | 11.68 |
The glycated albumin calibrator or quality control product prepared by the embodiment has a uniform state and stable measured value (which does not exceed the target value range) within 12 months of storage at-20 ℃, and is suitable for mass production.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (5)
1. A preparation method of a glycated albumin calibrator or quality control product is characterized by comprising the following steps:
(1) human recombinant albumin or serum is taken as a raw material to carry out glycosylation, and the glycosylation degree is determined according to the concentration of a calibrator or a quality control product;
(2) adding glycosylation reaction inhibitor or terminator to maintain the glycosylated protein at a certain concentration, wherein the inhibitor or terminator is one or more selected from metal chelating agent, lipoic acid or carnosine, the final concentration of the metal chelating agent is 0.1-3g/L, the final concentration of the lipoic acid is 0.1-1g/L, and the final concentration of the carnosine is 0.05-1 g/L;
(3) adding unfrozen liquid, wherein the volume ratio of the unfrozen liquid to the glycosylation reaction liquid is 1:3-1:1, and the unfrozen liquid is 1, 2-propylene glycol or 1, 3-propylene glycol or the combination thereof;
(4) storing the prepared glycated albumin calibrator or quality control product at a temperature below-20 deg.C.
2. The method of claim 1, wherein the final concentration of the metal chelating agent in step (2) is 0.5g/L, the final concentration of zinc sulfate is 0.35g/L, and the final concentration of carnosine is 0.75 g/L.
3. The method of claim 1, wherein the metal chelator of step (2) is nitrilotriacetic acid.
4. The method for preparing a glycated albumin calibrator or quality control according to any one of claims 1 to 3, further comprising the step of (4) storing the glycated albumin calibrator or quality control at a temperature ranging from-20 ℃ to-30 ℃.
5. The method of claim 4, wherein the glycated albumin calibrator or quality control product is stored in step (4) for a period of up to 12 months.
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