CN114755085A - Preparation and application of glycosylated hemoglobin quality control product - Google Patents
Preparation and application of glycosylated hemoglobin quality control product Download PDFInfo
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- CN114755085A CN114755085A CN202210467323.6A CN202210467323A CN114755085A CN 114755085 A CN114755085 A CN 114755085A CN 202210467323 A CN202210467323 A CN 202210467323A CN 114755085 A CN114755085 A CN 114755085A
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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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- 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
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Abstract
The invention discloses a preparation method and application of a glycosylated hemoglobin quality control product. The preparation method of the glycosylated hemoglobin quality control product is simple, and the glycosylated hemoglobin quality control product can be quickly prepared without complex operation and expensive instruments; the prepared glycosylated hemoglobin quality control product has good stability, the glycosylated hemoglobin value is within the range of +/-10% of the mean value within 24 months of storage at the temperature of 2-8 ℃, and the storage time is long.
Description
Technical Field
The invention belongs to the technical field of medical examination and determination, and particularly relates to preparation and application of a glycosylated hemoglobin quality control product.
Background
Glycated hemoglobin (HbA1c) is a product of hemoglobin in red blood cells combined with saccharides (mainly glucose) in serum by a non-enzymatic reaction. It is generally believed that glycated hemoglobin concentrations effectively reflect the average blood glucose levels over the past 8-12 weeks. The glycosylated hemoglobin consists of HbA1a, HbA1b and HbA1c, wherein HbA1c accounts for about 70%, and the glycosylated hemoglobin has a stable structure and is clinically used as a monitoring index for controlling diabetes.
Currently, there are various methods for clinically measuring the content of glycated hemoglobin (HbA1c), such as ion exchange chromatography (HPLC), affinity chromatography, electrophoresis, immunological methods, enzymatic methods, and the like. Before clinical measurement, each measurement method needs calibration by using a calibrator, and quality control is needed for a measurement system by using a quality control material. The existing whole blood calibrator and quality control product have the problems of short shelf life and poor stability, and are not beneficial to clinical use of the whole blood calibrator and quality control product. Generally speaking, the concentration of normal human glycosylated hemoglobin is less than 6.5%, and the different concentration values are obtained randomly and uncontrollably, which is not favorable for production.
The prior art discloses a method for preparing glycosylated hemoglobin quality control products with different concentrations, which comprises the following steps: the red blood cells are extracted from normal human whole blood, and the high-concentration glycosylated hemoglobin is further prepared by using the ion affinity chromatography, but the ion affinity chromatography is expensive in related instruments, slow in sample processing speed, not beneficial to actual amplification production, and incapable of meeting the actual application. Although the prior art provides a preparation method for preparing a glycosylated hemoglobin quality control product by quickly processing hemoglobin through glycosylation, the components of a reaction solution contain a reducing agent when the hemoglobin is glycosylated, and a step of removing the reducing agent is not carried out in the subsequent purification process, namely a great amount of reducing agent remains in a final product (the glycosylated hemoglobin quality control product); the product has no influence when being used as a reagent kit for determining the glycosylated red blood by a latex enhanced immunoturbidimetry method; however, in the case of quality control of a kit for assaying glycated red blood by a peroxidase method, a large amount of a reducing agent remains, and thus, the measurement cannot be performed. Both peroxidase method and latex enhanced immunoturbidimetry are commonly used methods for using a glycosylated hemoglobin determination kit, so that a new method for preparing glycosylated hemoglobin with different concentrations, which is simpler in preparation method, quicker in preparation and suitable for a glycosylated hemoglobin quality control product of peroxidase method and latex enhanced immunoturbidimetry, is very necessary.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing preparation of the glycosylated hemoglobin quality control product and provides preparation and application of the glycosylated hemoglobin quality control product.
The invention aims to provide a preparation method of a glycosylated hemoglobin quality control product.
The invention also aims to provide the glycosylated hemoglobin quality control product prepared by the preparation method.
The invention also aims to provide the application of the glycosylated hemoglobin quality control product in the quality control of glycosylated hemoglobin.
The above object of the present invention is achieved by the following technical means:
firstly, preparing glycosylated hemoglobin concentrated solutions with two concentrations, diluting the glycosylated hemoglobin concentrated solutions with the two concentrations, and mixing the diluted glycosylated hemoglobin concentrated solutions according to a specific proportion to prepare a high-value glycosylated hemoglobin quality control solution and a low-value glycosylated hemoglobin quality control solution; and finally, preparing the high-value glycosylated hemoglobin quality control freeze-dried powder and the low-value glycosylated hemoglobin quality control freeze-dried powder by adding mannitol and polyethylene glycol 6000 in a specific ratio. The freeze-dried powder prepared by the method has good stability, the glycosylated hemoglobin value is within +/-10% of the mean value within 24 months of storage at the temperature of 2-8 ℃, and the storage time is long.
A preparation method of a glycosylated hemoglobin quality control product comprises the following steps:
s1, obtaining whole blood erythrocytes of a healthy person, dissolving the erythrocytes to obtain a hemoglobin solution, and filtering to obtain a glycosylated hemoglobin solution 1; ultrafiltering, and concentrating to obtain concentrated solution of glycosylated hemoglobin solution 1;
s2, mixing the glycosylated hemoglobin solution 1 prepared in the step S1 with glucose, wherein the final concentration of the glucose in the mixed solution is 16-36 mmol/L, and incubating the mixed solution at 36-38 ℃ for 30-60 days; then removing unreacted glucose, and performing ultrafiltration and full concentration to obtain a concentrated solution of a glycosylated hemoglobin solution 2;
s3, diluting the concentrated solution 1 of the glycated hemoglobin solution prepared in the step S1 and the concentrated solution 2 of the glycated hemoglobin solution prepared in the step S2 respectively until the concentration of hemoglobin is 110-130 g/L, so as to obtain a diluted solution of the concentrated solution 1 of the glycated hemoglobin solution and a diluted solution of the concentrated solution 2 of the glycated hemoglobin solution; mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 in a volume ratio of 1: 1-1: 5 to obtain a high-value glycosylated hemoglobin solution; mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 in a volume ratio of 10: 1-15: 1 to obtain a low-value glycosylated hemoglobin solution;
s4, mixing the high-value glycosylated hemoglobin solution prepared in the step S3 with mannitol and polyethylene glycol 6000, and freeze-drying to obtain a high-value glycosylated hemoglobin quality control product; mixing the low-value glycosylated hemoglobin solution prepared in the step S3 with mannitol and polyethylene glycol 6000, and freeze-drying to obtain a low-value glycosylated hemoglobin quality control product; the final concentration of mannitol in the mixed solution is 70-100 g/L, and the final concentration of polyethylene glycol 6000 is 5-20 g/L.
The healthy person refers to a person who does not have viruses such as HIV, hepatitis B, hepatitis C and the like in a blood sample.
Preferably, the filtering in step S1 is filtering with a filter of 0.2-0.3 μ M.
Further preferably, the filtering in step S1 is filtering with a 0.22 μ M filter.
Preferably, the step S1 of ultrafiltration and full concentration is concentration by using an ultrafiltration centrifugal tube with 25-35 KD.
Further preferably, the ultrafiltration enrichment in step S1 is enrichment using a 30KD ultrafiltration centrifuge tube.
Preferably, in step S1, the volume ratio of the glycated hemoglobin solution 1 concentrate to the hemoglobin solution is 1:3 to 5.
Further preferably, in step S1, the volume ratio of the glycated hemoglobin solution 1 concentrate to the hemoglobin solution is 1: 4.
Preferably, in step S2, the volume ratio of the concentrated glycated hemoglobin solution 2 to the glycated hemoglobin solution 1 is 1:3 to 5.
Further preferably, in step S2, the volume ratio of the glycated hemoglobin solution 2 concentrate to the glycated hemoglobin solution 1 is 1: 4.
Preferably, in step S2, the final concentration of glucose in the mixture is 36mmol/L, and the mixture is incubated at 37 ℃ for 30 days.
Preferably, in step S2, the final concentration of glucose in the mixture is 18mmol/L, and the mixture is incubated at 37 ℃ for 60 days.
Preferably, in step S2, unreacted glucose is removed using glucose oxidase, and hydrogen peroxide is removed.
More preferably, the use concentration of the glucose oxidase is 8-12 KU/L.
Still more preferably, the glucose oxidase is used at a concentration of 10 KU/L.
Preferably, the ultrafiltration full concentration in the step S2 is performed by using an ultrafiltration centrifugal tube with 25-35 KD.
Further preferably, the ultrafiltration enrichment in step S2 is enrichment using a 30KD ultrafiltration centrifuge tube.
Preferably, in step S3, the concentrated solution of glycated hemoglobin solution 1 prepared in step S1 and the concentrated solution of glycated hemoglobin solution 2 prepared in step S2 are diluted to a hemoglobin concentration of 120g/L, respectively.
Preferably, in step S3, the diluted solution of the glycated hemoglobin solution 1 concentrated solution and the diluted solution of the glycated hemoglobin solution 2 concentrated solution are mixed at a volume ratio of 1:1 to obtain a high-value glycated hemoglobin solution; and mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 according to the volume ratio of 10:1 to obtain the low-value glycosylated hemoglobin solution.
Preferably, in step S4, the mixed solution has a final concentration of mannitol of 70-80 g/L and a final concentration of polyethylene glycol 6000 of 5-10 g/L.
Further preferably, in step S4, the mixed solution has a final concentration of mannitol of 70g/L and polyethylene glycol 6000 of 5 g/L.
The glycosylated hemoglobin quality control prepared by the preparation method is also within the protection scope of the invention.
The application of the glycosylated hemoglobin quality control product in the quality control of glycosylated hemoglobin is also within the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the glycosylated hemoglobin quality control product is simple, and the glycosylated hemoglobin quality control product can be quickly prepared without complex operation and expensive instruments; the prepared glycosylated hemoglobin quality control product has good stability, the glycosylated hemoglobin value is within the range of +/-10% of the mean value within 24 months of storage at the temperature of 2-8 ℃, and the storage time is long; and the method can be simultaneously used for the glycosylated red blood determination kit of the peroxidase method and the latex enhanced immunoturbidimetry, and has wider application.
Drawings
FIG. 1 is a stability monitoring chart of a glycated hemoglobin quality control substance lyophilized powder of the quality control substance 1 of the present invention, wherein A is a low value and B is a high value.
FIG. 2 is a graph showing the stability of a lyophilized powder of glycated hemoglobin quality control substance of quality control substance 2 according to the present invention, wherein A is a low value and B is a high value.
FIG. 3 is a graph showing the stability of a lyophilized powder of a glycated hemoglobin quality control substance of the quality control substance 3 of the present invention, wherein A is a low value and B is a high value.
FIG. 4 is a stability monitoring chart of a glycated hemoglobin quality control freeze-dried powder of the comparative quality control product 1 of the present invention, wherein A is a low value and B is a high value.
FIG. 5 is a stability monitoring chart comparing the quality control 2 glycated hemoglobin quality control lyophilized powder of the present invention, wherein A is a low value and B is a high value.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 preparation of a glycated hemoglobin solution 1 concentrate
1. Erythrocyte treatment
200mL of EDTA-anticoagulated whole blood samples of healthy persons were collected, and HIV (HIV1, HIV2) antibodies, hepatitis B surface antigen (HBsAg) and Hepatitis C Virus (HCV) antibodies were detected in each sample, and all of the samples were collected and used as negative results, and if one of the samples was positive, the samples were treated according to the treatment rules of the relevant medical waste. Centrifuging the collected whole blood sample at about 1900g, removing the upper plasma layer, adding physiological saline, repeatedly blowing and washing the red blood cells by using a pipette, centrifuging after washing, removing the upper physiological saline layer, and repeatedly washing for 3 times to prepare the washed red blood cells.
2. Preparation of a glycated hemoglobin solution 1 concentrate
(1) Preparation of treatment liquid
0.2g of 3-sulfopropyltetradecyldimethyl betaine and 0.028g of sodium nitrite were thoroughly dissolved in 100mL of pure water.
(2) Preparation of a glycated hemoglobin solution 1 concentrate
Fully mixing the washed red blood cells with the treatment solution according to the volume ratio of 1:1, dissolving the red blood cells to obtain a hemoglobin solution, and filtering the obtained hemoglobin solution (100mL) through a 0.22 mu M filter to obtain a glycosylated hemoglobin solution 1; and (3) concentrating the glycosylated hemoglobin solution 1 by using an ultrafiltration centrifugal tube with the retention rate of 30KD for 14000g of centrifugal force, centrifuging for 10min to obtain a concentrated solution (25mL) of the glycosylated hemoglobin solution 1, and storing at 2-8 ℃ for later use.
EXAMPLE 2 preparation of a glycated hemoglobin solution 2 concentrate
Glucose was added to and dissolved in the glycated hemoglobin solution 1(100mL) prepared in example 1 to a final concentration of 18mmol/L, and the mixed solution was incubated at 37 ℃ for 60 days. Adding glucose oxidase to remove unreacted glucose after incubation is finished, terminating the glycosylation reaction, wherein the final concentration of the glucose oxidase is 10KU/L, and placing the solution with glucose removed at 37 ℃ for 7 days to eliminate hydrogen peroxide; finally, an ultrafiltration centrifugal tube with the retention rate of 30KD is used for concentration treatment, the centrifugal force of 14000g is used, and the concentration solution (25mL) of the glycosylated hemoglobin solution 2 is obtained after centrifugation for 10 min; and storing at 2-8 ℃ for later use.
EXAMPLE 3 preparation of concentrated solution of glycated hemoglobin solution 2
Glucose was added to and dissolved in the glycated hemoglobin solution 1(100mL) prepared in example 1 at a final concentration of 36mmol/L, and the mixed solution was incubated at 37 ℃ for 30 days. Adding glucose oxidase to remove unreacted glucose after incubation is finished, terminating the glycosylation reaction, wherein the final concentration of the glucose oxidase is 10KU/L, and placing the solution with glucose removed at 37 ℃ for 7 days to eliminate hydrogen peroxide; finally, an ultrafiltration centrifugal tube with the retention rate of 30KD is used for concentration treatment, centrifugal force of 14000g is carried out, and the centrifugal force is carried out for 10min to obtain a concentrated solution (25mL) of the glycosylated hemoglobin solution 2; and storing at 2-8 ℃ for later use.
EXAMPLE 4 preparation of glycated hemoglobin quality control solution
A diluted solution (mother solution B) of the glycated hemoglobin solution 1 concentrated solution prepared in example 1 and a diluted solution (mother solution A) of the glycated hemoglobin solution 2 concentrated solution prepared in example 2 were each diluted with pure water to obtain a glycated hemoglobin solution 1 concentrated solution having a hemoglobin concentration of 120 g/L. The mother solutions a and B were set using a glycated hemoglobin kit (838 RHQ, a hydrographic medical technology (china) ltd.), and the glycated hemoglobin values of the mother solution a and the mother solution B were 15.9% and 4.5%, respectively, for setting using a fully automatic blood analyzer.
Mixing the mother liquor A with the mother liquor B, mixing the mother liquor B with the mother liquor A according to the volume ratio of 10:1 to obtain a low-value quality control liquid 1, and measuring to obtain a glycosylated hemoglobin value of the low-value quality control liquid of 5.5%;
and (3) mixing the mother liquor B and the mother liquor A in a volume ratio of 1:1 to obtain a high-value quality control liquid, wherein the glycosylated hemoglobin value of the high-value quality control liquid 1 is 11.7% through measurement.
Mixing the mother liquor A with the mother liquor B, mixing the mother liquor B with the mother liquor A according to the volume ratio of 15:1 to obtain a low-value quality control liquid 2, and measuring to obtain a glycosylated hemoglobin value of the low-value quality control liquid of 5.1%;
and (3) mixing the mother liquor B and the mother liquor A in a volume ratio of 1:5 to obtain a high-value quality control liquid, wherein the glycosylated hemoglobin value of the high-value quality control liquid 2 is 16.8% through measurement.
Example 5 preparation of glycated hemoglobin quality control lyophilized powder
1. Method of producing a composite material
(1) Preparation of lyophilized powder
Adding excipient mannitol and stabilizer polyethylene glycol 6000 into the high-value quality control solution 1 and the low-value quality control solution 1 prepared in example 4 respectively, fully dissolving, subpackaging the mixed solution with 0.5mL per bottle at-55 ℃ to-50 ℃, keeping the vacuum degree less than 3 Pa, freeze-drying for 24h, and then sealing for later use.
The freeze-dried powder prepared by using the mixed solution with the final concentration of 70g/L of mannitol and the final concentration of 5g/L of polyethylene glycol 6000 as a quality control product 1;
freeze-dried powder prepared by using the final concentration of mannitol in the mixed solution as 80g/L and the final concentration of polyethylene glycol 6000 as 10g/L is taken as a quality control substance 2;
the freeze-dried powder prepared by using the mixed solution with the final concentration of mannitol of 100g/L and the final concentration of polyethylene glycol 6000 of 20g/L is taken as a quality control product 3;
the freeze-dried powder prepared by using the mixed solution with the final concentration of 40g/L mannitol and the final concentration of 2g/L polyethylene glycol 6000 as a contrast quality control product 1;
the freeze-dried powder prepared by using the mixed solution with the final concentration of 70g/L of mannitol and the final concentration of 30g/L of polyethylene glycol 6000 as a contrast quality control material 2.
(2) Constant value of quality control
And (3) taking 10 bottles of freeze-dried powder, respectively adding 0.5mL of pure water for dissolving, testing the quality control of each bottle by using a glycosylated hemoglobin kit, and setting the value, wherein the target value is the average value of 10 bottles, and the quality control range is +/-10% of the target value.
(3) Determination of stability of lyophilized powder
And (3) storing the high-low value glycosylated hemoglobin quality control product freeze-dried powder at 2-8 ℃, randomly taking a bottle every month, testing by using a glycosylated hemoglobin determination kit, and monitoring the stability of the glycosylated hemoglobin quality control product freeze-dried powder.
2. Results
The definite value and the range of each glycosylated hemoglobin quality control freeze-dried powder are shown in table 1; the stability of each glycated hemoglobin quality control lyophilized powder is shown in table 2; the stability monitoring chart of the quality control product 1 glycated hemoglobin quality control product lyophilized powder is shown in figure 1, wherein A is a low value, and B is a high value; the stability monitoring chart of the lyophilized powder of the quality control product 2 glycated hemoglobin is shown in FIG. 2, wherein A is a low value and B is a high value; quality control product 3 stability monitoring chart of lyophilized powder of glycated hemoglobin quality control product is shown in FIG. 3, wherein A is low value and B is high value; the stability monitoring chart of the lyophilized powder of the control product 1 glycated hemoglobin is shown in FIG. 4, wherein A is a low value and B is a high value; a stability monitoring chart of the control 2 glycated hemoglobin control lyophilized powder is shown in FIG. 5, wherein A is a low value and B is a high value.
TABLE 1 fixed value and range of lyophilized powder of each glycated hemoglobin quality control product
Table 1 shows that, in the freeze-dried glycated hemoglobin finished product, the low-value fixed-value mean value of the quality control product 1 is 5.6, and the high-value fixed-value mean value is 11.5; the average value of the low fixed value and the average value of the high fixed value of the quality control product 2 is 6.3 and 12.2; the average value of the low fixed value of the quality control product 3 is 6.2, and the average value of the high fixed value is 12; the average value of the low fixed value and the average value of the high fixed value of the comparison quality control product 1 is 6.6 and 12.7; the comparison quality control product 2 had a low-value fixed-value mean value of 6.3 and a high-value fixed-value mean value of 12.2.
TABLE 2 stability of the glycated hemoglobin quality control lyophilized powder
As can be seen from the data in table 2 and fig. 1 to 5, the constant values of the quality control products 1, 2 and 3 after 24 months of storage are maintained at ± 10% of the target values, which indicates that the quality control products 1, 2 and 3 are stable after 24 months of storage; the fixed values of the control samples 1 and 2 after being stored for 14 months exceed the target value by +/-10%, which indicates that the control samples 1 and 2 cannot be stably stored within 24 months, and the stability of the control samples 1, 2 and 3 is superior to that of the control samples 1 and 2. Compared with the quality control products 1, 2 and 3, the concentration of mannitol and polyethylene glycol 6000 is only changed, which shows that the concentration of mannitol and polyethylene glycol 6000 has a remarkable influence on the stability of the lyophilized powder of the glycosylated hemoglobin quality control product.
In conclusion, the stable and good freeze-dried powder of the glycosylated hemoglobin quality control product can be prepared only by adding mannitol and polyethylene glycol 6000 in the concentration range of mannitol and polyethylene glycol 6000. The final concentration range of mannitol in the mixed solution is 70-100 g/L; the final concentration range of the polyethylene glycol 6000 is 5-20 g/L.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of a glycosylated hemoglobin quality control product is characterized by comprising the following steps:
s1, obtaining whole blood erythrocytes of a healthy person, dissolving the erythrocytes to obtain a hemoglobin solution, and filtering to obtain a glycosylated hemoglobin solution 1; ultrafiltering, and concentrating to obtain concentrated solution of glycosylated hemoglobin solution 1;
s2, mixing the glycosylated hemoglobin solution 1 prepared in the step S1 with glucose, wherein the final concentration of the glucose in the mixed solution is 16-36 mmol/L, and incubating the mixed solution at 36-38 ℃ for 30-60 days; then removing unreacted glucose, and performing ultrafiltration and full concentration to obtain a concentrated solution of a glycosylated hemoglobin solution 2;
s3, diluting the concentrated solution 1 of the glycosylated hemoglobin solution prepared in the step S1 and the concentrated solution 2 of the glycosylated hemoglobin solution prepared in the step S2 respectively until the concentration of hemoglobin is 110-130 g/L, and obtaining a diluent of the concentrated solution 1 of the glycosylated hemoglobin solution and a diluent of the concentrated solution 2 of the glycosylated hemoglobin solution; mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 in a volume ratio of 1: 1-1: 5 to obtain a high-value glycosylated hemoglobin solution; mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 in a volume ratio of 10: 1-15: 1 to obtain a low-value glycosylated hemoglobin solution;
s4, mixing the high-value glycosylated hemoglobin solution prepared in the step S3 with mannitol and polyethylene glycol 6000, and freeze-drying to obtain a high-value glycosylated hemoglobin quality control product; mixing the low-value glycosylated hemoglobin solution prepared in the step S3 with mannitol and polyethylene glycol 6000, and freeze-drying to obtain a low-value glycosylated hemoglobin quality control product; the final concentration of mannitol in the mixed solution is 70-100 g/L, and the final concentration of polyethylene glycol 6000 is 5-20 g/L.
2. The method according to claim 1, wherein the volume ratio of the glycated hemoglobin solution 1 concentrate to the hemoglobin solution in step S1 is 1:3 to 5; the volume ratio of the concentrated solution of the glycated hemoglobin solution 2 to the glycated hemoglobin solution 1 in the step S2 is 1: 3-5.
3. The method according to claim 1, wherein in step S2, unreacted glucose is removed by using glucose oxidase, and hydrogen peroxide is removed.
4. The preparation method according to claim 3, wherein the glucose oxidase is used at a concentration of 8 to 12 KU/L.
5. The method according to claim 1, wherein the final concentration of glucose in the mixture is 18mmol/L in step S2, and the mixture is incubated at 37 ℃ for 60 days.
6. The method of claim 1, wherein in step S3, the concentrated solution 1 of glycated hemoglobin prepared in step S1 and the concentrated solution 2 of glycated hemoglobin prepared in step S2 are diluted to a hemoglobin concentration of 120g/L, respectively.
7. The method according to claim 1, wherein in step S3, the diluted solution of the glycated hemoglobin solution 1 concentrated solution and the diluted solution of the glycated hemoglobin solution 2 concentrated solution are mixed at a volume ratio of 1:1 to obtain a high-value glycated hemoglobin solution; and mixing the diluent of the concentrated solution of the glycosylated hemoglobin solution 1 and the diluent of the concentrated solution of the glycosylated hemoglobin solution 2 according to the volume ratio of 10:1 to obtain the low-value glycosylated hemoglobin solution.
8. The method according to claim 1, wherein in step S4, the mixed solution has a final concentration of 70-80 g/L mannitol and a final concentration of 5-10 g/L polyethylene glycol 6000.
9. A glycated hemoglobin quality control product produced by the production method according to any one of claims 1 to 8.
10. Use of the glycated hemoglobin quality control product according to claim 9 for quality control of glycated hemoglobin.
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