CN116625777B - Low-level freeze-dried glycosylated hemoglobin control and preparation method thereof - Google Patents
Low-level freeze-dried glycosylated hemoglobin control and preparation method thereof Download PDFInfo
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Classifications
-
- 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
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of blood detection, in particular to a low-level freeze-dried glycosylated hemoglobin control and a preparation method thereof. The invention provides a reagent combination comprising a hemolysis agent and a freeze-drying protective agent, a low-level glycosylated hemoglobin control prepared by adopting the reagent combination, and a preparation method and application of the low-level glycosylated hemoglobin control. The low-level freeze-dried glycosylated hemoglobin control provided by the invention has high stability, normal human erythrocytes and porcine hemoglobin or fresh porcine blood are used as raw materials, the raw materials are convenient and easy to obtain, the test requirements outside a low value range at a medical decision level can be met, and meanwhile, the preparation method is simple, and the batch industrial production can be carried out.
Description
Technical Field
The invention relates to the technical field of blood detection, in particular to a low-level freeze-dried glycosylated hemoglobin control and a preparation method thereof.
Background
Normal hemoglobin (Hb) in human blood is composed of three hemoglobin subfractions: hemoglobin a (HbA), hemoglobin F (HbF), and hemoglobin A2 (HbA 2). Adult hemoglobin contains primarily hemoglobin a (HbA), which in turn consists of two subcomponents: glycosylated hemoglobin (HbA 1) (5% -7%) and non-glycosylated hemoglobin (HbA 0) (90%). Glycosylated hemoglobin (HbA 1) also has three subcomponents, respectively: hbA1a, hbA1b and HbA1c. Wherein HbA1c is about 75% to 80% of HbA1 and is structurally stable and can be used to react to an average blood glucose level of the first two to three months. And thus is used as a monitoring index for diabetes control. And is little disturbed by factors such as blood drawing time, whether the patient is empty or not, whether insulin is used, and the like.
Currently, the glycosylated hemoglobin detection methods used in clinical laboratories mainly include high performance liquid chromatography, electrophoresis, affinity chromatography, and immunization. Among them, high Performance Liquid Chromatography (HPLC) is recognized as a gold standard.
The quality control product is a substance widely applied in clinical examination work and has the function of evaluating whether the performances of in-vitro diagnostic equipment and reagents meet the original set standards. The control of glycosylated hemoglobin is required to cover the normal and abnormal test ranges in clinical tests. The test results for normal people are generally 4% -6%, and the test results for hyperglycemia and diabetes patients are generally 6% -16%, so most manufacturers of glycosylated hemoglobin control generally provide two levels of quality control products, namely a low level quality control product (4% -6%) and a high level quality control product (8% -10%), but the low level quality control product is a quality control product falling in a normal value reference range, is not a real low level (2% -4%) quality control product, is less in commercial quality control products below a normal range at present, has no measurement standard for detection limit, and needs to prepare the quality control product below the normal range in order to promote quality standard control of the whole detection range.
The glycosylated hemoglobin control is prepared from human whole blood or pig blood hemoglobin as raw material by centrifuging, washing, breaking cells, removing impurities, and lyophilizing. First, a blood sample is centrifuged to obtain red blood cells. Washing the red blood cells by using a washing liquid to form a red blood cell suspension. Adding reaction liquid into the washed red blood cells to carry out glycosylation or affinity chromatography to obtain high glycosylated hemoglobin solution, and preparing quality control products with different levels through operations such as purification, mixing, centrifugation and the like. And finally, adding a stabilizer and a freeze-drying protective agent into the processed red blood cell suspension to prepare the liquid or freeze-dried glycosylated hemoglobin control.
In summary, although the methods for preparing glycosylated hemoglobin control are different, the measurement value of HbA1c of low-level glycosylated hemoglobin control is more than 4% -6%, and a supporting reagent or a supporting instrument is needed to be used for quality control, so that the quality control test requirement beyond the lower limit of 4% of the reference value cannot be met.
Disclosure of Invention
In view of the above, the invention aims to provide a low-level freeze-dried glycosylated hemoglobin control and a preparation method thereof, and the low-level freeze-dried glycosylated hemoglobin control provided by the invention has the advantages of high stability, simple preparation method, convenient and easily obtained raw materials, and can meet the quality control test requirements beyond the lower limit range of normal reference values.
The invention provides a reagent combination for preparing a low-level glycosylated hemoglobin control, which comprises a hemolysis agent and a freeze-drying protective agent;
the hemolysis agent comprises phosphate buffer solution, sodium azide and surfactant, wherein the surfactant comprises at least one of triton, sodium deoxycholate and SDS;
the lyoprotectant comprises at least one of NaCl, BSA, tween-80, DTT, glucose, trehalose, lactose, vc, tris-HCl, hepes and glycine.
Compared with other reagent combinations, the reagent combination provided by the invention provides a more effective hemolysis effect and a more stable freeze-drying system for the preparation of low-level glycosylated hemoglobin control, and can ensure the stability, sensitivity and specificity of the lyophilized hemoglobin control to a greater extent, thereby obtaining more accurate technical effects.
In some embodiments, the hemolytic agent comprises phosphate buffer, sodium azide, and triton, the phosphate buffer comprising sodium dihydrogen phosphate and disodium hydrogen phosphate;
the lyoprotectant comprises NaCl, BSA, tween-80, trehalose, hepes and glycine.
Compared with other reagents, the components are tightly matched with each other, so that the compatibility is strong, and the preparation of the hemoglobin control can be more effectively matched, thereby obtaining more accurate technical effects.
In some embodiments, the hemolytic agent comprises 0.5 to 3mmol/L phosphate buffer, 0.5 to 1.5mmol/L sodium azide, and 0.2 to 1.0mmol/L triton;
the freeze-drying protective agent comprises 0.1-0.3 mol/L NaCl, 2-5wt% BSA, 0.1-0.2wt% Tween-80, 2-5wt% trehalose, 20-100 mmol/L Hepes and 2-5wt% glycine.
In some embodiments, the hemolytic agent comprises 0.4mmol/L triamcinolone, 1.0mmol/L sodium dihydrogen phosphate, 2.5mmol/L disodium hydrogen phosphate, and 0.5mmol/L sodium azide;
the lyoprotectant comprises 0.15mol/LNaCl, 2wt% BSA, 0.1 wt% Tween-80, 5wt% trehalose, 60mmol/LHepes and 4wt% glycine.
Experiments show that the reagent combination has the best preparation effect when being matched with the hemoglobin control at the concentration.
The invention provides a preparation method of a low-level glycosylated hemoglobin control, which comprises the following steps:
step 1: taking a normal blood sample, centrifuging, removing blood plasma and blood platelets to obtain a solution, and washing with PBS buffer solution, centrifuging, and removing supernatant to obtain red blood cell suspension;
step 2: mixing the red blood cell suspension with the hemolytic agent in the reagent combination, and centrifuging and removing sediment to obtain a hemoglobin solution;
step 3: and mixing the hemoglobin solution with the freeze-drying protective agent in the reagent combination to obtain the low-level glycosylated hemoglobin control.
In some embodiments, the blood sample comprises a human blood sample and a pig blood sample;
the red blood cell suspension in the step 1 comprises a human red blood cell suspension and a pig red blood cell suspension;
the hemoglobin solution in the step 2 comprises a human hemoglobin solution and a pig blood hemoglobin solution;
in the step 3, the hemoglobin solution includes a volume ratio of 1: (0.1-10) human hemoglobin solution and pig blood hemoglobin solution.
Experiments show that the peak time of bovine blood samples and bovine hemoglobin and human blood samples and the difference of target proteins are large, the detection results of clinical samples cannot be truly reflected, and the bovine blood samples and the bovine hemoglobin cannot be used as control substances for product stability in quality control analysis and use. From the point of view of the number of peaks and the total peak area, the number of peaks of the human hemoglobin, the pig blood hemoglobin and the bovine hemoglobin are less than those of corresponding blood samples, the peak area is lower, and the peak area at the target protein is small, so that the preparation of the low-level glycosylated hemoglobin control can be more effectively realized by selecting the human blood sample and the pig blood sample for mixed test, and the more accurate technical effect is obtained.
Further, the concentration of hemoglobin in the human hemoglobin solution and the porcine hemoglobin solution is 0.1-0.2g/mL, the result of detecting NGSP glycosylated hemoglobin HbA1c by the human hemoglobin solution is 4-6%, and the result of detecting NGSP by the porcine hemoglobin solution is 1-3%.
Further, in the step 1, the centrifugation conditions are as follows: centrifuging at 2000-4000rpm for 10-20min, wherein the volume ratio of the solution to the PBS buffer is 1 (1-5), and the washing times are 2-5 times;
in the step 2, the volume ratio of the red blood cell suspension to the hemolytic agent is 1: (0.5-2);
in the step 3, the volume ratio of the hemoglobin solution to the lyoprotectant is 1: (1-5).
In some embodiments, the human blood sample and the pig blood sample require an anticoagulant to be used during collection to prevent the collected blood from quickly solidifying into a solid that cannot be used in subsequent centrifugation and other procedures. The anticoagulant includes at least one of EDTA, sodium citrate, and heparin.
In other embodiments, the method further comprises the step of lyophilizing the low-level glycosylated hemoglobin control, wherein the low-level glycosylated hemoglobin control is obtained in a lyophilized state by vacuum lyophilization.
The invention provides a low-level glycosylated hemoglobin control product prepared by the preparation method.
The invention also provides application of the low-level glycosylated hemoglobin control prepared by the preparation method in preparation of glycosylated hemoglobin detection reagents or kits.
The invention provides a reagent combination comprising a hemolysis agent and a freeze-drying protective agent, a low-level glycosylated hemoglobin control prepared by adopting the reagent combination, and a preparation method and application of the low-level glycosylated hemoglobin control. The low-level freeze-dried glycosylated hemoglobin control provided by the invention has high stability, normal human erythrocytes and porcine hemoglobin or fresh porcine blood are used as raw materials, the raw materials are convenient and easy to obtain, the test requirements outside a low value range at a medical decision level can be met, and meanwhile, the preparation method is simple, and the batch industrial production can be carried out. The low-level freeze-dried glycosylated hemoglobin control provided by the invention can be stably stored for 2 years at 2-8 ℃, can be stably stored for 15 days at 2-8 ℃ after being opened for redissolution, and can be stably stored for 60 days at-20 ℃.
Drawings
FIG. 1 shows a high performance liquid chromatogram of a low level lyophilized glycosylated hemoglobin control.
Detailed Description
The invention provides a low-level freeze-dried glycosylated hemoglobin control and a preparation method thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
Glycosylated hemoglobin can respond to the average blood glucose level in the human body for two to three months before measurement, and glycosylated hemoglobin monitoring is used as a "gold index" for judging and adjusting the treatment scheme of diabetes. The glycosylated hemoglobin control is prepared from hemoglobin, and contains normal hemoglobin and freeze-drying protective agent. Three-gradient elution is carried out by using an ion exchange High Performance Liquid Chromatography (HPLC), hbA1a (glycosylated hemoglobin a), hbA1b (glycosylated hemoglobin b), hbF (hemoglobin F), LA1c (unstable glycosylated hemoglobin), A1c (stable glycosylated hemoglobin) and HbA0 (hemoglobin A0) are gradually eluted, and the absorbance of the eluate is continuously measured on line by a single-wavelength (415 nm) visible light color comparison method, so that a corresponding hemoglobin chromatographic chart is obtained.
The quality control product is mainly used for detecting glycosylated hemoglobin analyzer and matched reagent, and is used for controlling indoor quality when detecting glycosylated hemoglobin in human anticoagulated whole blood sample.
The main purpose of this embodiment is to screen the optimal raw materials to meet the design performance index, and the manufacturer can supply the materials stably
The main raw materials in the glycosylated hemoglobin control kit comprise hemoglobin and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises trehalose, bovine serum albumin, salt substances and the like. The research data require verification of screening human hemoglobin and lyoprotectant combinations. The main equipment is a glycosylated hemoglobin analyzer SGH-200.
1. Raw material screening
Screening of hemoglobin the effectiveness of screening hemoglobin of different sources and ratios is shown in table 1 below.
TABLE 1
| Raw material name | Manufacturing factories |
| Human blood sample | Volunteer donation |
| Human hemoglobin | Sigma |
| Human hemoglobin | Living beings in He Wu |
| Pig blood sample | Zhengzhou Jiulong (nine-Dragon) |
| Porcine hemoglobin | Source leaf organisms |
| Bovine blood sample | Zhengzhou Jiulong (nine-Dragon) |
| Bovine hemoglobin | Source leaf organisms |
Screening of the major components of the hemolytic agents the effect of different hemolytic agents on the results of the product was tested as shown in table 2 below.
TABLE 2
Screening of lyoprotectants the effect of the addition of different lyoprotectants on the product results was tested as shown in table 3 below.
TABLE 3 Table 3
2. Screening results
2.1 screening of hemoglobin of different origins
a. The human blood sample, human hemoglobin, pig blood sample, pig blood hemoglobin and bovine-derived hemoglobin are detected. The results of screening for feasibility as a major raw material in comparison with human blood samples are shown in Table 4.
TABLE 4 Table 4
The test results show that the peak time and the target protein of the bovine blood sample and the bovine hemoglobin and the human blood sample have larger difference, can not truly reflect the detection result of the clinical sample, and can not be used as a control substance for product stability in quality control analysis and use. From the point of view of the number of peaks and the total peak area, the number of peaks of the human hemoglobin, the pig hemoglobin and the bovine hemoglobin are less than those of corresponding blood samples, the peak area is lower, the peak area at the target protein is small, and from the point of view of comprehensive detection results and cost, the human blood samples and the pig blood samples can be selected for mixed test.
b. The results of the test of human blood samples (human hemoglobin) mixed with pig blood samples (pig blood hemoglobin) in different ratios are shown in table 5.
TABLE 5
The results show that after the glycosylated hemoglobin analysis is carried out by preparing the hemoglobin solutions with different mixing ratios, the number of the obtained peaks is only 6 peaks which can be the same as that of a clinical sample after being mixed with a human blood sample, and the mixing ratio can reach the low-value quality control requirement of 2% -4% from 10:1-1:10.
2.2 Effect of different hemolytic Agents on the product outcome
The sample hemolysis test was performed using three different hemolytic agents, respectively, which act to rupture red blood cells and release hemoglobin in the bleeding cells, so that the effect of the hemolytic agents reflects the total peak area in the test results under the same conditions. The same human blood sample was taken in 1ml and 1ml of each of the different hemolytic agents was added thereto, and then the resultant was subjected to measurement by a glycosylated hemoglobin analyzer, and the results are shown in Table 6.
TABLE 6
| Name of the name | Saccharification value | Number of peaks | Total peak area |
| Hemolytic agent 1 | 4.9％ | 6 | 1096.4 |
| Hemolytic agent 2 | 5.3％ | 6 | 738.1 |
| Hemolytic agent 3 | 4.7％ | 5 | 1150.5 |
From the total peak area results, it was shown that hemolysis of hemolytic agents 1 and 3 was more sufficient than 2, and analysis of the number of peaks from hemolytic agent 3 had an effect on the number of peaks, probably because it was an ionic surfactant, on the elution process. Therefore, hemolytic agent 1, that is, a hemolytic agent containing triton as a main component is preferable.
4.3 analysis of the effects of lyoprotectants on results
The prepared human blood sample and pig blood sample mixed solution are respectively mixed with a freeze-drying protective agent A, B, C and PT according to the proportion of 1:1, the influence of the human blood sample and pig blood sample mixed solution on the effectiveness and stability of the product before and after freeze-drying is tested, and the results are shown in tables 7 and 8.
TABLE 7 detection results before lyophilization
From the comparison and detection results of the stock solution before lyophilization and for adding the lyoprotectant in table 7, the effect of adding the lyoprotectant on the saccharification value is not great, the relative deviation is not more than +/-8%, the lyoprotectant A is closest to the stock solution from the point of peak time and peak number, and the peak area is reduced by half compared with the stock solution because of the reduction of the concentration of hemoglobin after dilution. After mixing, lyophilization was performed following the same lyophilization procedure: the pre-freezing temperature is-40 ℃ for 4 hours, the temperature is raised and sublimated according to a gradient of 5-10 ℃ after the pre-freezing is finished, the vacuum degree is controlled to be 150bar, the temperature is-30 ℃, the temperature is-20 ℃, the temperature is-10 ℃, the temperature is 0 ℃ and the temperature is 10 ℃ respectively for 2 hours, the temperature is 20 ℃ for 2 hours, and the vacuum degree is reduced to 50bar.
TABLE 8 detection results after lyophilization
The freeze-dried and redissolved products are in a uniform red liquid state in appearance, no precipitation and undissolved state appears, and the re-dissolved products meet the requirements in appearance, and the test results of the machine show that the repeated freeze-dried protective agents A in the table 8 are best in three aspects of saccharification value, peak-out time and total peak area after four freeze-dried protective agents are respectively used for freeze-drying, and meet the product design requirements.
Through the screening experimental study of raw materials, the main raw materials of the glycosylated hemoglobin control are finally determined to be mixed with a pig blood sample, the freeze-drying protective agent is selected as a freeze-drying protective agent A, and the hemolytic agent takes triatomic acid as a main component.
Experiments prove that when the hemolytic agent comprises 0.5-3 mmol/L phosphate buffer solution, 0.5-1.5 mmol/L sodium azide and 0.2-1.0 mmol/L triton, and the freeze-drying protective agent comprises 0.1-0.3 mol/L NaCl, 2-5wt% BSA, 0.1-0.2wt% Tween-80, 2-5wt% trehalose, 20-100 mmol/L Hepes and 2-5wt% glycine, the difference between the hemolytic agent and the low-level glycosylated hemoglobin control prepared by the freeze-drying protective agent is small.
EXAMPLE 2 preparation of Low level lyophilized glycosylated hemoglobin control
The method specifically comprises the following steps:
in the first step, phosphate Buffer (PBS) is prepared, and the pH is adjusted to be in the range of 6-8. And preparing a hemolytic agent, adding a surfactant and a preservative except PBS, wherein the surfactant is trialaton, and the preservative is sodium azide. The freeze-drying protective agent is prepared and comprises NaCl, BSA, tween-80, trehalose, hepes, glycine and the like.
Secondly, collecting normal human citric acid anticoagulated whole blood samples and citric acid anticoagulated pig blood samples, respectively placing the samples into a 2-200ml centrifuge tube according to the sample amount, centrifuging for 10-20min at 2000-4000rpm, and carefully sucking the supernatant plasma and the middle platelet layer by using a pipette to obtain a lower-layer red blood cell suspension.
Thirdly, adding PBS (phosphate buffer solution) according to the volume ratio of 1:1-1:5, uniformly mixing and washing upside down, centrifuging for 10-20min according to 2000-4000rpm, carefully sucking the supernatant by a pipette, adding PBS again for washing, repeating for 2-5 times, and discarding the supernatant to obtain human red blood cell suspension and pig red blood cell suspension;
fourth, according to the volume ratio of 1:0.5-1:2 respectively adding hemolytic agent into human and pig erythrocyte suspensions, shaking and mixing uniformly, centrifuging at 4000-8000rpm for 10-20min, and discarding the precipitate to obtain human hemoglobin solution and pig hemoglobin solution; the concentration of hemoglobin is measured to be 0.1-0.2g/ml, the result of testing human glycosylated hemoglobin is 4% -6%, and the result of pig glycosylated hemoglobin is 1% -3%.
Fifthly, mixing human hemoglobin and pig blood hemoglobin according to a ratio of 1:10-10:1;
sixth step: and uniformly mixing the mixed hemoglobin solution and the freeze-drying protective agent according to the volume ratio of 1:1-1:5, and measuring HbA1c to obtain 2% -4% of HbA1c, thereby obtaining the low-level glycosylated hemoglobin control.
Seventh step: the prepared quality control solution is packaged in penicillin bottles, and is placed in a freeze dryer for vacuum freeze drying, so as to obtain a low-level freeze-dried glycosylated hemoglobin control product, and the low-level freeze-dried glycosylated hemoglobin control product is stored at 2-8 ℃.
The detection result of the high performance liquid chromatogram of the prepared low-level freeze-dried glycosylated hemoglobin control is shown in figure 1.
Example 3 measurement of Properties of quality control Material prepared according to the present invention
The low-level freeze-dried glycosylated hemoglobin control prepared in example 2 is subjected to uniformity detection, bottle opening stability at 4 ℃ and bottle opening stability at minus 20 ℃ in sequence, accelerated stability investigation at 37 ℃, and specific measurement data are shown in tables 9-11 below.
TABLE 9SGH-200 glycosylated hemoglobin analyzer measurement quality control uniformity
Table 10SGH-200 glycosylated hemoglobin analyzer measuring quality control product stability in opening bottle
TABLE 11SGH-200 glycosylated hemoglobin analyzer measurement quality control acceleration stability
Conclusion: the prepared low-level quality control material has stable quality, uniformity variation coefficient within 5%, stable storage at 2-8deg.C for 2 years, stable storage at 4deg.C for 15 days after bottle opening and re-dissolution, and stable storage at-20deg.C for 60 days after re-dissolution; the relative deviation of HbA1c measured after acceleration at 37 ℃ for 14 days is within 6%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A reagent combination for preparing a low-level glycosylated hemoglobin control, comprising a hemolysis agent, a lyoprotectant, and a blood sample;
the hemolytic agent consists of 1.0mmol/L sodium dihydrogen phosphate, 2.5mmol/L disodium hydrogen phosphate, 0.5mmol/L sodium azide and 0.4mmol/L triamcinolone;
the freeze-drying protective agent consists of 0.15mol/L NaCl, 2wt% BSA, 0.1 wt% Tween-80, 5wt% trehalose, 60mmol/L Hepes and 4wt% glycine;
the blood sample consists of a human blood sample and a pig blood sample.
2. The preparation method of the low-level glycosylated hemoglobin control is characterized by comprising the following steps:
step 1: taking the human blood sample and the pig blood sample in the reagent combination of claim 1, centrifuging, removing the blood plasma and the blood platelets to obtain a solution, and washing with PBS buffer solution, centrifuging, removing the supernatant to obtain human red blood cell suspension and pig red blood cell suspension;
step 2: the human red blood cell suspension and the pig red blood cell suspension are mixed with the hemolysis agent in the reagent combination of claim 1, and human hemoglobin solution and pig blood hemoglobin solution are obtained after centrifugation and precipitation removal;
step 3: the low-level glycosylated hemoglobin control is obtained after the human hemoglobin solution, the pig hemoglobin solution, and the lyoprotectant in the reagent combination of claim 1 are mixed.
3. The method according to claim 2, wherein,
in the step 3, the volume ratio of the human hemoglobin solution to the pig blood hemoglobin solution is 1: (0.1 to 10).
4. The method according to claim 3, wherein the concentration of hemoglobin in the human hemoglobin solution and the porcine hemoglobin solution is 0.1-0.2g/mL, the result of detecting NGSP glycosylated hemoglobin HbA1c by the human hemoglobin solution is 4% -6%, and the result of detecting NGSP by the porcine hemoglobin solution is 1% -3%.
5. The method for producing a ceramic green sheet according to any one of claim 2 to 4,
in the step 1, the centrifugation conditions are as follows: centrifuging at 2000-4000rpm for 10-20min, wherein the volume ratio of the solution to the PBS buffer is 1 (1-5), and the washing times are 2-5 times;
in the step 2, the volume ratio of the human red blood cell suspension or the pig red blood cell suspension to the hemolytic agent is 1: (0.5-2);
in the step 3, the volume ratio of the mixed solution of the human hemoglobin solution and the pig blood hemoglobin solution to the freeze-drying protective agent is 1: (1-5).
6. The method of preparing according to claim 5, further comprising the step of freeze-drying, wherein the freeze-drying comprises taking the low-level glycosylated hemoglobin control, and obtaining the low-level glycosylated hemoglobin control in a freeze-dried state by vacuum freeze-drying.
7. The low-level glycosylated hemoglobin control product produced by the production method according to any one of claims 2 to 6.
8. The use of the low-level glycosylated hemoglobin control prepared by the preparation method of any one of claims 2 to 6 in the preparation of glycosylated hemoglobin detection reagent or kit.
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