CN114019175A - Antifreeze agent for glycosylated hemoglobin kit and application method thereof - Google Patents
Antifreeze agent for glycosylated hemoglobin kit and application method thereof Download PDFInfo
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- CN114019175A CN114019175A CN202111093834.8A CN202111093834A CN114019175A CN 114019175 A CN114019175 A CN 114019175A CN 202111093834 A CN202111093834 A CN 202111093834A CN 114019175 A CN114019175 A CN 114019175A
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- antifreeze agent
- kit
- glycated hemoglobin
- hemoglobin kit
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- 239000007798 antifreeze agent Substances 0.000 title claims abstract description 47
- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 35
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229920000609 methyl cellulose Polymers 0.000 claims abstract description 14
- 239000001923 methylcellulose Substances 0.000 claims abstract description 14
- 235000010981 methylcellulose Nutrition 0.000 claims abstract description 14
- SZYJELPVAFJOGJ-UHFFFAOYSA-N trimethylamine hydrochloride Chemical compound Cl.CN(C)C SZYJELPVAFJOGJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- ZSZRUEAFVQITHH-UHFFFAOYSA-N 2-(2-methylprop-2-enoyloxy)ethyl 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CC(=C)C(=O)OCCOP([O-])(=O)OCC[N+](C)(C)C ZSZRUEAFVQITHH-UHFFFAOYSA-N 0.000 claims abstract description 9
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims description 44
- 239000003381 stabilizer Substances 0.000 claims description 35
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 16
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 16
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 16
- 108091005995 glycated hemoglobin Proteins 0.000 claims description 15
- 235000011187 glycerol Nutrition 0.000 claims description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 7
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 229920000858 Cyclodextrin Polymers 0.000 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- 229920002307 Dextran Polymers 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000001116 FEMA 4028 Substances 0.000 claims description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 3
- 235000004279 alanine Nutrition 0.000 claims description 3
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 3
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 3
- 229960004853 betadex Drugs 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 235000010356 sorbitol Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000000811 xylitol Substances 0.000 claims description 3
- 235000010447 xylitol Nutrition 0.000 claims description 3
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 3
- 229960002675 xylitol Drugs 0.000 claims description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 229960002086 dextran Drugs 0.000 claims description 2
- 125000000647 trehalose group Chemical group 0.000 claims description 2
- 239000002577 cryoprotective agent Substances 0.000 claims 2
- 150000001298 alcohols Chemical class 0.000 claims 1
- 238000010257 thawing Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 6
- 229940127554 medical product Drugs 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 description 33
- 238000012360 testing method Methods 0.000 description 22
- 230000000052 comparative effect Effects 0.000 description 13
- 238000007710 freezing Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 238000005303 weighing Methods 0.000 description 12
- 230000008014 freezing Effects 0.000 description 11
- 238000002835 absorbance Methods 0.000 description 8
- 230000002528 anti-freeze Effects 0.000 description 8
- 238000003908 quality control method Methods 0.000 description 6
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 5
- 229950004354 phosphorylcholine Drugs 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000000306 component Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NJNWCIAPVGRBHO-UHFFFAOYSA-N 2-hydroxyethyl-dimethyl-[(oxo-$l^{5}-phosphanylidyne)methyl]azanium Chemical group OCC[N+](C)(C)C#P=O NJNWCIAPVGRBHO-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
- B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
- B65D81/00—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents
- B65D81/18—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents providing specific environment for contents, e.g. temperature above or below ambient
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an antifreeze agent for a glycosylated hemoglobin kit and an application method thereof, belonging to the field of medical products and technologies, wherein the antifreeze agent comprises the following components: trimethyl ammonium hydrochloride, glycerol, methyl cellulose and 2-methacryloyloxyethyl phosphorylcholine. The antifreeze agent can resist low-temperature freeze thawing when being used in a glycosylated hemoglobin kit, can be transported and stored at the temperature of lower than-0 ℃ and can be repeatedly stored and taken for 5 times in the environment of-20 ℃, so that the low-temperature stable storage time is more than one month, and meanwhile, the antifreeze agent has the advantages of high detection sensitivity, strong specificity, good precision and lower cost, and can realize accurate detection of the glycosylated hemoglobin.
Description
Technical Field
The invention belongs to the technical field of medical products, and particularly relates to an antifreeze agent for a glycosylated hemoglobin kit and an application method thereof.
Background
Glycated hemoglobin (HbA1c) is a product of the non-enzymatic reaction between hemoglobin in red blood cells and saccharides in serum, and it is generally considered that the glycated hemoglobin concentration effectively reflects the average blood glucose level over the past 8 to 12 weeks. Application range of the glycosylated hemoglobin determination kit the kit is used for in vitro quantitative determination of glycosylated hemoglobin in serum or plasma.
In order to ensure effectiveness, the glycosylated hemoglobin determination kit needs special cold chain transportation, but a large-scale cold chain transportation tool has a complex structure and extremely high cost, is only suitable for mass transportation, and is generally stored and transported by an independent foam insulation box with the characteristics of heat insulation, water resistance, shock resistance, ageing resistance, low price and the like in the small-batch transportation process; or the reagent kit is difficult to transport for a long time in a high-latitude cold area, so that the reagent in the reagent kit is inactivated, thereby influencing the determination result. Therefore, the development of an antifreeze for glycated hemoglobin and an antifreeze assay kit thereof is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the antifreeze agent for the glycosylated hemoglobin kit and the application method thereof are provided, the antifreeze agent is added into the glycosylated hemoglobin kit, the kit can be transported and stored at the temperature of lower than-0 ℃, and can be repeatedly stored and taken for 5 times in the environment from-20 ℃ to room temperature, the low-temperature stable storage time can be more than one month, meanwhile, the antifreeze agent has the advantages of high detection sensitivity, strong specificity, good precision and lower cost, and can realize the accurate detection of the glycosylated hemoglobin.
In order to achieve the purpose, the invention provides the following technical scheme:
an antifreeze agent for a glycated hemoglobin kit, the antifreeze agent comprising the following components: trimethyl ammonium hydrochloride, methyl cellulose and 2-methacryloyloxyethyl phosphorylcholine.
Further, the antifreeze agent comprises the following raw materials in parts by weight: 1-5 parts of trimethylammonium hydrochloride, 0-5 parts of glycerol, 1-5 parts of methyl cellulose and 1-5 parts of 2-methacryloyloxyethyl phosphorylcholine.
Further, the antifreeze agent comprises the following raw materials in parts by weight: 2-5 parts of trimethylammonium hydrochloride, 1-2 parts of glycerol, 3-5 parts of methylcellulose and 1-4 parts of 2-methacryloyloxyethyl phosphorylcholine.
The invention provides an application method of the antifreeze agent, which is used for preparing a glycosylated hemoglobin kit.
Further, the mass concentration of the anti-freezing agent in the glycosylated hemoglobin kit is 0.5-5 g/L.
The invention provides a glycosylated hemoglobin kit, which contains glycosylated hemoglobin, a stabilizer and the antifreeze agent as described in claim 1 to 3.
Further, the mass concentration of the stabilizer is 0.5-3 g/L.
Further, the stabilizer contains any one or more of amino acid, saccharide, alcohol and polymer.
Further, the amino acid is citric acid, serine, lysine or alanine;
the saccharide is trehalose or sucrose;
the alcohol is xylitol, sorbitol or mannitol;
the polymer is polyvinylpyrrolidone PVP, bovine serum albumin, dextran or beta-cyclodextrin.
Further, the stabilizer comprises the following raw materials in parts by weight: 1-5 parts of citric acid, 1-5 parts of trehalose and 1-5 parts of polyvinylpyrrolidone (PVP).
The invention has the following beneficial effects:
1. the invention provides a formula of an antifreeze agent, which contains trimethylammonium hydrochloride, glycerol, methyl cellulose and 2-methacryloyloxyethyl phosphorylcholine, wherein the 2-methacryloyloxyethyl phosphorylcholine consists of phosphorylcholine groups and methacrylic acid groups, and the phosphorylcholine groups are hydrophilic parts of phospholipid and exist on cell membranes of organisms, have high biocompatibility, high hydrophilicity, high lubricity and high anticoagulation performance, can eliminate non-specific adsorption of biomolecules, cells or blood components, can improve the compatibility between hemoglobin and various materials, and improve the freeze-thaw resistance and stability of a kit; and the freeze-thaw resistance and the stability of the kit are further improved by combining the common use of trimethyl ammonium hydrochloride, glycerol and methyl cellulose.
2. The antifreeze agent is added into the glycosylated hemoglobin kit together, so that the freeze-thaw resistance stability of the kit can be remarkably improved, the kit can be transported and stored at the temperature of lower than-0 ℃, and can be repeatedly stored and taken for 5 times in the environment from-20 ℃ to room temperature, and the low-temperature stable storage time can be more than one month.
3. The invention also discloses a formula of the stabilizer, in the glycosylated hemoglobin kit, the stabilizer and the anti-freezing agent act together, so that the freeze-thaw resistance stability can be further enhanced, and the performance of the kit after low-temperature storage is not affected.
Drawings
FIG. 1 is a graph of freeze-thaw resistance experiments for example 5, 6, 7 and comparative example kits.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the application of the present invention is not limited thereto. The following raw materials used in the invention are all purchased from chemical and biological reagent raw materials companies and can be directly used.
Example 1
Preparing an antifreeze agent: weighing an antifreeze agent raw material, dissolving the antifreeze agent raw material in water to prepare an antifreeze agent solution containing the following raw materials in mass concentration: 1mg/L trimethyl ammonium hydrochloride, 5mg/L methylcellulose and 1 mg/L2-methacryloyloxyethyl phosphorylcholine.
Example 2
Preparing an antifreeze agent: weighing an antifreeze agent raw material, dissolving the antifreeze agent raw material in water to prepare an antifreeze agent solution containing the following raw materials in mass concentration: 5mg/L trimethylammonium hydrochloride, 5mg/L glycerin, 1mg/L methylcellulose and 5 mg/L2-methacryloyloxyethyl phosphorylcholine.
Example 3
Preparing an antifreeze agent: weighing an antifreeze agent raw material, dissolving the antifreeze agent raw material in water to prepare an antifreeze agent solution containing the following raw materials in mass concentration: 2mg/L trimethylammonium hydrochloride, 2mg/L glycerol, 3mg/L methylcellulose and 2 mg/L2-methacryloyloxyethyl phosphorylcholine.
Example 4
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 3g/L of citric acid, 2g/L of trehalose and 4g/L of polyvinylpyrrolidone PVP.
Example 5
Preparing a glycosylated hemoglobin kit: the reagent comprises a reagent R1 and a reagent R2, and is prepared according to the following mass concentration;
the reagent R1:
| components | Mass concentration |
| Glycine buffer | 50mmol/L |
| Latex microspheres | 1.5g/L |
| Preservative | 1g/L |
The reagent R2:
| components | Mass concentration |
| Glycine buffer | 50mmol/L |
| Glycated hemoglobin antibody | 25mg/L |
| Goat anti-mouse polyclonal antibody | 75mg/L |
| EXAMPLE 1 anti-freeze agent | 2g/L |
| EXAMPLE 4 stabilizers | 1g/L |
| Preservative | 1g/L |
Example 6
The difference from example 5 is that: "example 1 antifreeze" was adjusted to "0.5 g/L example 2 antifreeze"; the amount of the stabilizer was adjusted to 3 g/L.
Example 7
The difference from example 5 is that: "example 1 antifreeze" was adjusted to "5 g/L example 3 antifreeze"; the amount of the stabilizer was adjusted to 0.5 g/L.
Comparative example:
the difference from example 5 is that: no antifreeze is added.
Application examples
The kits obtained in each of examples 5 to 7 and comparative example were prepared; placing them in-20 deg.C environment for 4 days, taking out, placing in 2-8 deg.C environment for 2 days, and testing their performances (analysis sensitivity, linear range, precision, accuracy); the above ambient temperature was then switched repeatedly 5 times, i.e. freeze-thaw was repeated 5 times, and the stability of the 5 kits was recorded.
(1) Assay sensitivity validation
The quality control substance test reagent (kit) with the concentration of 5.0-6.0% is used, the absorbance difference of the reagent (kit) under the specified parameters is recorded, and the absorbance difference (delta A) is converted into 5.5% according to the formula (1). The difference of absorbance (delta A) before and after reaction is not less than 0.01. The test calculation data are shown in table 1.
In the formula:
Δ A-absorbance difference;
A1-absorbance value at start-up;
A2-starting the absorbance value after 5min (t).
X is the mean concentration of the samples to be measured.
TABLE 1
| Example 5 | Example 6 | Example 7 | Comparison ofExample (b) | |
| Initial absorbance A1 | 0.3951 | 0.3945 | 0.3945 | 0.3966 |
| End Point Absorbance A2 | 0.4380 | 0.4369 | 0.4378 | 0.3993 |
| Converting the difference | 0.0421 | 0.0416 | 0.0441 | 0.0028 |
| Concentration of sample | 5.6 | 5.6 | 5.4 | 5.6 |
As can be seen from Table 1, the analytical sensitivity of the reagents of example 5, example 6 and example 7 still met the requirements after freezing and thawing of the reagents, while the analytical sensitivity of the comparative reagents did not meet the requirements, indicating that the sensitivity of the comparative reagents was greatly affected after freezing and thawing of the reagents.
(2) Linear range
Diluting the high concentration sample near the upper limit of the linear region with the low concentration sample near the lower limit of the linear region, and mixing to 6 diluted concentrations (x)i) (see Table 2). By means of respective reagent (kit)The samples were tested 3 times for each dilution concentration, and the mean (y) of the results of each dilution concentration test was determinedi). In diluted concentration (x)i) As independent variable, the mean value (y) of the detection results is usedi) Linear regression equations were solved for the dependent variables. The correlation coefficient (r) of the linear regression was calculated according to equation (2) and should be in accordance with the linear range of the reagent (kit) of [ 3.8%, 14.0% ]]Internal; wherein,
a) the linear correlation coefficient (r) should be not less than 0.990;
b) within the range of [ 3.8%, 7.0% ], the absolute deviation of linearity should not exceed plus or minus 0.5%;
within the range (7.0%, 14.0% >), the linear relative deviation should not exceed ± 7%.
In the formula:
r is the correlation coefficient;
xi-diluting the concentration;
n is the number of dilution concentrations;
yi-mean of 3 replicate test results corresponding to dilution concentration;
i——1,2,3……n。
TABLE 2
As can be seen from Table 2, the linear range of the reagents of example 5, example 6 and example 7 still met after freezing and thawing of the reagents, whereas the linear range of the comparative reagents did not meet the requirements and, during the test, fluctuations occurred indicating unstable testing of the reagents after freezing and thawing.
(3) Precision degree
Under the repeated condition, testing 2 quality control substances with different concentrations, repeating the test for 10 times, and respectively calculating the average value of the measured values according to the formula (3) and the formula (4) ((
) And Standard Deviation (SD). Calculating Coefficient of Variation (CV) according to formula (5), and calculating data shown in tables 3 and 4; the resulting Coefficient of Variation (CV) should be no more than 3%.
In the formula:
CV-coefficient of variation;
SD-Standard deviation;
n is the number of tests;
i——1,2,3……n。
TABLE 3 quality control level 1
| Example 5 | Example 6 | Example 7 | Comparative example | |
| 1 | 5.23 | 5.09 | 5.30 | 5.10 |
| 2 | 5.25 | 5.27 | 5.20 | 4.23 |
| 3 | 5.14 | 5.16 | 5.29 | 5.67 |
| 4 | 5.08 | 5.27 | 5.21 | 5.09 |
| 5 | 5.22 | 5.26 | 5.11 | 5.11 |
| 6 | 5.10 | 5.33 | 5.25 | 5.27 |
| 7 | 5.22 | 5.26 | 5.14 | 5.88 |
| 8 | 5.11 | 5.24 | 5.31 | 5.14 |
| 9 | 5.22 | 5.28 | 5.24 | 5.02 |
| 10 | 5.29 | 5.23 | 5.26 | 5.07 |
| X | 5.19 | 5.24 | 5.23 | 5.16 |
| SD | 0.072 | 0.068 | 0.067 | 0.434 |
| CV | 1.39％ | 1.30％ | 1.28％ | 8.41％ |
TABLE 4 quality control level 2
As can be seen from the data in tables 3 and 4, the precision of the reagents of examples 5, 6 and 7 was still satisfactory after freezing and thawing of the reagents, while the precision of the reagents of the comparative examples was not satisfactory, indicating that the reagents became unstable after freezing and thawing.
(4) Accuracy of
3 tests with a certified reference substance (CRM) or other accepted reference substance test kit, the test results are labeled (x)i) The relative deviation (B) is calculated according to the formula (6), and the calculated data are shown in Table 5, so that the accuracy relative deviation should not exceed +/-7%.
In the formula:
b-relative deviation;
xi-a test result;
t-certified reference substance index value;
i——1,2,3……n。
TABLE 5
| Example 5 | Example 6 | Example 7 | | |
| Test | ||||
| 1 | 6.20 | 6.05 | 6.03 | 5.42 |
| |
5.84 | 5.92 | 6.03 | 5.56 |
| |
5.89 | 5.94 | 5.92 | 5.80 |
| Mean value | 5.98 | 5.97 | 5.99 | 5.59 |
| Target value | 6.02 | 6.02 | 6.02 | 6.02 |
| Deviation of | -0.66％ | -0.83％ | -0.50％ | -7.14％ |
As can be seen from table 5, the reagent tests of examples 5, 6 and 7 demonstrated a high consistency of the reference substance after freezing and thawing of the reagent, while the reagent tests of the comparative examples demonstrated a large deviation of the reference substance, indicating that the reagent test was inaccurate after freezing and thawing and that the calibration curve needs to be corrected.
(5) Freeze thaw stability
And (5) repeatedly freezing and thawing, taking out, testing the quality control product, observing the concentration change condition, and calculating data as shown in table 6 and figure 1.
TABLE 6
| Example 5 | Example 6 | Example 7 | Comparative example | |
| For the first time | 10.73 | 10.99 | 10.91 | 8.36 |
| For the second time | 10.94 | 10.90 | 10.61 | 6.52 |
| The third time | 10.60 | 11.01 | 10.94 | 4.21 |
| Fourth time | 10.77 | 10.56 | 10.58 | 2.12 |
| Fifth time | 10.85 | 10.76 | 10.74 | 0.55 |
FIG. 1 is a graph of freeze-thaw resistance experiments for example 5, 6, 7 and comparative example kits. As can be seen from Table 6 and FIG. 1, the reagent test quality control of example 5, example 6 and example 7 is still in a stable state after repeated freezing and thawing of the reagent 5, while the comparative example has lower and lower detection activity under continuous freezing and thawing, and the performance of the reagent can not be normally used for clinical application.
Example 8
Preparing an antifreeze agent: weighing an antifreeze agent raw material, dissolving the antifreeze agent raw material in water to prepare an antifreeze agent solution containing the following raw materials in mass concentration: 3mg/L trimethylammonium hydrochloride, 5mg/L glycerol, 3mg/L methylcellulose and 4 mg/L2-methacryloyloxyethyl phosphorylcholine.
Example 9
Preparing an antifreeze agent: weighing an antifreeze agent raw material, dissolving the antifreeze agent raw material in water to prepare an antifreeze agent solution containing the following raw materials in mass concentration: 4mg/L trimethylammonium hydrochloride, 4mg/L glycerin, 4mg/L methylcellulose and 2 mg/L2-methacryloyloxyethyl phosphorylcholine.
Example 10
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 1g/L citric acid, 5g/L trehalose and 1g/L polyvinylpyrrolidone PVP.
Example 11
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 5g/L of citric acid, 1g/L of trehalose and 5g/L of polyvinylpyrrolidone PVP.
Example 12
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 3g/L serine, 1g/L trehalose, 2g/L mannitol, 2g/L dextran.
Example 13
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 3g/L lysine, 1g/L xylitol and 5g/L bovine serum albumin.
Example 14
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 4g/L of sucrose, 1g/L of sorbitol and 7g/L of polyvinylpyrrolidone PVP.
Example 15
Preparing a stabilizer: weighing raw materials of a stabilizer, dissolving the raw materials in water to prepare a stabilizer solution containing the following raw materials in mass concentration: 2g/L alanine, 1g/L trehalose and 3g/L beta-cyclodextrin.
The antifreeze and the stabilizer of the embodiment 8-15 are used for preparing kits according to the method of the embodiment 5, and the kits are tested according to the application examples, and the obtained data are similar to the data of the embodiment 5, 6 and 7, which shows that the kit obtained by the method of the invention has good reproducibility.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. An antifreeze agent for a glycosylated hemoglobin kit, which is characterized by comprising the following components: trimethyl ammonium hydrochloride, methyl cellulose and 2-methacryloyloxyethyl phosphorylcholine.
2. The antifreeze agent for the glycated hemoglobin kit according to claim 1, wherein the antifreeze agent comprises the following raw materials in parts by weight: 1-5 parts of trimethylammonium hydrochloride, 0-5 parts of glycerol, 1-5 parts of methyl cellulose and 1-5 parts of 2-methacryloyloxyethyl phosphorylcholine.
3. The antifreeze agent for the glycated hemoglobin kit according to claim 2, wherein the antifreeze agent comprises the following raw materials in parts by weight: 2-5 parts of trimethylammonium hydrochloride, 1-2 parts of glycerol, 3-5 parts of methylcellulose and 1-4 parts of 2-methacryloyloxyethyl phosphorylcholine.
4. A method of using the cryoprotectant of claims 1-3, wherein the cryoprotectant is used in the preparation of a glycated hemoglobin kit.
5. The method for using the antifreeze agent according to claim 4, wherein the mass concentration of the antifreeze agent in the glycated hemoglobin kit is 0.5-5 g/L.
6. A glycated hemoglobin kit comprising glycated hemoglobin, a stabilizer, and the antifreeze agent according to any one of claims 1 to 3.
7. The glycated hemoglobin kit according to claim 6, wherein the mass concentration of the stabilizer is 0.5 to 3 g/L.
8. The glycated hemoglobin kit according to claim 6, wherein the stabilizer comprises one or more of amino acids, saccharides, alcohols, and polymers.
9. The glycated hemoglobin kit of claim 8, wherein the amino acid is citric acid, serine, lysine, or alanine;
the saccharide is trehalose or sucrose;
the alcohol is xylitol, sorbitol or mannitol;
the polymer is polyvinylpyrrolidone PVP, bovine serum albumin, dextran or beta-cyclodextrin.
10. The glycated hemoglobin kit as in claim 9, wherein the stabilizer comprises the following raw materials in parts by weight: 1-5 parts of citric acid, 1-5 parts of trehalose and 1-5 parts of polyvinylpyrrolidone (PVP).
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