CN116794332A - Preparation method of three-item combined detection card for anemia and kit - Google Patents
Preparation method of three-item combined detection card for anemia and kit Download PDFInfo
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- CN116794332A CN116794332A CN202210265233.9A CN202210265233A CN116794332A CN 116794332 A CN116794332 A CN 116794332A CN 202210265233 A CN202210265233 A CN 202210265233A CN 116794332 A CN116794332 A CN 116794332A
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- detection
- fluorescent microsphere
- coating liquid
- pad
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- 238000001514 detection method Methods 0.000 title claims abstract description 167
- 208000007502 anemia Diseases 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 113
- 239000004005 microsphere Substances 0.000 claims abstract description 89
- 239000011248 coating agent Substances 0.000 claims abstract description 65
- 238000000576 coating method Methods 0.000 claims abstract description 65
- 239000007788 liquid Substances 0.000 claims abstract description 62
- 238000008416 Ferritin Methods 0.000 claims abstract description 61
- 102000008857 Ferritin Human genes 0.000 claims abstract description 59
- 108050000784 Ferritin Proteins 0.000 claims abstract description 59
- 239000011724 folic acid Substances 0.000 claims abstract description 59
- 239000011715 vitamin B12 Substances 0.000 claims abstract description 59
- 229960000304 folic acid Drugs 0.000 claims abstract description 58
- 229930003779 Vitamin B12 Natural products 0.000 claims abstract description 57
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims abstract description 57
- 235000019152 folic acid Nutrition 0.000 claims abstract description 57
- 235000019163 vitamin B12 Nutrition 0.000 claims abstract description 57
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 56
- 238000012360 testing method Methods 0.000 claims abstract description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 29
- 241000283707 Capra Species 0.000 claims abstract description 24
- 238000003908 quality control method Methods 0.000 claims abstract description 24
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 21
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 239000003365 glass fiber Substances 0.000 claims abstract description 17
- 238000005507 spraying Methods 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 238000002156 mixing Methods 0.000 claims description 26
- 239000003085 diluting agent Substances 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 23
- 239000007853 buffer solution Substances 0.000 claims description 21
- 238000010521 absorption reaction Methods 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 230000001681 protective effect Effects 0.000 claims description 16
- 238000007789 sealing Methods 0.000 claims description 13
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 241001529936 Murinae Species 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 8
- 230000003213 activating effect Effects 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000003761 preservation solution Substances 0.000 claims description 7
- 239000008213 purified water Substances 0.000 claims description 7
- 241001494479 Pecora Species 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- -1 proclin300 Chemical compound 0.000 claims description 5
- 241000287828 Gallus gallus Species 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 230000003167 anti-vitamin Effects 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 55
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 16
- 229940098773 bovine serum albumin Drugs 0.000 description 16
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 6
- 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 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 239000003146 anticoagulant agent Substances 0.000 description 4
- 229940127219 anticoagulant drug Drugs 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000002274 desiccant Substances 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 238000007639 printing Methods 0.000 description 4
- 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 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 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 description 3
- 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 description 3
- 229910021538 borax Inorganic materials 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000004328 sodium tetraborate Substances 0.000 description 3
- 235000010339 sodium tetraborate Nutrition 0.000 description 3
- 206010002065 Anaemia megaloblastic Diseases 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 208000000682 Megaloblastic Anemia Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 231100001016 megaloblastic anemia Toxicity 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000013074 reference sample Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 206010022971 Iron Deficiencies Diseases 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 238000010304 firing Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 208000002670 vitamin B12 deficiency Diseases 0.000 description 1
- 229940082632 vitamin b12 and folic acid Drugs 0.000 description 1
Classifications
-
- 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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The embodiment of the application belongs to the technical field of in-vitro diagnosis and rapid detection, and relates to a preparation method of an anemia three-item joint detection card and a kit, wherein the preparation method comprises the steps of immersing glass fibers in sample pad treatment liquid for treatment to prepare a sample pad; respectively marking the vitamin B12 marked antibody, the folic acid marked antibody, the ferritin marked antibody and the goat anti-chicken IgY marked antibody by using fluorescent microspheres to prepare fluorescent microsphere markers; spraying fluorescent microsphere markers on glass fibers to prepare a bonding pad; preparing a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid, and respectively spraying and scribing the first detection line, the second detection line, the third detection line and the quality control line on the nitrocellulose membrane; sequentially overlapping the sample pad, the combining pad and the water absorbing pad on the bottom plate to prepare a test strip; and installing the test strip in a card shell of the detection card to obtain the detection card. The application can improve the detection efficiency and reduce the detection cost.
Description
Technical Field
The application relates to the technical field of in-vitro diagnosis and rapid detection, in particular to a preparation method of a three-item combined detection card for anemia and a kit.
Background
The measurement of vitamin B12, folic acid and ferritin in serum is a main index for diagnosing anemia and classifying anemia at present. Vitamin B12 and folic acid are two water-soluble vitamins necessary for erythrocyte maturation, and the deficiency of these two vitamins causes DNA synthesis disorders, which unbalance the development of cell nuclei and cytoplasm, forming megaloblastic erythrocytes. Thus, serum folate, vitamin B12 deficiency is a major marker of megaloblastic anemia. Ferritin is an iron-containing protein, can store and provide iron elements for organisms, and serum ferritin is the most sensitive index for detecting iron deficiency in the bodies, and is clinically used for diagnosing iron deficiency anemia.
The combined detection of vitamin B12, folic acid and ferritin is helpful for early finding out the anemia state of human body, and can also more comprehensively judge the cause of anemia, thus having certain value for early prevention of anemia and treatment of anemia. The three are combined for detection, so that the detection rate of early anemia can be improved, iron deficiency anemia and megaloblastic anemia can be identified, and early detection, early treatment and symptom aggravation prevention can be realized in the early stage of anemia symptom occurrence.
However, currently, the existing immunochromatography products of vitamin B12/folic acid/ferritin in the market can only be used for single-item detection, if all three items of anemia need to be detected simultaneously, three different immunochromatography test strips need to be used, so that the detection cost and the operation time can be increased, and the detection efficiency is reduced.
Disclosure of Invention
The embodiment of the application aims to provide a preparation method of a three-item combined detection card for anemia, which aims to solve the technical problems of high detection cost, long operation time and low detection efficiency of simultaneous detection of three-item vitamin B12, folic acid and ferritin in anemia in related technologies.
In order to solve the technical problems, the embodiment of the application provides a preparation method of an anemia three-item joint detection card, which adopts the following technical scheme:
preparing a marking buffer solution, a marking activating solution, a marking preserving solution, a coating diluting solution and a sample pad treatment solution in advance;
immersing glass fibers into a sample pad treatment liquid for treatment, and drying the immersed glass fibers to obtain a sample pad;
respectively marking the vitamin B12 marked antibody, the folic acid marked antibody, the ferritin marked antibody and the goat anti-chicken IgY marked antibody by using fluorescent microspheres to respectively form a fluorescent microsphere vitamin B12 antibody complex, a fluorescent microsphere folic acid antibody complex, a fluorescent microsphere ferritin antibody complex and a fluorescent microsphere goat anti-chicken IgY antibody complex;
mixing the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex to prepare a fluorescent microsphere marker;
spraying fluorescent microsphere markers on glass fibers to prepare a bonding pad;
respectively diluting the vitamin B12 coupled BSA antigen, the folic acid coupled BSA antigen, the ferritin monoclonal antibody and the chicken IgY antibody by using the coating diluent to prepare a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid;
attaching a nitrocellulose membrane to a bottom plate, and respectively spraying a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid onto the nitrocellulose membrane;
sequentially overlapping the sample pad, the combination pad and the water absorption pad on a bottom plate, assembling to obtain a reaction plate, and manufacturing the reaction plate into a test strip;
and installing the test strip in a detection card shell to obtain the detection card.
Further, the steps of labeling the anti-vitamin B12 murine monoclonal IgG, the anti-folic acid murine monoclonal IgG, the anti-ferritin murine monoclonal IgG and the goat anti-chicken IgY goat antibody with fluorescent microspheres respectively to form a fluorescent microsphere vitamin B12 antibody complex, a fluorescent microsphere folic acid antibody complex, a fluorescent microsphere ferritin antibody complex and a fluorescent microsphere goat anti-chicken IgY antibody complex respectively comprise:
taking four clean centrifuge tubes with the numbers of 1, 2, 3 and 4, and respectively adding a marking buffer solution into each centrifuge tube;
vortex mixing fluorescent microsphere uniformly, taking fluorescent microsphere with solid content of 1% in a marking buffer solution, and vortex mixing uniformly again;
adding the marked activating solution into the centrifuge tube, and uniformly mixing;
after the uniform mixing is finished, placing the centrifuge tube on a rotary mixing instrument for rotary reaction;
after the reaction is completed, centrifuging the centrifuge tube, and reserving a precipitate;
adding a marking buffer solution into a centrifuge tube, and performing ultrasonic scattering;
adding a vitamin B12 labeled antibody, a folic acid labeled antibody, a ferritin labeled antibody and a goat anti-chicken IgY labeled antibody into centrifugal tubes with the numbers of 1, 2, 3 and 4 respectively, uniformly mixing, and performing a rotation reaction;
after the rotary reaction, taking the marked sealing liquid into the centrifuge tube, performing ultrasonic scattering, performing the rotary reaction, and then centrifuging the centrifuge tube to leave a precipitate;
and (3) respectively adding the marked preservation solution into centrifuge tubes with the numbers of 1, 2, 3 and 4, and scattering by using ultrasound to prepare the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere goat anti-chicken IgY antibody complex.
Further, the mixing volume ratio of the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex in the fluorescent microsphere marker is 1:1:1:1.
Further, the concentration of the first detection line coating liquid, the second detection line coating liquid and the third detection line coating liquid is 0.5mg/mL, and the concentration of the quality control line coating liquid is 1.0mg/mL.
Further, the distance between the first detection line and the upper edge of the nitrocellulose membrane, the distance between the first detection line and the second detection line, the distance between the second detection line and the third detection line, the distance between the third detection line and the quality control line, and the distance between the quality control line and the lower edge of the nitrocellulose membrane are all 4.5-5.5 mm.
Further, the step of sequentially overlapping the sample pad, the bonding pad and the water absorbing pad on the bottom plate to assemble the reaction plate comprises the following steps:
cutting the water absorption pad into a preset size, spreading the bottom plate on a workbench surface, uncovering a protective film at the pasting position of the water absorption pad on the bottom plate, and adhering the water absorption pad on the bottom plate, wherein one end of the water absorption pad is aligned with the top end of the bottom plate, and the other side of the water absorption pad is partially covered on the nitrocellulose film;
the protective film at the bonding pad pasting part on the bottom plate is uncovered, the bonding pad is adhered to the upper end of the protective film, the bonding pad faces upwards, and one end of the bonding pad is covered on the nitrocellulose film;
and uncovering the protective film at the adhering position of the sample pad on the bottom plate, adhering the sample pad on the protective film, aligning the lower end of the sample pad with the adhering position of the sample pad on the bottom plate, and covering the upper end of the sample pad on the combining pad to prepare the reaction plate.
Further, the spray pad amount of the fluorescent microsphere marker is 4.0 mu L/cm; the film dividing amounts of the first detection line coating liquid, the second detection line coating liquid, the third detection line coating liquid and the quality control line coating liquid are all 1 mu L/cm.
In order to solve the technical problems, the embodiment of the application also provides an anemia three-item joint detection kit, which comprises a sample diluent, a detection chip and a detection card prepared by adopting the anemia three-item joint detection card preparation method.
Further, the detection chip is prepared by the following method:
and (3) respectively detecting the vitamin B12, folic acid and ferritin enterprise calibrator by using a detection card to obtain linear data of signal values and corresponding preparation concentrations, and writing the linear data, a fitting algorithm and ID information into a chip to obtain the detection chip.
Further, the components of the sample diluent include sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, proclin300, and purified water.
Compared with the prior art, the embodiment of the application has the following main beneficial effects:
according to the detection card prepared by the preparation method of the three-item combined detection card for anemia, the collected blood sample sequentially reacts with the vitamin B12 labeled antibody, the folic acid labeled antibody and the ferritin labeled antibody which are coated on the binding pad by spraying the fluorescent microsphere, and the vitamin B12 coupled BSA antigen, the folic acid coupled BSA antigen and the ferritin monoclonal antibody which are coated on the first detection line, the second detection line and the third detection line on the nitrocellulose membrane, so that the content of the three-item vitamin B12, folic acid and ferritin can be detected simultaneously, the detection time is shortened, the detection efficiency is improved, and the detection cost is reduced.
Drawings
In order to more clearly illustrate the solution of the present application, a brief description will be given below of the drawings required for the description of the embodiments of the present application, it being apparent that the drawings in the following description are some embodiments of the present application, and that other drawings may be obtained from these drawings without the exercise of inventive effort for a person of ordinary skill in the art.
FIG. 1 is a flow chart of one embodiment of a method of preparing a three-item combined anemia test card of the present application;
FIG. 2 is a schematic diagram of the structure of the test strip of the present application;
fig. 3 is a schematic structural view of the test card of the present application.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the applications herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having" and any variations thereof in the description of the application and the claims and the description of the drawings above are intended to cover a non-exclusive inclusion. The terms first, second and the like in the description and in the claims or in the above-described figures, are used for distinguishing between different objects and not necessarily for describing a sequential or chronological order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
In order to make the person skilled in the art better understand the solution of the present application, the technical solution of the embodiment of the present application will be clearly and completely described below with reference to the accompanying drawings.
The embodiment of the application provides a preparation method of an anemia three-item joint detection card, which is shown in fig. 1, and comprises the following steps:
step S10, preparing a marking buffer solution, a marking activating solution, a marking preservation solution, a coating dilution solution and a sample pad treatment solution in advance.
In this example, the labeling buffer composition comprises MES (2-morpholinoethanesulfonic acid) in an optimum amount of 1.067g and purified water in an optimum amount of 80mL.
After MES is dissolved, adjusting the pH value to be 6.20+/-0.05 by using 1mol/L HCl or 1mol/L NaOH, and fixing the volume to be 100mL; the storage period is 3 months at the temperature of 2-8 ℃.
The components of the labeling-activating solution include EDC (1-ethyl-carbodiimide hydrochloride) and purified water, wherein the optimum amount of EDC is 0.020g. EDC was dissolved by adding 1mL of purified water and used within 10 min.
The components of the label stock solution include BSA (bovine serum albumin), sucrose, casein sodium salt, trehalose, proclin300 and 0.2M pH8.0Tris buffer. Wherein, the optimal usage amount of BSA is 2.000g, the optimal usage amount of sucrose is 2.000g, the optimal usage amount of casein sodium salt is 0.500g, the optimal usage amount of trehalose is 5.000g, and the optimal usage amount of Proclin300 is 0.05mL.
The preparation of the mark preservation solution comprises the following steps: adding 100mL0.2M pH8.0Tris buffer solution to dissolve BSA, sucrose, casein sodium salt and trehalose, adding Proclin300 solution, mixing well, and preserving at 2-8deg.C for 3 months.
The components of the coating diluent comprise sucrose and 0.01M PBS buffer solution with pH of 7.4, specifically, 100mL 0.01M pH7.4 PBS buffer solution is added to dissolve 1.000g sucrose, so as to prepare the coating diluent, and the coating diluent is stored at the temperature of 2-8 ℃ and has the effective period of 3 months.
The sample pad treatment solution comprises BSA, tween 20 (Tween-20) and 0.2M borax borate buffer solution (pH 8.0), wherein the optimal usage amount of BSA is 10.000g, the optimal usage amount of Tween-20 is 5mL, and the optimal usage amount of 0.2M borax borate buffer solution is 1000mL.
Specifically, the preparation method of the sample pad treatment fluid comprises the following steps: dissolving BSA with 0.2M borax borate buffer solution, adding Tween-20, stirring until the BSA is completely dissolved, and storing at 2-8deg.C for 3 months.
And step S20, immersing the glass fiber into a sample pad treatment liquid for treatment, and drying the immersed glass fiber to obtain the sample pad.
Specifically, the glass fiber is put into a stainless steel container, and after the sample pad treatment liquid is added to fully infiltrate the glass fiber, the treated glass fiber is taken out and placed on a clean, dry and flat airing frame. And (3) drying the airing rack overnight, drying to obtain a sample pad, placing the sample pad into a self-sealing bag, sealing the self-sealing bag, and placing a proper amount of drying agent.
And S30, respectively marking the vitamin B12 marked antibody, the folic acid marked antibody, the ferritin marked antibody and the goat anti-chicken IgY marked antibody by using fluorescent microspheres to respectively form a fluorescent microsphere vitamin B12 antibody complex, a fluorescent microsphere folic acid antibody complex, a fluorescent microsphere ferritin antibody complex and a fluorescent microsphere goat anti-chicken IgY antibody complex.
Wherein the vitamin B12 labeled antibody is a vitamin B12 murine monoclonal IgG antibody, the folic acid labeled antibody is a folic acid murine monoclonal IgG antibody, the ferritin labeled antibody is a ferritin murine monoclonal IgG antibody, the goat anti-chicken IgY labeled antibody is a goat anti-chicken IgY goat antibody, and the fluorescent microsphere is a latex particle with rare earth element europium as a fluorescent matrix material.
In this example, the preparation method of the four complexes comprises the following steps:
(1) Taking four clean centrifuge tubes with the numbers of 1, 2, 3 and 4, and respectively adding a marking buffer solution into each centrifuge tube;
(2) Vortex mixing fluorescent microsphere uniformly, taking fluorescent microsphere with solid content of 1% in a marking buffer solution, and vortex mixing uniformly again;
(3) Adding the marked activating solution into the centrifuge tube, and uniformly mixing;
(4) After the uniform mixing is finished, placing the centrifuge tube on a rotary mixing instrument for rotary reaction;
(5) After the reaction is completed, centrifuging the centrifuge tube, and reserving a precipitate;
(6) Adding a marking buffer solution into a centrifuge tube, and performing ultrasonic scattering;
(7) Adding a vitamin B12 labeled antibody, a folic acid labeled antibody, a ferritin labeled antibody and a goat anti-chicken IgY labeled antibody into centrifugal tubes with the numbers of 1, 2, 3 and 4 respectively, uniformly mixing, and performing a rotation reaction;
(8) After the rotary reaction, taking the marked sealing liquid into the centrifuge tube, performing ultrasonic scattering, performing the rotary reaction, and then centrifuging the centrifuge tube to leave a precipitate;
(9) And (3) respectively adding the marked preservation solution into centrifuge tubes with the numbers of 1, 2, 3 and 4, and scattering by using ultrasound to prepare the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere goat anti-chicken IgY antibody complex.
In the step (1), centrifuge tubes with the numbers of 1, 2, 3 and 4 are 2mL centrifuge tubes, and each tube is added with 1mL of marking buffer solution by using a pipette; taking 100 mu L of fluorescent microspheres in the step (2); in the step (3), 100 mu L of marked activating solution is taken and placed in the centrifuge tube, a cover is tightly covered, and the mixture is slowly turned upside down for 10 to 20 times and uniformly mixed; in the step (4), rotating reaction is carried out for 30min; in the step (5), the centrifugation conditions are: centrifuging at 4 ℃ at 10000rpm for 30min; in the step (6), 1000 mu L of the marking buffer solution is sucked by a liquid-transfering gun and added into a centrifuge tube, and the marking buffer solution is completely and ultrasonically dispersed by an ultrasonic cell crusher; in the step (7), centrifuge tubes numbered 1, 2, 3 and 4 are respectively added with 100 mug of vitamin B12 labeled antibody, 50 mug of folic acid labeled antibody, 50 mug of ferritin labeled antibody and 80 mug of goat anti-chicken IgY labeled antibody, and the mixture is evenly mixed up and down for 20 times, placed on a rotary mixer and subjected to rotary reaction for 30 minutes; in the step (8), 100 mu L of marked sealing liquid is sucked into the centrifuge tube by using a liquid-transfering gun, and is completely scattered by using an ultrasonic cell crusher, and is placed on a rotary mixer again for rotary reaction for 2 hours; centrifuging at 4deg.C at 10000rpm for 30min; in the step (9), 1mL of the labeled preservation solution is sucked up by a pipette and added into a centrifuge tube, and the labeled preservation solution is completely and ultrasonically dispersed by an ultrasonic cell crusher.
In the embodiment, after the step (9), the compound with the number of 1, 2, 3 and 4 is respectively moved into a 10mL centrifuge tube with the number of 5, 6, 7 and 8, a 2mL centrifuge tube with the number of 1, 2, 3 and 4 used for sucking 4mL cleaning mark of the mark preservation liquid is sucked, and after cleaning, the compound is moved into the 10mL centrifuge tube, and vortex mixing is carried out; after being evenly mixed, the mixture is labeled, and the solution is preserved at the temperature of 2-8 ℃.
And S40, mixing the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere goat anti-chicken IgY antibody complex to prepare the fluorescent microsphere marker.
Wherein, the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex are uniformly mixed according to the volume ratio of 1:1:1:1 (V: V), and the ultrasonic cell grinder is used for uniformly mixing the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex.
And S50, spraying fluorescent microsphere markers on the glass fibers to obtain the bonding pad.
In this example, glass fibers were cut to (10.+ -. 0.5 mm) × (300.+ -.10 mm) according to the production lot calculation, fluorescent microsphere markers were loaded into a metal-spraying streaking machine, sprayed onto the cut glass fibers at a spray-mat amount of 4.0. Mu.L/cm, and the sprayed glass fibers were dried overnight to produce a conjugate pad.
Taking out the dried bonding pad, putting a proper amount of drying agent, and sealing in a self-sealing bag. Labels are attached, and names, production dates, lot numbers and related information are noted.
And S60, respectively diluting the vitamin B12 coupled BSA antigen, the folic acid coupled BSA antigen, the ferritin monoclonal antibody and the chicken IgY antibody by using a coating diluent to prepare a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid.
Diluting the vitamin B12 coupled BSA antigen to 0.5mg/mL by using a coating diluent, namely a first detection line coating liquid; diluting folic acid coupled BSA antigen to 0.5mg/mL by using coating diluent, namely coating liquid of a second detection line; diluting the ferritin monoclonal antibody to 0.5mg/mL by using a coating diluent to obtain a third detection line coating liquid; diluting the chicken IgY antibody to 1.0mg/mL by using coating diluent, namely the quality control line coating liquid; and (3) preserving the prepared solution at the temperature of 2-8 ℃.
And S70, sticking a nitrocellulose membrane on a bottom plate, and respectively spraying a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid onto the first detection line, the second detection line, the third detection line and the quality control line on the nitrocellulose membrane.
Specifically, cutting the nitrocellulose membrane into 25mm× (300+/-5 mm), slightly uncovering a protective film on the sticking position of the nitrocellulose membrane on the bottom plate, and sticking the nitrocellulose membrane on the bottom plate, wherein the bottom plate is a PVC bottom plate.
Starting a metal spraying film drawing machine, setting the film drawing amount to be 1 mu L/cm, changing the film drawing length to be 300mm, adjusting the film drawing needle position, respectively loading a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid to the metal spraying film drawing machine, spraying to the corresponding positions of the first detection line, the second detection line, the third detection line and the quality control line on the nitrocellulose film, drying the nitrocellulose film in an electrothermal blowing drying oven after finishing film drawing, sealing in a self-sealing bag, putting a proper amount of drying agent, and pasting a label.
The distance between the first detection line and the upper edge of the nitrocellulose membrane is 4.5-5.5 mm, the distance between the first detection line and the second detection line is 4.5-5.5 mm, the distance between the second detection line and the third detection line is 4.5-5.5 mm, the distance between the third detection line and the quality control line is 4.5-5.5 mm, and the distance between the quality control line and the lower edge of the nitrocellulose membrane is 4.5-5.5 mm.
And S80, sequentially overlapping the sample pad, the bonding pad and the water absorbing pad on the bottom plate, assembling to obtain a reaction plate, and manufacturing the reaction plate into a test strip.
Wherein, the assembly steps of the reaction plate are as follows:
(1) Cutting the water absorption pad into a preset size, spreading the bottom plate on a workbench surface, uncovering a protective film at the pasting position of the water absorption pad on the bottom plate, and adhering the water absorption pad on the bottom plate, wherein one end of the water absorption pad is aligned with the top end of the bottom plate, and the other side of the water absorption pad is partially covered on the nitrocellulose film;
(2) The protective film at the bonding pad pasting part on the bottom plate is uncovered, the bonding pad is adhered to the upper end of the protective film, the bonding pad faces upwards, and one end of the bonding pad is covered on the nitrocellulose film;
(3) And uncovering the protective film at the adhering position of the sample pad on the bottom plate, adhering the sample pad on the protective film, aligning the lower end of the sample pad with the adhering position of the sample pad on the bottom plate, and covering the upper end of the sample pad on the combining pad to prepare the reaction plate.
Specifically, in the step (1), the water absorption pad is cut into the size of (23+/-1 mm) x (300+/-10 mm), and the other side part of the water absorption pad is covered on the nitrocellulose membrane for 2-3 mm; in the step (2), the front surface of the bonding pad is a surface sprayed with fluorescent microsphere markers, and one end of the bonding pad is covered on the nitrocellulose membrane for 1-2 mm; the sample pad in step (3) was cut to a size of (10.+ -. 0.5 mm) × (300.+ -. 10 mm).
The assembled reaction plate can be placed into an aluminum foil bag, and a drying agent is added, a sealing machine is used for sealing, and a label is attached.
In this example, the obtained reaction plate was cut into test strips 3.85.+ -. 0.05mm wide, and the schematic structure of the test strips is shown in FIG. 2.
As shown in fig. 2, 1 is a base plate, 2 is a sample pad, 3 is a bonding pad, 4 is a nitrocellulose membrane, 5 is a first detection line, 6 is a second detection line, 7 is a third detection line, 8 is a quality control line, and 9 is a water absorbing pad.
And step S90, mounting the test strip in a detection card shell to obtain the detection card.
Specifically, according to the production batch calculation demand, preparing corresponding quantity of detection card shell upper covers, determining jet printing projects and batch numbers by operators, jet printing the projects and batch numbers on the card shell upper covers, loading each test strip into the detection card shell lower covers, covering the upper covers after loading, placing the test strip on a card pressing machine, and adjusting the height of the card pressing machine to be 3.5mm so as to tightly combine the upper covers and the lower covers of the card shells.
Referring to fig. 3, fig. 3 is a schematic structural diagram of the detection card of the present application, specifically, the detection card 10 includes a sample adding hole 11, a detection area 12 and a hand-held area 13, wherein the sample adding hole 11 is located in a sample pad area of a test strip, the detection area 12 is located in a nitrocellulose membrane area of the test strip, and the hand-held area 13 is located at a water absorbing pad end of the test strip.
The embodiment of the application also provides an anemia three-joint detection kit which is characterized by comprising sample diluent, a detection chip and a detection card prepared by the anemia three-joint detection card preparation method.
The sample diluent is phosphate buffer solution, and the components of the sample diluent comprise sodium chloride, potassium chloride, disodium hydrogen phosphate, monopotassium phosphate, proclin300 and purified water; wherein, the optimal dosage of sodium chloride is 8g, the optimal dosage of potassium chloride is 0.2g, the optimal dosage of disodium hydrogen phosphate is 1.14g, the optimal dosage of monopotassium phosphate is 0.27g,Proclin 300, the optimal dosage of purified water is 800mL.
The preparation method of the sample diluent comprises the following steps: after the components are dissolved, the pH value is regulated to be 7.40+/-0.05 by using 1mol/L HCl or 1mol/LNaOH, the volume is fixed to 1000mL, and the storage life is 3 months at the temperature of 4-30 ℃.
After preparation, the sample diluent to be split charged is split charged into the diluent tube by a dispenser according to the specification of 0.3 mL/tube after checking the label and the batch number, and the bottle cap is closed and the label is attached to the diluent tube.
In some alternative implementations, the detection chip is prepared as follows:
and (3) respectively detecting the vitamin B12, folic acid and ferritin enterprise calibrator by using a detection card to obtain linear data of signal values and corresponding preparation concentrations, and writing the linear data, a fitting algorithm and ID information into a chip to obtain the detection chip.
The method comprises the following specific steps of:
(1) Preparing curve data: taking a vitamin B12 enterprise calibrator, a folic acid enterprise calibrator and a ferritin enterprise calibrator, and operating according to a reagent instruction; the mean of T/C values (ratio of detection line to quality control line) was tested on a fluorescence immunoassay management tool. The prepared concentration and the corresponding T/C mean value are respectively used as vitamin B12, folic acid and ferritin as linear data.
(2) Configuring chip data: on a fluorescence immunoassay experiment management tool, selecting curve generation, filling vitamin B12/folic acid/ferritin curve data prepared in the first step into a table, and selecting a fitting algorithm to perform MMF Model four-parameter growth curve fitting; and (5) finishing the preparation of the vitamin B12/folic acid/ferritin curve after the accurate fitting result is determined.
And selecting an ID file to be generated on a fluorescence immunoassay experiment management tool, sequentially setting the production year, the production month and the production lot in a basic parameter area, automatically generating a bar code at the bar code after setting, clicking the ID file to be generated after confirming no errors, and completing the chip data configuration.
(3) Firing a chip: and finding the latest HEX file corresponding to the bar code data in the Item file of the fluorescence immunoassay management tool. The chip data file is burned out 2-3 chips in a programmer, and after the fluorescent analyzer reads the chip data and the data and parameters are confirmed, the chips can be burned in a large scale.
And (5) calculating the required quantity according to the production batch, printing a certain number of chip labels, and labeling the chip labels.
And (3) filling the prepared detection card, the labeled sample diluent, the labeled detection chip and the instruction book into labeled finished product packaging boxes according to the specification number of the kit, and thus completing the kit assembly.
Based on the kit, the embodiment of the application also provides a using method of the three-item combined detection kit for anemia, which comprises the following steps:
(1) Sample collection, wherein the sample types include whole blood samples, plasma samples, and serum samples.
Specifically, the whole blood sample collection method comprises the following steps: collecting venous blood of a subject by using an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant, and shaking the collected blood sample for later use; the collection method of the plasma sample comprises the following steps: collecting venous blood of a tested person by using an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant, and separating plasma as soon as possible after blood collection so as to avoid hemolysis; the serum sample collection method comprises the following steps: venous blood is collected from a subject using a serum tube or a rapid serum tube containing a coagulant, and the serum should be separated as soon as possible after collection to avoid hemolysis.
The sample should be used as immediately as possible after collection.
(2) Chip reading: the instrument was opened, the same chip as the reagent lot number was inserted, and the sample type (serum/plasma/whole blood) was selected in the instrument.
(3) Sample testing: the detection card is horizontally placed on the desktop; 100 mu L of the sample is sucked by a pipette and added into one sample diluent to be uniformly mixed, and then 100 mu L of the uniformly mixed sample is added into a sample adding hole of a detection card. After reacting for 15min at room temperature, placing the detection card into an instrument card slot, selecting an 'instant test' mode, clicking a 'test', clicking a 'print' after the test is finished, and printing a detection result report.
The embodiment of the application also provides an analysis performance example of the three-item combined anemia detection kit.
Analytical performance one: assessment and analysis of the accuracy of the kit
(1) Performance requirements: the relative deviation of the detection results of the enterprise reference for detecting the vitamin B12 by the kit is not more than +/-15%; determining folic acid enterprise reference, wherein the relative deviation of the determination result is not more than +/-15%; the relative deviation of the measurement results of the ferritin enterprise reference is not more than +/-10%.
(2) The detection method comprises the following steps: and (3) measuring vitamin B12, folic acid or ferritin enterprise references, detecting according to the steps of the specification, recording test results (Xi) each time after repeated measurement for 3 times, respectively calculating relative deviation (Bi) according to the following formula, and judging to be qualified if the 3 results meet the accuracy performance requirement. If the result of more than or equal to 2 times is not consistent, the result is judged to be unqualified. If 1 test result is not in accordance with the requirement, the test should be repeated for 20 times, and the relative deviation (Bi) is calculated according to the following formula, if the test result is greater than or equal to 19 times, the accuracy meets the performance requirement.
Wherein, the concentration of the vitamin B12 enterprise reference substance is about 200pg/mL, 500pg/mL, the folic acid concentration is about 5ng/mL, 20ng/mL, the concentration of the ferritin enterprise reference substance is about 40ng/mL, 400ng/mL, and the specific concentration is based on the marking result of enterprise standardized record.
Wherein B is i In order for the relative deviation to be a function of,X i for each measurement of concentration, T represents the indicated concentration.
The results of the detection are shown in Table 1.
Table 1 results of the accuracy test of the kit
Analysis performance II: evaluation and test of the detection limit of the kit of the present application
(1) Performance requirements: the detection limit of vitamin B12 should not be higher than 145pg/mL; the detection limit of folic acid is not higher than 2.5ng/mL; the limit of detection of ferritin should be no higher than 5ng/mL.
(2) The inspection method comprises the following steps: and detecting 5 low-value samples with approximate detection limits of concentration, repeatedly detecting each sample for 5 times, and sequencing the detection results according to the size, wherein the detection results meet the following requirements, namely the blank Limit (LOB) and the detection Limit (LOD) are basically and reasonably set.
a) The number of detection results below the blank limit in the 25 detection steps is less than or equal to 3;
b) When applicable, all of the 25 detection values are not higher than the detection limit. Wherein the blank limit of vitamin B12 is 130pg/mL, the blank limit of folic acid is 2ng/mL, and the blank limit of ferritin is 4ng/mL.
The test results are shown in Table 2.
TABLE 2 detection results of detection limits of the kit
Analysis performance three: evaluation and examination of the Linear Performance of the kit of the application
(1) Performance requirements: vitamin B12 in the range of [150, 1000] pg/mL, the linear correlation coefficient (r) should be not lower than 0.9900; folic acid is in the range of [2.5, 50.0] ng/mL, and the linear correlation coefficient (r) should be not lower than 0.9900; ferritin should have a linear correlation coefficient (r) of not less than 0.9900 in the range of [10, 500] ng/mL.
(2) The inspection method comprises the following steps: and (3) operating the fluorescent immunity quantitative analyzer according to a reagent instruction, repeatedly detecting the enterprise linear reference sample of each concentration for 3 times, calculating the average value of the enterprise linear reference sample, performing linear fitting on the average value of the measured concentration and the theoretical concentration or dilution ratio by using a least square method, and calculating a linear correlation coefficient r according to the following formula, wherein the result meets the linear performance requirement. Wherein the vitamin B12 linear reference concentration is about 1000pg/mL, 700pg/mL, 500pg/mL, 300pg/mL, 150pg/mL; the folic acid linear reference concentration is about 50ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 2.5ng/mL; the concentration of ferritin linear reference was approximately 500ng/mL,250ng/mL,100ng/mL,40ng/mL,10ng/mL. The specific concentration is based on the marking result of enterprise standardized records.
Wherein n is the number of measurement samples; xi represents the measured sample concentration; yi represents the mean of measured values corresponding to the measured sample concentrations for 3 replicates; r represents a linear correlation coefficient.
The test results are shown in Table 3, and the linearity performance meets the requirements.
TABLE 3 test results of the linear correlation coefficient of the kit
Analysis performance four: testing the reproducibility of the kits of the application
(1) Performance requirements: the variation Coefficient (CV) of vitamin B12 in the batch is less than or equal to 10 percent; the variation Coefficient (CV) in folic acid batch is less than or equal to 15 percent; the Coefficient of Variation (CV) in ferritin batches is less than or equal to 15 percent.
(2) The inspection method comprises the following steps: the same batch of reagent is randomly extracted, vitamin B12 with low concentration and vitamin B with high concentration or ferritin enterprise reference are respectively detected, the detection is repeated 10 times, the average value M and the standard deviation SD of 10 measurement results are respectively calculated, the Coefficient of Variation (CV) is obtained according to the following formula, and the results all meet the requirement of repeatability. Wherein the vitamin B12 enterprise reference concentration is about 200pg/mL, 500pg/mL, folic acid concentration is about 5ng/mL, 20ng/mL, ferritin enterprise reference concentration is about 40ng/mL, 400ng/mL, and the specific concentration is based on the marking result of enterprise standardized record.
CV＝SD/M×100％
Wherein CV represents the coefficient of variation; SD represents the standard deviation of the 10 measurements; m represents the average of 10 measurements.
The test results are shown in Table 4, and the results show that the test results of the repeatability of the kit meet the requirements.
Table 4 results of the kit repeatability test
Analysis performance five: testing the batch to batch differences of the kits of the application
(1) Performance requirements: the inter-batch Coefficient of Variation (CV) of vitamin B12 should be less than or equal to 15%; the variation Coefficient (CV) between folic acid batches is less than or equal to 20 percent; the Coefficient of Variation (CV) between ferritin batches should be less than or equal to 15%.
(2) The inspection method comprises the following steps: three batches of reagents are randomly extracted, vitamin B12, folic acid or ferritin enterprise references with low and high concentrations are detected respectively, the steps are repeated 10 times respectively, the average value M and the standard deviation SD of 30 measurement results of each concentration sample are calculated respectively, the Coefficient of Variation (CV) is obtained according to the formula, and the result meets the performance requirement of the difference between batches. Wherein the vitamin B12 enterprise reference concentration is about 200pg/mL, 500pg/mL, folic acid concentration is about 5ng/mL, 20ng/mL, ferritin enterprise reference concentration is about 40ng/mL, 400ng/mL, and the specific concentration is based on the marking result of enterprise standardized record.
The test results are shown in tables 5 to 7, and the results show that the batch-to-batch difference test results of the kit are satisfactory.
TABLE 5 results of the vitamin B12 assay for inter-batch differences
TABLE 6 results of the folic acid detection inter-batch difference test
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TABLE 7 results of the batch-to-batch differential assay for ferritin
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It is apparent that the above-described embodiments are only some embodiments of the present application, but not all embodiments, and the preferred embodiments of the present application are shown in the drawings, which do not limit the scope of the patent claims. This application may be embodied in many different forms, but rather, embodiments are provided in order to provide a thorough and complete understanding of the present disclosure. Although the application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing description, or equivalents may be substituted for elements thereof. All equivalent structures made by the content of the specification and the drawings of the application are directly or indirectly applied to other related technical fields, and are also within the scope of the application.
Claims (10)
1. The preparation method of the three-item combined detection card for anemia is characterized by comprising the following steps of:
preparing a marking buffer solution, a marking activating solution, a marking preserving solution, a coating diluting solution and a sample pad treatment solution in advance;
immersing glass fibers into a sample pad treatment liquid for treatment, and drying the immersed glass fibers to obtain a sample pad;
respectively marking the vitamin B12 marked antibody, the folic acid marked antibody, the ferritin marked antibody and the goat anti-chicken IgY marked antibody by using fluorescent microspheres to respectively form a fluorescent microsphere vitamin B12 antibody complex, a fluorescent microsphere folic acid antibody complex, a fluorescent microsphere ferritin antibody complex and a fluorescent microsphere goat anti-chicken IgY antibody complex;
mixing the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex to prepare a fluorescent microsphere marker;
spraying fluorescent microsphere markers on glass fibers to prepare a bonding pad;
respectively diluting the vitamin B12 coupled BSA antigen, the folic acid coupled BSA antigen, the ferritin monoclonal antibody and the chicken IgY antibody by using the coating diluent to prepare a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid;
attaching a nitrocellulose membrane to a bottom plate, and respectively spraying a first detection line coating liquid, a second detection line coating liquid, a third detection line coating liquid and a quality control line coating liquid onto the nitrocellulose membrane;
sequentially overlapping the sample pad, the combination pad and the water absorption pad on a bottom plate, assembling to obtain a reaction plate, and manufacturing the reaction plate into a test strip;
and installing the test strip in a detection card shell to obtain the detection card.
2. The method for preparing the three-item combined detection card for anemia according to claim 1, wherein the steps of labeling the anti-vitamin B12 murine monoclonal IgG, the anti-folic acid murine monoclonal IgG, the anti-ferritin murine monoclonal IgG, the goat anti-chicken IgY goat antibody with fluorescent microspheres to form a fluorescent microsphere vitamin B12 antibody complex, a fluorescent microsphere folic acid antibody complex, a fluorescent microsphere ferritin antibody complex, and a fluorescent microsphere goat anti-chicken IgY antibody complex, respectively, comprise:
taking four clean centrifuge tubes with the numbers of 1, 2, 3 and 4, and adding a marking buffer solution into each centrifuge tube;
vortex mixing fluorescent microsphere uniformly, taking fluorescent microsphere with solid content of 1% in a marking buffer solution, and vortex mixing uniformly again;
adding the marked activating solution into the centrifuge tube, and uniformly mixing;
after the uniform mixing is finished, placing the centrifuge tube on a rotary mixing instrument for rotary reaction;
after the reaction is completed, centrifuging the centrifuge tube, and reserving a precipitate;
adding a marking buffer solution into a centrifuge tube, and performing ultrasonic scattering;
adding a vitamin B12 labeled antibody, a folic acid labeled antibody, a ferritin labeled antibody and a goat anti-chicken IgY labeled antibody into centrifugal tubes with the numbers of 1, 2, 3 and 4 respectively, uniformly mixing, and performing a rotation reaction;
after the rotary reaction, taking the marked sealing liquid into the centrifuge tube, performing ultrasonic scattering, performing the rotary reaction, and then centrifuging the centrifuge tube to leave a precipitate;
and (3) respectively adding the marked preservation solution into centrifuge tubes with the numbers of 1, 2, 3 and 4, and scattering by using ultrasound to prepare the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere goat anti-chicken IgY antibody complex.
3. The method for preparing the three-item combined detection card for anemia according to claim 1, wherein the mixing volume ratio of the fluorescent microsphere vitamin B12 antibody complex, the fluorescent microsphere folic acid antibody complex, the fluorescent microsphere ferritin antibody complex and the fluorescent microsphere sheep anti-chicken IgY antibody complex in the fluorescent microsphere marker is 1:1:1:1.
4. The method for preparing the three-item combined anemia detection card according to claim 1, wherein the concentration of the first detection line coating liquid, the concentration of the second detection line coating liquid and the concentration of the third detection line coating liquid are all 0.5mg/mL, and the concentration of the quality control line coating liquid is 1.0mg/mL.
5. The method for preparing the three-item combined anemia detection card according to claim 1, wherein the distance between the first detection line and the upper edge of the nitrocellulose membrane, the distance between the first detection line and the second detection line, the distance between the second detection line and the third detection line, the distance between the third detection line and the quality control line and the distance between the quality control line and the lower edge of the nitrocellulose membrane are all 4.5-5.5 mm.
6. The method for preparing the three-item combined anemia detection card according to claim 1, wherein the step of sequentially overlapping the sample pad, the binding pad and the water absorbing pad on the bottom plate, and assembling the reaction plate comprises the steps of:
cutting the water absorption pad into a preset size, spreading the bottom plate on a workbench surface, uncovering a protective film at the pasting position of the water absorption pad on the bottom plate, and adhering the water absorption pad on the bottom plate, wherein one end of the water absorption pad is aligned with the top end of the bottom plate, and the other side of the water absorption pad is partially covered on the nitrocellulose film;
the protective film at the bonding pad pasting part on the bottom plate is uncovered, the bonding pad is adhered to the upper end of the protective film, the bonding pad faces upwards, and one end of the bonding pad is covered on the nitrocellulose film;
and uncovering the protective film at the adhering position of the sample pad on the bottom plate, adhering the sample pad on the protective film, aligning the lower end of the sample pad with the adhering position of the sample pad on the bottom plate, and covering the upper end of the sample pad on the combining pad to prepare the reaction plate.
7. The method for preparing the three-item combined detection card for anemia of claim 1, wherein the spraying amount of the fluorescent microsphere marker is 4.0 mu L/cm; the film dividing amounts of the first detection line coating liquid, the second detection line coating liquid, the third detection line coating liquid and the quality control line coating liquid are all 1 mu L/cm.
8. An anemia three-joint detection kit, which is characterized by comprising a sample diluent, a detection chip and a detection card prepared by the anemia three-joint detection card preparation method according to any one of claims 1 to 7.
9. The three-item combined anemia test kit of claim 8, wherein the test chip is prepared by the following method:
and (3) respectively detecting the vitamin B12, folic acid and ferritin enterprise calibrator by using a detection card to obtain linear data of signal values and corresponding preparation concentrations, and writing the linear data, a fitting algorithm and ID information into a chip to obtain the detection chip.
10. The three-item combined anemia test kit of claim 8, wherein the sample diluent comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, proclin300, and purified water.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117214445A (en) * | 2023-11-09 | 2023-12-12 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection reagent for joint inspection of folic acid, vitamin B12 and ferritin and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117214445A (en) * | 2023-11-09 | 2023-12-12 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection reagent for joint inspection of folic acid, vitamin B12 and ferritin and application thereof |
| CN117214445B (en) * | 2023-11-09 | 2024-01-26 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection reagent for joint inspection of folic acid, vitamin B12 and ferritin and application thereof |
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