CN108956982B - Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof - Google Patents
Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof Download PDFInfo
- Publication number
- CN108956982B CN108956982B CN201810746013.1A CN201810746013A CN108956982B CN 108956982 B CN108956982 B CN 108956982B CN 201810746013 A CN201810746013 A CN 201810746013A CN 108956982 B CN108956982 B CN 108956982B
- Authority
- CN
- China
- Prior art keywords
- microspheres
- rheumatoid arthritis
- reaction
- test paper
- arthritis marker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 206010039073 rheumatoid arthritis Diseases 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 51
- 238000012360 testing method Methods 0.000 title claims abstract description 42
- 239000003550 marker Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims description 13
- 239000004005 microsphere Substances 0.000 claims abstract description 56
- 108010074051 C-Reactive Protein Proteins 0.000 claims abstract description 28
- 102100032752 C-reactive protein Human genes 0.000 claims abstract description 28
- 229920001184 polypeptide Polymers 0.000 claims abstract description 23
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 23
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 21
- 150000002910 rare earth metals Chemical class 0.000 claims abstract description 21
- 239000011248 coating agent Substances 0.000 claims abstract description 17
- 238000000576 coating method Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000003908 quality control method Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 17
- 239000004793 Polystyrene Substances 0.000 claims description 12
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 229920002223 polystyrene Polymers 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 239000007993 MOPS buffer Substances 0.000 claims description 8
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 239000002244 precipitate Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 239000003365 glass fiber Substances 0.000 claims description 7
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 7
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- 238000002604 ultrasonography Methods 0.000 claims description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 6
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 claims description 5
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 229960002173 citrulline Drugs 0.000 claims description 5
- 235000013477 citrulline Nutrition 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 229920001220 nitrocellulos Polymers 0.000 claims description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 4
- 230000002745 absorbent Effects 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims 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 claims description 3
- 239000000020 Nitrocellulose Substances 0.000 claims description 3
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 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
- 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 3
- 230000004913 activation Effects 0.000 claims 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 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000010030 laminating Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 3
- 239000011265 semifinished product Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000004328 sodium tetraborate Substances 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 3
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 claims description 2
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004342 Benzoyl peroxide Substances 0.000 claims description 2
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- 229910016644 EuCl3 Inorganic materials 0.000 claims description 2
- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 241001363516 Plusia festucae Species 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 229910052786 argon Inorganic materials 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 2
- 230000021523 carboxylation Effects 0.000 claims description 2
- 238000006473 carboxylation reaction Methods 0.000 claims description 2
- 239000006185 dispersion Substances 0.000 claims description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- 239000006249 magnetic particle Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 239000003761 preservation solution Substances 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 11
- 210000004369 blood Anatomy 0.000 abstract description 10
- 239000008280 blood Substances 0.000 abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 8
- 238000010521 absorption reaction Methods 0.000 abstract description 6
- 238000004587 chromatography analysis Methods 0.000 abstract description 4
- 238000003317 immunochromatography Methods 0.000 abstract description 4
- 238000012123 point-of-care testing Methods 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 238000003745 diagnosis Methods 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000004393 prognosis Methods 0.000 description 6
- 238000003759 clinical diagnosis Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000003628 erosive effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 208000025747 Rheumatic disease Diseases 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- -1 amino, carboxyl Chemical group 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010023203 Joint destruction Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 208000018631 connective tissue disease Diseases 0.000 description 1
- RKHQGWMMUURILY-UHRZLXHJSA-N cortivazol Chemical compound C([C@H]1[C@@H]2C[C@H]([C@]([C@@]2(C)C[C@H](O)[C@@H]1[C@@]1(C)C2)(O)C(=O)COC(C)=O)C)=C(C)C1=CC1=C2C=NN1C1=CC=CC=C1 RKHQGWMMUURILY-UHRZLXHJSA-N 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 208000018937 joint inflammation Diseases 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000003571 opsonizing effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/10—Magnetic particle immunoreagent carriers the magnetic material being used to coat a pre-existing polymer particle but not being present in the particle core
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
A rheumatoid arthritis marker combined quantitative detection test paper is provided, wherein the rheumatoid arthritis marker is a rheumatoid factor, an anti-cyclic citrullinated polypeptide antibody and a C-reactive protein, the test paper comprises a sample pad, a rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere label pad, a coating film and a water absorption pad, three rheumatoid arthritis marker quantitative detection lines and a quality control area C line are arranged on the coating film, a magnetic field is used for concentrating a detected substance in a sample, and then a low-background high-sensitivity fluorescence immunochromatography method is used for detecting the detected substance, so that the problem of low sensitivity of POCT products is solved. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to a rheumatoid arthritis marker combined quantitative detection test paper based on a magnetic rare earth fluorescent microsphere as a tracer marker and a preparation method thereof, in particular to a detection reagent card for determining the content of rheumatoid factors/anti-cyclic citrullinated polypeptide antibodies/C-reactive protein in blood (serum, plasma and whole blood).
Background
Rheumatoid Arthritis (RA) is a multi-system inflammatory autoimmune disease mainly involving peripheral joints, the specific cause of the disease is not clear and may be related to two factors, namely an infection factor and a genetic tendency, the RA is a rheumatic disease characterized by the degeneration of joint inflammation, joint destruction and progressive dysfunction, the prevalence rate of China is about 0.3-0.6%, and the RA is one of the main causes of the loss of labor and disability of people in China. Early stage of the disease is often misdiagnosed due to symptoms or failure to diagnose timely, and delayed treatment makes the prognosis of the disease poor, and if the disease can be diagnosed early and reasonable treatment can be given timely, the prognosis can be improved obviously. Clinical diagnosis of RA Rheumatoid Factor (RF) is a commonly used classic serological indicator, but lacks specificity, and is found in other diseases such as connective tissue disease, hepatitis, tumor, infectious disease, and normal healthy people, in addition to patients. Although RF is not specific for RA, high titers of RF are closely associated with RA disease. In particular, the higher the RF titer, the worse the prognosis of the patient, the most powerful prognostic factor in the lesion index, and other autoantibodies cannot replace the status in diagnosis and prognosis judgment. Therefore, RF has the advantages of early appearance, high sensitivity and the like in RA diseases, and is still one of the main bases of clinical early diagnosis.
The anti-Cyclic Citrullinated Polypeptide (CCP) appears in the early stage of rheumatoid arthritis and has close correlation with the joint erosion condition and prognosis of RA patients, the sensitivity of the anti-CCP to the rheumatoid arthritis is lower than that of rheumatoid factors, but the diagnosis specificity is close to 95 percent and is obviously higher than other common indexes of clinical diagnosis such as the rheumatoid factors, the anti-CCP of the RA patients is detected by an ELISA method, the anti-CCP has high specificity and sensitivity to the RA diagnosis, and the anti-CCP is gradually applied to the clinical diagnosis of RA at home. The anti-CCP antibody is a good early diagnosis marker of RA and is also a sensitive index for identifying non-erosive and erosive RA.
C-reactive protein (CRP) is a protein (acute protein) which rises sharply in plasma when a body is infected or tissues are damaged, activates complement and strengthens phagocytosis of phagocytes to play an opsonizing role, and eliminates pathogenic microorganisms invading the body and damaged, necrotic and apoptotic tissue cells. CRP rises several hours after the onset of inflammation and peaks at 48 hours, with regression of the lesion and reduction of tissue, structure and function to normal levels. The reaction is not affected by radiotherapy, chemotherapy and corticoid. Therefore, the detection of CRP is quite widely used in clinical applications, including diagnosis and differential diagnosis of acute infectious diseases, monitoring of postoperative infections; observation of antibiotic efficacy; disease course detection, prognosis judgment and the like.
When the three indexes of RF, anti-CCP antibody and CRP are jointly used for diagnosing RA, the sensitivity is obviously improved, and the missed diagnosis rate is greatly reduced compared with other single indexes; meanwhile, the negative predictive value is also improved, if the indexes of RF, anti-CCP antibody and CRP are all negative, the possibility of suffering from RA can be almost eliminated, and the method has quite high clinical application value.
At present, the most widely used clinical diagnosis is to adopt imported reagents to detect the contents of rheumatoid factors/anti-cyclic citrullinated polypeptide antibodies/C-reactive proteins in serum and plasma. The existing kit on the market is complex in detection operation, time-consuming, not suitable for common screening and only can be used for a definite diagnosis test. Meanwhile, the sensitivity of the sample on the market is low because the sample is diluted and then tested.
Disclosure of Invention
In order to solve the problems and conveniently test the human serum rheumatoid factor/anti-cyclic citrullinated polypeptide antibody/C-reactive protein, the invention provides the rheumatoid arthritis marker combined quantitative test paper which is simple, reliable, low in cost and high in sensitivity; the detection method utilizes a magnetic field to concentrate the detected substance in the sample, and utilizes a low-background and high-sensitivity fluorescence immunochromatography to detect the detected substance, thereby solving the problem of low sensitivity of POCT products. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
In order to achieve the purpose of the invention, the technical scheme provided by the invention is as follows: the test paper for joint quantitative detection of the rheumatoid arthritis markers comprises a box body, wherein the box body comprises a bottom plate and a box cover, the detection test paper is arranged in the box body, the bottom plate is arranged below the detection test paper, and the upper part of the detection test paper is connected with the box cover; the box cover is provided with a through hole area; the through hole area is provided with a sample adding hole and a result detection window; a detection area is arranged above the detection test paper and corresponds to the through hole area; the detection area comprises a sample pad, a rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere labeling pad, a coating film and a water absorption pad, wherein three rheumatoid arthritis marker combined quantitative detection lines and a quality control area C line are arranged on the coating film, and the detection lines and the quality control area C line are arranged in parallel.
Further, the rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere is formed by wrapping a magnetic rare earth fluorescent compound by a high polymer material, the sphere diameter of the magnetic rare earth fluorescent microsphere is 10-500nm, the fluorescence emission wavelength range is 400-800nm, and the surface modification active group of the nano microsphere is amino, carboxyl or sulfydryl.
Furthermore, two ends of the coating film are respectively connected with the water absorption pad and the rheumatoid arthritis marker antibody marking pad in an overlapping manner, and the coating film is a nitrocellulose film; a sample pad is pressed and stuck on the rheumatoid arthritis marker antibody marking pad, and the sample pad is a glass fiber sample pad.
Further, the rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere label pad is glass fiber or polyester fiber.
A preparation method of a rheumatoid arthritis marker combined quantitative detection test paper comprises the steps of marking a rheumatoid arthritis marker antibody and preparing the detection test paper, wherein the rheumatoid arthritis marker antibody marking step comprises the following steps:
firstly, coating a magnetic rare earth fluorescent microsphere prepared by Eu3+ -Fe3O4 compound by using a carboxylated polystyrene microsphere, wherein the particle size is 200nm, and the solid content is 1%;
adding 1mL of the microspheres into 5mL of pH 7.00.02M 3- (N-Malinio) propanesulfonic acid buffer solution (MOPS), uniformly mixing, centrifuging at 25000rpm for 10min, and discarding the supernatant; then adding 5mL of MOPS, performing 90W ultrasound, working for 2s, pausing for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and removing the supernatant;
thirdly, adding 5mL of MOPS again, performing 90W ultrasonic treatment, working for 2s, performing intermittent operation for 5s, and repeating for 2 times to complete the cleaning of the microspheres;
adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
fifthly, after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
sixthly, respectively adding 1mg of each of the rheumatoid factor, the anti-cyclic citrullinated polypeptide antibody and the C-reactive protein corresponding antibody, placing the mixture on a rotary incubator, and reacting for 90-120 min at 37 ℃;
seventhly, adding 100 mu L of 5% BSA and 100 mu L of 1% PEG20000, sealing unmarked sites, placing on a rotary incubator, and reacting at 37 ℃ for 60 min;
after reaction is finished, the liquid is centrifuged at 2000rpm for 15min, the supernatant is discarded, and a preservation solution (pH 8.00.2M boric acid borax buffer solution containing 1% BSA, 10% sucrose and 2% trehalose) is added into the supernatant for 5mL and is subjected to ultrasound for 2 s;
ninthly, diluting the marked microspheres by 50-100 times, subpackaging each microsphere by 200 mu L to obtain a single-person semi-finished product, and matching with rheumatoid factors, anti-cyclic citrullinated polypeptide antibodies and C-reactive protein test paper for use.
The preparation method of the rheumatoid arthritis marker combined quantitative detection test paper comprises the following steps:
coating three detection lines of rheumatoid factors, anti-cyclic citrulline polypeptide antibodies and antibodies corresponding to C-reactive protein and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold spot membrane spraying instrument according to the parameter of 1 mu L/cm, and drying for 16 hours at 37 ℃ to serve as a reaction membrane.
② soaking the glass fiber by 0.1M PBS solution containing 0.5 percent Tween-20, then drying for 16 hours at 37 ℃, and cutting 17mm multiplied by 300mm to be used as a sample pad.
Thirdly, the sample pad, the reaction film and the absorbent paper (22 mm multiplied by 300 mm) are adhered and assembled on the bottom plate in a mode of laminating layer by layer.
Compared with the prior art, the invention has the following beneficial effects: the detection area comprises a sample pad, a rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere labeling pad, a coating film and a water absorption pad, three rheumatoid arthritis marker combined quantitative detection lines and a quality control area C line are arranged on the coating film, the detected substance in a sample is concentrated by a magnetic field, and then the detected substance is detected by a low-background and high-sensitivity fluorescence immunochromatography, so that the problem of low sensitivity of POCT products is solved. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
Drawings
FIG. 1 is a test curve for rheumatoid factor of the present invention;
FIG. 2 is a test curve for an anti-cyclic citrullinated polypeptide antibody of the present invention;
FIG. 3 is a test curve for C-reactive protein of the present invention;
FIG. 4 shows the results of the precision test of the method for measuring rheumatic factor of the present invention;
FIG. 5 shows the results of the precision test of the determination method of the anti-cyclic citrullinated polypeptide antibody of the present invention;
FIG. 6 shows the results of the precision test of the method for measuring C-reactive protein according to the present invention;
FIG. 7 is a schematic structural view of the present invention;
in the figure: 1. a base plate; 2. a sample pad; 3. a marker antibody magnetic rare earth fluorescent microsphere label pad for rheumatoid arthritis; 4. a coating film; 5. a water absorbent pad; 6. a box body; 61. a box cover; 71. a sample application hole; 72. a result observation window; 8. a combined quantitative detection line for rheumatoid arthritis markers; 9. and (4) a quality control area C line.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Unless the context clearly indicates otherwise, the names and parameters of the detection substances in the present invention may be present in a single form or in a plurality of forms, and the present invention is not limited thereto. Although the steps in the present invention are arranged by using reference numbers, the order of the steps is not limited, and the relative order of the steps can be adjusted unless the order of the steps is explicitly stated or other steps are required for the execution of a certain step. It is to be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The test paper for joint quantitative detection of the rheumatoid arthritis markers comprises a box body, wherein the box body comprises a bottom plate and a box cover, the detection test paper is arranged in the box body, the bottom plate is arranged below the detection test paper, and the upper part of the detection test paper is connected with the box cover; the box cover is provided with a through hole area; the through hole area is provided with a sample adding hole and a result detection window; a detection area is arranged above the detection test paper and corresponds to the through hole area; the detection area comprises a sample pad, a rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere labeling pad, a coating film and a water absorption pad, wherein three rheumatoid arthritis marker combined quantitative detection lines and a quality control area C line are arranged on the coating film, and the detection lines and the quality control area C line are arranged in parallel. The rheumatoid arthritis marker antibody magnetic rare earth fluorescent microsphere is formed by wrapping a magnetic rare earth fluorescent compound by a high polymer material, the sphere diameter of the magnetic rare earth fluorescent microsphere is 10-500nm, the fluorescence emission wavelength range is 400-800nm, and the surface modification active group of the nano microsphere is amino, carboxyl or sulfydryl. The two ends of the coating film are respectively connected with the water absorption pad and the rheumatoid arthritis marker antibody marking pad in an overlapping manner, and the coating film is a nitrocellulose film; a sample pad is pressed and stuck on the rheumatoid arthritis marker antibody marking pad, and the sample pad is a glass fiber sample pad. The marker antibody magnetic rare earth fluorescent microsphere marker pad for rheumatoid arthritis is glass fiber or polyester fiber.
Example 1, establishment of detection method:
1.1 preparation of polystyrene microsphere seeds
1.1.1 weighing 100ml of styrene and a separating funnel, repeatedly washing with 1M NaOH for 3 times to remove impurities in the styrene, then washing with purified water to be neutral, and then placing in a drying dish for drying for more than 24 hours;
1.1.2 filling argon into a reaction device, and removing oxygen in the device;
1.1.3 adding 100ml of ethanol into a reaction device, then adding 5g of PVP, and stirring for 30 min;
1.1.4 adding 40ml of washed styrene and 5ml of Azobisisobutyronitrile (AIBN), heating to 45 ℃ in a water bath, reacting for 30min, then heating to 75 ℃ and reacting for 12 h;
1.1.5 after the reaction, filtering the reaction solution by using a mesh screen, washing and centrifuging for 3-5 times by using absolute ethyl alcohol, then putting the polymer subjected to preliminary impurity removal into 40% ethyl alcohol to naturally precipitate, and weighing the precipitate weight.
1.2 carboxylation modification of polystyrene surface
1.2.1 dispersing the polystyrene microspheres obtained by the above dispersion as seeds 1g with 10ml of 0.25% SDS;
1.2.2 adding cyclohexane of which the weight is 2 times that of the seeds into the system, and swelling for 16-18 h at 35 ℃;
1.2.3 weighing 0.02g of benzoyl peroxide, 10 mu l of ethylene glycol monomethyl ether and 10 mu l of ethylene glycol dimethacrylate, respectively adding the weighed materials into the reaction solution, and continuing swelling for 12 hours;
1.2.4, after swelling, adding 2.5mg of PVA, raising the reaction temperature to 75 ℃, and continuing to react for 10-12 hours;
1.2.5 repeated washing with ethanol 3 times to remove unreacted monomers and oligomers, followed by centrifugation at 20000rpm to settle the microspheres.
1.3 preparation of magnetic-fluorescent nanospheres
1.3.1 FeCl2、FeCl3、EuCl3NaOH solution, in molar proportion Fe2+：Fe3+：Eu3+：Na+=1:1:2:5, the solution is mixed and stirred for reaction to obtain a precipitate, the precipitate is aged for 3-5 h and then filtered, the filtrate is washed with purified water for 2-3 times, and the Eu is obtained after drying and grinding3+-Fe3O4And (c) a complex.
1.3.2 adding 0.1g of carboxyl modified polystyrene microspheres into 20ml of isopropanol, and performing ultrasonic dispersion for 10 min;
1.3.3 weighing Eu3+-Fe3O4Adding 0.5mg of the compound into 5ml of chloroform, and performing ultrasonic dispersion for 20 min;
1.3.4 mixing the two liquids of 1.3.2 and 1.3.3, placing the mixture in a vacuum environment for 5 to 6 hours, and then placing the mixture in normal pressure for 2 to 3 hours;
1.3.5 washing the swelled microspheres with ethanol for 3 times, removing the microspheres which are not successfully wrapped with the magnetic particles by using a magnetic field, resuspending the microspheres with a proper amount of aqueous solution containing 0.05 percent of sodium azide to obtain the magnetic rare earth fluorescent microspheres, storing the magnetic rare earth fluorescent microspheres at the temperature of 2-8 ℃, and strictly freezing the microspheres.
1.4 labeling of antibodies
1.4.1 taking 1ml of the prepared microspheres with the particle size of 200nm and the solid content of 1%, adding 5ml of 7.00.02M 3- (N-Malinio) propanesulfonic acid buffer solution (MOPS), uniformly mixing, centrifuging at 25000rpm for 10min, and discarding the supernatant;
1.4.2 adding 5ml MOPS, performing 90W ultrasound for 2s, intermittently repeating for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and discarding the supernatant;
1.4.3, adding 5ml of MOPS again, performing 90W ultrasound, working for 2s, intermittently performing for 5s, repeating for 2 times, and finishing the cleaning of the microspheres;
1.4.4 adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
1.4.5 after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
1.4.6 adding 1mg of rheumatoid factor, 1mg of anti-cyclic citrullinated polypeptide antibody and 1mg of corresponding antibody of C-reactive protein respectively, placing on a rotary incubator, and reacting for 90-120 min at 37 ℃;
1.4.7 then 100. mu.l of 5% BSA and 100. mu.l of 1% PEG20000 were added, the unlabelled sites were blocked, placed on a rotary incubator and reacted for 60min at 37 ℃;
1.4.8 after the reaction, centrifuging the above liquid at 2000rpm for 15min, discarding the supernatant, adding preserving solution (pH 8.00.2M borax borate buffer solution containing 1% BSA, 10% sucrose, 2% trehalose), 5ml, and performing ultrasound for 2 s;
1.4.9 diluting the marked microspheres by 50-100 times, and subpackaging each microsphere by 200 μ l to obtain a single semi-finished product, which is used in combination with test paper for determination of rheumatoid factor/anti-cyclic citrullinated polypeptide antibody/C-reactive protein.
1.5 preparation of test paper
1.5.1 coating a rheumatoid factor antibody, an anti-cyclic citrullinated polypeptide antibody, three detection lines of antibodies corresponding to C-reactive protein and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold-spraying membrane instrument according to the parameter of 1 mul/cm, and drying for 16 hours at 37 ℃ to serve as a reaction membrane.
1.5.2 soaking glass fiber with 0.1M PBS solution containing 0.5% Tween-20, then drying for 16 hours at 37 ℃, cutting 17mm × 300mm as sample pad.
1.5.3 sticking and assembling the sample pad, the reaction membrane and the absorbent paper (22 mm multiplied by 300 mm) on a PVC bottom plate in a mode of laminating layer by layer, cutting to obtain the test paper for determining the rheumatoid factor/anti-cyclic citrulline polypeptide antibody/C-reactive protein, and placing the test paper into a plastic card box to obtain the reagent card for determining the rheumatoid factor/anti-cyclic citrulline polypeptide antibody/C-reactive protein.
Example 2 detection of rheumatoid factor, anti-cyclic citrullinated polypeptide antibody, C-reactive protein
2.1 Linear Range
2.1.1, respectively diluting the high-value samples close to the upper limit of the linear range to at least 5 concentrations according to a certain proportion, wherein the low-value samples are required to be close to the lower limit of the linear range;
2.1.2 taking 100 mul of each concentration sample, respectively adding the samples into the packaged marked microspheres, and uniformly mixing by vortex;
2.1.3 placing the above reaction solution on a magnetic field, after 1min, discarding the supernatant, and then adding 200. mu.l PBS (0.02M, pH7.4) to each tube;
2.1.4 removing the magnetic field, uniformly mixing by vortexing again, respectively taking 100 mu l of each concentration sample, adding the samples into test paper for measuring the rheumatoid factor/anti-cyclic citrullinated polypeptide antibody/C-reactive protein, and repeatedly testing for 3 times at each concentration;
2.1.5 after reacting for 10min, putting the test paper into a dry immunofluorescence analyzer, reading the T/C value, calculating the T/C mean value of each concentration, fitting a concentration-reaction curve (the X axis is lg (concentration); the Y axis is lg (T/C)) by using a double-logarithmic straight line, wherein the rheumatoid factor reaction curve is Y = 1.0581X-1.1134, and R =0.9964 as shown in the figure; the anti-cyclic citrullinated polypeptide antibody response curve is y = 1.3833 x-2.4438, R = 0.9905; the C-reactive protein response curve was y = 0.5475x-0.1391, R = 0.9907.
2.2 minimum detection Limit
The negative serum is operated according to 2.1.2-2.1.4, repeated testing is carried out for 20 times, the T/C mean value is calculated and is substituted into a 2.1.5 test curve to obtain the lowest detection limit of the method, wherein the lowest detection limit of the rheumatoid factor is 3 IU/mL; the lowest detection limit of the anti-cyclic citrulline polypeptide antibody is 7U/mL; the minimum detection limit of C-reactive protein is 0.2 mg/L.
2.3 precision
Diluting the calibrator to high, medium and low concentrations, operating according to 2.1.2-2.1.4, and repeating the test for 10 times per concentration. The results are shown in fig. 1-3, and it can be seen that the coefficient of variation measured by the method is less than 10%, and thus the method has high precision.
2.4 comparison with other products
The method tests 92 clinical sera, and the results of rheumatoid factors are as follows: y = 0.9631x + 0.5134 with a correlation coefficient of 0.975, the anti-cyclic citrullinated polypeptide antibody results: y = 0.9426x + 0.7012 with a correlation coefficient of 0.984, C-reactive protein results: y = 0.9755x + 0.6312 with a correlation coefficient of 0.967, indicating good agreement of the reagent with the detection method already on the market.
Comprehensively, the method can detect the rheumatoid factor, the anti-cyclic citrullinated polypeptide antibody and the C-reactive protein, has better precision and has the coefficient of variation less than 10 percent. The product is clinically tested and has good correlation with the marketed product. The invention uses the magnetic field to concentrate the detected substance in the sample, and uses the fluorescence immunochromatography with low background and high sensitivity to detect the detected substance, thereby solving the problem of low sensitivity of POCT products. The problem of low sensitivity caused by testing the sample after diluting the sample in order to reduce the interference of other substances in blood when the immune lateral chromatography reagent detects the blood sample can be avoided.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (1)
1. A preparation method of a rheumatoid arthritis marker combined quantitative detection test paper is characterized by comprising a rheumatoid arthritis marker antibody labeling step and a detection test paper preparation step, wherein the rheumatoid arthritis marker antibody labeling step comprises the following steps:
firstly, wrapping Eu by carboxylated polystyrene microspheres3+-Fe3O4The magnetic rare earth fluorescent microsphere prepared from the compound has the particle size of 200nm and the solid content of 1 percent;
adding 5mL of 0.02M3- (N-Malinio) propanesulfonic acid buffer solution with the pH value of 7.0 into 1mL of the microspheres, uniformly mixing, centrifuging at 25000rpm for 10min, and removing supernatant; then adding 5mL of MOPS, performing 90W ultrasound, working for 2s, pausing for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and discarding the supernatant;
thirdly, adding 5mL of MOPS again, performing 90W ultrasonic treatment, working for 2s, performing intermittent operation for 5s, and repeating for 2 times to complete the cleaning of the microspheres;
adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
fifthly, after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
sixthly, respectively adding 1mg of each of the rheumatoid factor, the anti-cyclic citrullinated polypeptide antibody and the C-reactive protein corresponding antibody, placing the mixture on a rotary incubator, and reacting for 90-120 min at 37 ℃;
seventhly, adding 100 mu L of 5% BSA and 100 mu L of 1% PEG20000, sealing the unmarked sites, placing on a rotary incubator, and reacting at 37 ℃ for 60 min;
after the reaction is finished, centrifuging at 2000rpm for 15min, removing supernatant, and adding a preservation solution; 5mL of borax borate buffer solution (pH8.00.2M) containing 1% BSA, 10% sucrose and 2% trehalose, and performing ultrasonic treatment for 2 s;
ninthly, diluting the marked microspheres by 50-100 times, and subpackaging 200 mu L of each microsphere to obtain a single-part semi-finished product;
the preparation method of the rheumatoid arthritis marker combined quantitative detection test paper comprises the following steps:
coating three detection lines of rheumatoid factors, anti-cyclic citrulline polypeptide antibodies and antibodies corresponding to C-reactive protein and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold spot membrane spraying instrument according to the parameter of 1 mu L/cm, and drying for 16 hours at 37 ℃ to serve as a reaction membrane;
soaking glass fiber in 0.1M PBS solution containing 0.5% Tween-20, drying at 37 deg.c for 16 hr, and cutting into 17mm × 300mm sample pad;
thirdly, sticking and assembling the sample pad, the reaction film and the absorbent paper with the thickness of 22mm multiplied by 300mm on a bottom plate in a mode of laminating layer by layer;
the preparation of the carboxylated polystyrene microspheres in the marking step of the rheumatoid arthritis marker antibody is as follows:
1.1 preparation of polystyrene microsphere seeds
1.1.1 weighing 100ml of styrene and a separating funnel, repeatedly washing with 1M NaOH for 3 times to remove impurities in the styrene, then washing with purified water to be neutral, and then placing in a drying dish for drying for more than 24 hours;
1.1.2 filling argon into a reaction device, and removing oxygen in the device;
1.1.3 adding 100ml of ethanol into a reaction device, then adding 5g of PVP, and stirring for 30 min;
1.1.4 adding 40ml of washed styrene and 5ml of azobisisobutyronitrile, heating to 45 ℃ in a water bath, reacting for 30min, then heating to 75 ℃ and reacting for 12 h;
1.1.5 after the reaction is finished, filtering the reaction solution by using a mesh screen, washing and centrifuging for 3-5 times by using absolute ethyl alcohol, then putting the polymer subjected to preliminary impurity removal into 40% ethyl alcohol to naturally precipitate, and weighing the precipitate weight;
1.2 carboxylation modification of polystyrene surface
1.2.1 the polystyrene microspheres obtained by dispersion were used as seeds 1g, which were dispersed with 10ml of 0.25% SDS;
1.2.2 adding cyclohexane of which the weight is 2 times that of the seeds into the system, and swelling for 16-18 h at 35 ℃;
1.2.3 weighing 0.02g of benzoyl peroxide, 10 mu l of ethylene glycol monomethyl ether and 10 mu l of ethylene glycol dimethacrylate, respectively adding the weighed materials into the reaction solution, and continuing swelling for 12 hours;
1.2.4 after swelling, adding 2.5mg of PVA, raising the reaction temperature to 75 ℃, and continuing to react for 10-12 h;
1.2.5 repeatedly washing with ethanol for 3 times to remove unreacted monomers and oligomers, and then centrifugally settling microspheres at 20000 rpm;
in the marking step of the rheumatoid arthritis marker antibody, carboxylated polystyrene microspheres wrap Eu3+-Fe3O4The steps of preparing the magnetic rare earth fluorescent microsphere by the compound are as follows:
1.3.1 FeCl2、FeCl3、EuCl3NaOH solution, in molar proportion Fe2+：Fe3+：Eu3+：Na+=1:1:2:5, the solution is mixed and stirred for reaction to obtain a precipitate, the precipitate is aged for 3-5 h and then filtered, the filtrate is washed with purified water for 2-3 times, and the Eu is obtained after drying and grinding3+-Fe3O4A complex;
1.3.2 adding 0.1g of carboxyl modified polystyrene microspheres into 20ml of isopropanol, and performing ultrasonic dispersion for 10 min;
1.3.3 weighing Eu3+-Fe3O40.5mg of compound is added into 5ml of chloroform and ultrasonically dispersed for 20 min;
1.3.4 mixing the two liquids of 1.3.2 and 1.3.3, placing the mixture in a vacuum environment for 5 to 6 hours, and then placing the mixture in normal pressure for 2 to 3 hours;
1.3.5 washing the swelled microspheres with ethanol for 3 times, removing the microspheres which are not successfully wrapped with the magnetic particles by using a magnetic field, and suspending the microspheres with an aqueous solution containing 0.05% sodium azide to obtain the magnetic rare earth fluorescent microspheres, and storing the magnetic rare earth fluorescent microspheres at the temperature of 2-8 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810746013.1A CN108956982B (en) | 2018-07-09 | 2018-07-09 | Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810746013.1A CN108956982B (en) | 2018-07-09 | 2018-07-09 | Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108956982A CN108956982A (en) | 2018-12-07 |
| CN108956982B true CN108956982B (en) | 2022-05-20 |
Family
ID=64482509
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810746013.1A Active CN108956982B (en) | 2018-07-09 | 2018-07-09 | Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108956982B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470836A (en) * | 2019-05-14 | 2019-11-19 | 广州维佰生物科技有限公司 | A kind of detection avian leukosis virus fluorescent micro-ball immune chromatography test paper and preparation method thereof |
| CN111868073B (en) * | 2019-05-31 | 2021-04-30 | 广州市雷德生物科技有限公司 | Specific polypeptide related to rheumatoid arthritis and application thereof |
| CN112285364A (en) * | 2020-11-05 | 2021-01-29 | 南京申基医药科技有限公司 | Kit for combined detection of three items of rheumatism and preparation method thereof |
| CN114062666A (en) * | 2021-11-24 | 2022-02-18 | 山东康华生物医疗科技股份有限公司 | Preparation method of detection plate for quantitatively detecting RF, ASO, CRP and CCP in one card mode |
| CN115420899B (en) * | 2022-10-31 | 2023-03-24 | 迪亚莱博（张家港）生物科技有限公司 | Preparation method of fluorescence immunochromatographic kit for combined detection of RF-IgG/RF-IgM/RF-IgA |
| CN116027021B (en) * | 2023-02-17 | 2025-06-10 | 江苏省苏微微生物研究有限公司 | Antibody-fluorescent nanoparticle conjugate for synchronous detection of antibiotics enrofloxacin and ciprofloxacin and preparation method thereof |
| CN117110603B (en) * | 2023-07-14 | 2024-07-02 | 深圳市睿盟创新生物科技有限公司 | Fluorescent immunochromatographic test strip for combined detection of anti-MCV antibodies, anti-CCP antibodies and RF |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5292697A (en) * | 1992-11-23 | 1994-03-08 | Amoco Corporation | Nondestructive trivalent cation exchange of molecular sieves |
| AUPM807094A0 (en) * | 1994-09-09 | 1994-10-06 | Commonwealth Scientific And Industrial Research Organisation | Polymer beads and method for preparation thereof |
| CN101392172B (en) * | 2008-11-01 | 2012-04-18 | 厦门大学 | Carboxylic fluorescent encoding microsphere and synthetic method thereof |
| CN102565386B (en) * | 2011-12-29 | 2014-11-05 | 北京康美天鸿生物科技有限公司 | Magnetic fluorescent microsphere immunochromatography quantitative detection method |
| CN104237522B (en) * | 2012-12-03 | 2016-12-21 | 武汉生之源生物科技股份有限公司 | Adiponectin reagent box for detecting content and preparation method thereof |
| US9770418B2 (en) * | 2013-01-07 | 2017-09-26 | Bar-Ilan University | Dopamine nanocapsules and uses thereof |
| CN104262812B (en) * | 2014-09-28 | 2017-01-25 | 湖北工业大学 | Magnetic fluorescent polymer microsphere with high load stability and preparation method of magnetic fluorescent polymer microsphere |
| CN104459140B (en) * | 2014-12-05 | 2016-06-01 | 重庆乾德生物技术有限公司 | The detection kit of a kind of detection by quantitative Rheumatoid factors, polyclonal, antistreptolysin O, cyclic citrullinated peptid, c reactive protein |
| CN104721844B (en) * | 2015-02-02 | 2017-11-03 | 湖北大学 | A kind of new CT, MRI, the functional microsphere of rare-earth fluorescent three and its preparation method and application |
| CN104707544A (en) * | 2015-02-16 | 2015-06-17 | 天津大学 | Preparation method of polygenetic upconversion magnetic coding microspheres for screening pathogenic bacteria |
| CN105092861A (en) * | 2015-09-14 | 2015-11-25 | 广州市微米生物科技有限公司 | Immunofluorescence chromatography test paper for CRP (C-reaction protein)/SAA (Serum amyloid A protein) quantitative combined detection and preparation method of immunofluorescence chromatography test paper |
| CN205749529U (en) * | 2015-11-25 | 2016-11-30 | 深圳市金准生物医学工程有限公司 | A kind of three combined immunization chromatography detecting test paper strips of renal function |
| CN105950148A (en) * | 2016-04-29 | 2016-09-21 | 中国计量大学 | Preparation method for preparing ferroferric oxide hollow ball-based fluorescent magnetic composite material |
| CN105842464B (en) * | 2016-06-12 | 2017-10-03 | 吉林大学 | Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr device and preparation method thereof |
| CN107271669A (en) * | 2016-09-23 | 2017-10-20 | 清华大学深圳研究生院 | Propepsin, helicobacter pylori antibody and G17 detection method and its kit |
| CN106771207A (en) * | 2016-11-03 | 2017-05-31 | 重庆高圣生物医药有限责任公司 | A kind of cancer of pancreas dry type fluorescence quantum joint inspection diagnostic kit |
| CN106947026A (en) * | 2017-03-31 | 2017-07-14 | 中国科学院宁波材料技术与工程研究所 | A kind of method that utilization Dual Surfactants prepare monodisperse polystyrene microsphere |
| CN106896094B (en) * | 2017-04-11 | 2023-07-14 | 中国农业科学院饲料研究所 | A method for simultaneous detection of CLE, RAC and SBL and its special paper chip |
-
2018
- 2018-07-09 CN CN201810746013.1A patent/CN108956982B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108956982A (en) | 2018-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108956982B (en) | Rheumatoid arthritis marker combined quantitative detection test paper and preparation method thereof | |
| CN102759631B (en) | The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit | |
| CN109001464B (en) | Inflammation marker combined quantitative detection test paper and preparation method thereof | |
| CN103308698B (en) | A coupling method for covalently coupling amino-containing molecules to microspheres | |
| CN105548565A (en) | Kit for detecting trypanosoma cruzi antibody as well as preparation and application thereof | |
| JP5249576B2 (en) | Endotoxin measuring method and endotoxin measuring reagent kit | |
| CN110187104A (en) | Preparation method, sensor and its application of lateral relaxation time immunosensor based on bio-orthogonal reaction | |
| JPWO2003081240A1 (en) | Judgment method of viral infection | |
| JPS63229367A (en) | Immuno-reactive reagent, manufacture thereof and application thereof for measuring immuno-reactive specy | |
| WO2005071416A1 (en) | Method of enhancing signal detection of cell-wall components of cells | |
| JPH0782021B2 (en) | Non-immunochemical binding of lipopolysaccharide and its sandwich analysis method | |
| CN202916286U (en) | Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) | |
| WO2024001044A1 (en) | Biomarker combination related to lung cancer, kit containing same, and use thereof | |
| WO2021160121A1 (en) | Use of granulocyte population basophilic granulocytes as diagnostic markers for allergic diseases | |
| CN106556703A (en) | A kind of chronic kidney disease mark suPAR detection kit and preparation method | |
| CN106198962B (en) | Method for blocking biomagnetic beads | |
| CN102834719A (en) | Method and reagent for avoiding the influence of endogenous lipoprotein | |
| CN110940815A (en) | Latex immunoturbidimetric assay kit for quantitatively determining cat serum amyloid protein A | |
| CN109633163B (en) | procalcitonin/C reactive protein two-in-one detection kit | |
| JP2997520B2 (en) | Direct chemical bonding method of D-dimer from biological samples for diagnosis and testing of thrombotic and hypercoagulable states | |
| CN114137208A (en) | A three-in-one time-resolved fluorescence immunochromatography kit and preparation method thereof | |
| JPS60177265A (en) | Antibody immunoglobulin class detection method | |
| JP3328058B2 (en) | Method for producing immunodiagnostic drug | |
| Sobanski et al. | Measurement of serum antigen concentration by ultrasound-enhanced immunoassay and correlation with clinical outcome in meningococcal disease | |
| JP2000046828A (en) | Immunoassay reagent and production thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |