CN117890576A - Reagent and method for detecting CD89-IgA immune complex and application thereof - Google Patents
Reagent and method for detecting CD89-IgA immune complex and application thereof Download PDFInfo
- Publication number
- CN117890576A CN117890576A CN202311703460.6A CN202311703460A CN117890576A CN 117890576 A CN117890576 A CN 117890576A CN 202311703460 A CN202311703460 A CN 202311703460A CN 117890576 A CN117890576 A CN 117890576A
- Authority
- CN
- China
- Prior art keywords
- iga
- detection
- immune complex
- reagent
- detecting
- 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.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 124
- 208000010159 IgA glomerulonephritis Diseases 0.000 claims abstract description 26
- 206010021263 IgA nephropathy Diseases 0.000 claims abstract description 26
- 239000012502 diagnostic product Substances 0.000 claims abstract description 15
- 201000010099 disease Diseases 0.000 claims abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 14
- 208000015446 immunoglobulin a vasculitis Diseases 0.000 claims abstract description 11
- 208000031814 IgA Vasculitis Diseases 0.000 claims abstract description 9
- 201000004331 Henoch-Schoenlein purpura Diseases 0.000 claims abstract description 7
- 206010019617 Henoch-Schonlein purpura Diseases 0.000 claims abstract description 7
- 101000878602 Homo sapiens Immunoglobulin alpha Fc receptor Proteins 0.000 claims description 38
- 102100038005 Immunoglobulin alpha Fc receptor Human genes 0.000 claims description 38
- 210000002966 serum Anatomy 0.000 claims description 21
- 238000009739 binding Methods 0.000 claims description 18
- 230000027455 binding Effects 0.000 claims description 17
- 238000011161 development Methods 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 12
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 239000000975 dye Substances 0.000 claims description 8
- 229940088598 enzyme Drugs 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000004005 microsphere Substances 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 3
- 239000005081 chemiluminescent agent Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- 102000012440 Acetylcholinesterase Human genes 0.000 claims description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 claims description 2
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 2
- 102000003846 Carbonic anhydrases Human genes 0.000 claims description 2
- 108090000209 Carbonic anhydrases Proteins 0.000 claims description 2
- 241000238424 Crustacea Species 0.000 claims description 2
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 claims description 2
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 claims description 2
- 108010015776 Glucose oxidase Proteins 0.000 claims description 2
- 239000004366 Glucose oxidase Substances 0.000 claims description 2
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 claims description 2
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 2
- 229940022698 acetylcholinesterase Drugs 0.000 claims description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 2
- 208000026935 allergic disease Diseases 0.000 claims description 2
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 2
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 2
- ADRVRLIZHFZKFE-UHFFFAOYSA-N ethanediperoxoic acid Chemical compound OOC(=O)C(=O)OO ADRVRLIZHFZKFE-UHFFFAOYSA-N 0.000 claims description 2
- 229940116332 glucose oxidase Drugs 0.000 claims description 2
- 235000019420 glucose oxidase Nutrition 0.000 claims description 2
- 229960002247 lomustine Drugs 0.000 claims description 2
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 claims description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 229910052707 ruthenium Inorganic materials 0.000 claims description 2
- 230000009870 specific binding Effects 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims 1
- 238000010168 coupling process Methods 0.000 claims 1
- 238000005859 coupling reaction Methods 0.000 claims 1
- 239000011546 protein dye Substances 0.000 claims 1
- 239000001022 rhodamine dye Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 16
- 238000003745 diagnosis Methods 0.000 abstract description 9
- 239000000090 biomarker Substances 0.000 abstract description 6
- 230000000405 serological effect Effects 0.000 abstract description 5
- 238000001647 drug administration Methods 0.000 abstract description 2
- 229940099472 immunoglobulin a Drugs 0.000 description 98
- 239000000523 sample Substances 0.000 description 38
- 239000000243 solution Substances 0.000 description 21
- 238000005406 washing Methods 0.000 description 20
- 238000002965 ELISA Methods 0.000 description 19
- 239000000872 buffer Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 16
- 238000007789 sealing Methods 0.000 description 15
- 210000002381 plasma Anatomy 0.000 description 11
- 206010037549 Purpura Diseases 0.000 description 10
- 241001672981 Purpura Species 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 10
- 238000000576 coating method Methods 0.000 description 10
- 230000005965 immune activity Effects 0.000 description 9
- 238000012417 linear regression Methods 0.000 description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 210000002700 urine Anatomy 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000012470 diluted sample Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 5
- 210000002865 immune cell Anatomy 0.000 description 5
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000010998 test method Methods 0.000 description 4
- 102000009109 Fc receptors Human genes 0.000 description 3
- 108010087819 Fc receptors Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108010004469 allophycocyanin Proteins 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000003317 immunochromatography Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- 239000012898 sample dilution Substances 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- QCPFFGGFHNZBEP-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 QCPFFGGFHNZBEP-UHFFFAOYSA-N 0.000 description 2
- IKYJCHYORFJFRR-UHFFFAOYSA-N Alexa Fluor 350 Chemical compound O=C1OC=2C=C(N)C(S(O)(=O)=O)=CC=2C(C)=C1CC(=O)ON1C(=O)CCC1=O IKYJCHYORFJFRR-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 2
- 206010018372 Glomerulonephritis membranous Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010053210 Phycocyanin Proteins 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 208000033679 diabetic kidney disease Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 201000008350 membranous glomerulonephritis Diseases 0.000 description 2
- 231100000855 membranous nephropathy Toxicity 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000013102 re-test Methods 0.000 description 2
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000016918 Complement C3 Human genes 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 241000125183 Crithmum maritimum Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000860173 Myxococcus xanthus C-factor Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 208000008675 hereditary spastic paraplegia Diseases 0.000 description 1
- 229940096329 human immunoglobulin a Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/6854—Immunoglobulins
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to the field of immunodetection, and particularly discloses a reagent and a method for detecting a CD89-IgA immune complex and application thereof. The detection reagent of the CD89-IgA immune complex comprises a capture antibody and a detection secondary antibody, wherein the capture antibody comprises an anti-CD 89 antibody, and the detection secondary antibody comprises an anti-IgA antibody. The invention also provides diagnostic products for IgA nephropathy, allergic purpura, methods of detecting CD89-IgA immune complexes and uses thereof. The invention can detect CD89-IgA-IC with high specificity and high sensitivity, and combines detection of IgA immune complex combined with non-CD 89 to make the detection of the whole IgA immune complex tend to perfect, thereby improving the sensitivity of using serological immune complex as biomarker in the auxiliary diagnosis process, and having important significance for auxiliary diagnosis, disease tracking, drug administration assessment and the like of IgA related diseases.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a reagent and a method for detecting a CD89-IgA immune complex and application thereof.
Background
In the autoimmune reaction process of human body, human immunoglobulin A (IgA) is combined with pathogen to start immune reaction, and together with downstream antibody IgG, igM, C3 complement of complement pathway and other molecules, igA forms IgA-IgG, igA-IgM, igA-C3, igA-IgG-IgM and other complexes, collectively called IgA complex. Aberrant clearance of IgA complexes in the immune system may lead to several autoimmune diseases: deposition of IgA complexes that are not efficiently cleared in the kidneys results in common IgA nephropathy (IgA nehpropath, igAN), a number of young adults; deposition of non-efficiently cleared IgA complexes in capillaries leads to IgA vasculitis (IgAV), also known as Henoch-Schonlein purpura, HSP, a common occurrence in children. Therefore, the circulatory IgA complex can be used as an important biomarker for the diseases.
CD89 is the Fc receptor for IgA, a type I transmembrane glycoprotein expressed on the surface of myeloid cells (monocytes/macrophages, dendritic cells, kupffer cells, neutrophils and eosinophils), CD89 binds to IgA1 and IgA2 molecules through its amino terminal end interacting with the cα2/cα3 junction site of IgA, the immune system utilizing CD89 as a typical function of IgA Fc receptors on monocytes, responsible for clearance of IgA pathogens or multimeric IgA complexes in the circulation, CD89 having a higher affinity for multimeric IgA than for monomeric IgA, enabling phagocytes to selectively capture and clear multimeric IgA complexes. Studies have shown that, on the one hand, in addition to attaching to immune cells, some cells secrete soluble CD89 (sCD 89) into the circulatory system, these sCD89 can bind to IgA complexes before they trigger the clearance of immune cells, forming CD89-IgA immune complexes, denoted CD89-IgA-IC, which can deposit in the kidneys for IgA nephropathy susceptible populations, together with other IgA immune complexes capable of binding CD89, known as an inducer of IgA nephropathy; on the other hand, igA immune complexes captured by CD89, a transmembrane protein of immune cells (including circulating immune cells and infiltrating immune cells on kidney tissue), may exfoliate during the immune response, enter the circulatory system in the form of CD89-IgA-IC, and recirculate and deposit with the kidneys, and become an inducer of IgA nephropathy along with other IgA immune complexes capable of binding CD 89.
In the patent application of the reagent for detecting IgA immune complex (patent number: 2022102944568), the reagent for detecting IgA complex using CD89 as the capture probe and the application are described in detail, however, since CD89 is used as the capture probe, the solution cannot capture and detect IgA or IgA complex which has already bound to CD89, that is, cannot capture and detect CD89-IgA-IC. Therefore, in addition to detecting IgA or IgA complexes that are not bound by CD89, there is a need for a method that can detect CD89-IgA-IC that, as a complementary method, will perfect the overall IgA immune complex detection and thereby increase the sensitivity of using serological immune complexes as biomarkers in IgA nephropathy and purpura assisted diagnosis.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the CD89-IgA immune complex (CD 89-IgA-IC) detection reagent provided by the invention can detect CD89-IgA-IC with high specificity and high sensitivity, and the detection of the IgA immune complex combined with non-CD 89 by using the CD89-IgA immune complex detection reagent provided by the invention can enable the detection of the whole IgA immune complex to be perfect, thereby improving the sensitivity of using the serological immune complex as a biomarker in the auxiliary diagnosis process of diseases such as IgA nephropathy, purpura and the like, and having high application value.
The invention also provides a diagnostic product for IgA nephropathy, allergic purpura and/or IgA immune complex related diseases.
The invention also provides a method for detecting the CD89-IgA immune complex.
The invention also provides an application of the CD89-IgA immune complex detection reagent and a method for detecting the CD89-IgA immune complex.
In a first aspect of the invention there is provided a detection reagent for a CD89-IgA immune complex, the reagent comprising a capture antibody and a detection secondary antibody;
wherein the capture antibody comprises an anti-CD 89 antibody and the detection secondary antibody comprises an anti-IgA antibody.
The capture antibody of the detection reagent can bind to and capture CD89-IgA-IC, and the secondary antibody is further bound to the CD89-IgA immune complex, so that the CD89-IgA-IC in a sample can be specifically detected.
In some embodiments of the invention, the detection secondary antibody is labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
By capturing the capture antibody and detecting the combination of the secondary antibody, the CD89-IgA-IC in the sample can be specifically detected, a detectable marker is connected or coupled or marked on the detection secondary antibody, and the marker is subjected to targeted treatment, so that the content information of the CD89-IgA-IC in the sample can be represented, and the detection of the CD89-IgA-IC is realized.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In some embodiments of the invention, the detectable label is selected from the group consisting of an enzyme that catalyzes the development of a substrate, a chemiluminescent reagent, a fluorescent microsphere, a radioisotope, or a nanoparticle-based label.
In some preferred embodiments of the invention, the enzyme that catalyzes the development of a substrate is selected from horseradish peroxidase (HRP), alkaline phosphatase (ALP), β -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, or glucose 6-phosphate deoxygenase.
In some more preferred embodiments of the invention, the enzyme that catalyzes the development of a substrate comprises horseradish peroxidase.
In some preferred embodiments of the present invention, the chemiluminescent agent is selected from the group consisting of acridinium esters and derivatives thereof, luminol and derivatives thereof, ruthenium bipyridine and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, dioxane and derivatives thereof, lomustine and derivatives thereof, or peroxyoxalate and derivatives thereof.
In some more preferred embodiments of the invention, the chemiluminescent agent comprises an acridinium ester and derivatives thereof.
In some preferred embodiments of the present invention, the fluorescent substance on the fluorescent microsphere is selected from fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC), hydroxy-Fluorescein (FAM), tetrachloro-fluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, etc. or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa-Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), or protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazochrome (APC), etc.).
In some embodiments of the invention, the detection reagent further comprises a calibrator.
In some preferred embodiments of the invention, the calibrator comprises at least one of a CD89-IgA complex formed by binding of recombinant CD89 to recombinant multimeric IgA or multimeric IgA purified from commercial human serum IgA, a CD89-IgA complex formed by directed conjugation of CD89 to multimeric IgA, a CD89-IgA complex naturally occurring in commercial human serum IgA.
In some embodiments of the invention, the sample to be tested by the reagent comprises serum, plasma, urine.
In a second aspect of the invention there is provided a diagnostic product of IgA nephropathy, allergic purpura and/or IgA immune complex related diseases, said product comprising a detection reagent for CD89-IgA immune complex according to the first aspect of the invention.
The diagnostic product provided by the invention can be used as a complementary method and combined with the diagnostic product of the application of the reagent for detecting IgA immune complex (patent number: 2022102944568), so that the detection of the integral IgA immune complex is improved, and the sensitivity of the serological immune complex used as a biomarker in the auxiliary diagnostic process of IgA nephropathy and purpura is improved; the detection indexes of the two diagnostic products are used for carrying out linear regression to establish the comprehensive index of the IgA immune complex, and the method has important significance for carrying out auxiliary diagnosis, disease tracking, drug administration evaluation and the like on IgA nephropathy and purpura. In addition, the statistical characteristics, clinical characteristics, treatment effects and the like of different crowds can be established, so that the personal accurate medical treatment and disease management of IgA nephropathy can be performed.
In some embodiments of the invention, the reagents of the diagnostic product further comprise an enzyme label plate, a coating buffer, a calibrator, a chromogenic solution, a stop solution, a wash solution, a sample diluent, a blocking buffer, a sealing membrane and self-sealing bag, a positive control, a negative control, a standard curve card.
In a third aspect of the invention there is provided a method of detecting a CD89-IgA immune complex of non-diagnostic interest using a detection reagent for a CD89-IgA immune complex according to the first aspect of the invention or a diagnostic product according to the second aspect of the invention.
In some embodiments of the invention, the method comprises the steps of:
s1, capturing a CD89-IgA immune complex by using a capture antibody;
s2, detecting the secondary antibody specific binding of the CD89-IgA immune complex captured in the step S1;
s3, detecting a luminescence value or a fluorescence value after processing the detectable marker for detecting the secondary antibody mark;
s4, calculating the content of the CD89-IgA immune complex in the sample according to a standard curve.
In some preferred embodiments of the invention, the method comprises the steps of:
s1, dissolving a capture antibody in a coating buffer solution, adding the coating buffer solution into an ELISA plate for coating, washing the ELISA plate by using a washing solution after coating, adding a sealing solution for sealing treatment, adding a sample after sealing, and capturing CD89-IgA-IC in the sample by the capture antibody;
s2, washing the ELISA plate by using a washing solution, and adding the CD89-IgA-IC captured in the step S1 for detecting the secondary antibody;
s3, washing the ELISA plate by using a washing liquid, adding a color development liquid, developing in a dark place, adding a stop solution after the color development is finished, and detecting a luminescence value or a fluorescence value by using an ELISA instrument or a chemiluminescent detector or a fluorescent detector;
s4, calculating the content of the CD89-IgA immune complex in the sample according to a standard curve.
In the practical use process, a person skilled in the art can select a full-automatic detector according to detection conditions or actual needs to realize the method for detecting the CD89-IgA-IC, and if the detection principle is consistent with that of the detection method or the detection method obtained by the person skilled in the art without creative labor on the basis of the principle of the detection method, the method belongs to the protection scope of the invention.
In a fourth aspect, the invention provides the use of a detection reagent for a CD89-IgA immune complex according to the first aspect of the invention for the preparation of a diagnostic product for IgA nephropathy, allergic purpura and/or diseases associated with the IgA immune complex.
Since the application provided by the invention uses the detection reagent of the CD89-IgA immune complex according to the first aspect of the invention, at least all the beneficial effects of the detection reagent are provided.
In some embodiments of the invention, the product comprises any one of a kit, a chip, or a test strip.
In some embodiments of the invention, the kit comprises a capture antibody, a detection secondary antibody, an elisa plate, a coating buffer, a calibrator, a chromogenic solution, a stop solution, a wash solution, a sample diluent, a blocking buffer, a sealing membrane and self-sealing bag, a positive control, a negative control, a standard curved line card.
In some embodiments of the invention, the capture antibodies are coated on the chip.
In some embodiments of the invention, the test strip comprises a detection card and an antibody binding pad.
In some embodiments of the invention, the antibody-binding pad contains the detection secondary antibody thereon.
In some embodiments of the present invention, the detection line and the quality control line are disposed in parallel on the detection card, and the capture antibody is coated on the detection line.
In some preferred embodiments of the invention, the kit comprises an ELISA detection kit, a chemiluminescent detection kit, a time-resolved immunochromatographic detection kit, an immune colloidal gold detection kit, an immunoturbidimetry detection kit.
In some embodiments of the invention, the diagnostic product further comprises an IgA complex detection reagent that is not CD89 binding.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram showing the detection flow of an ELISA detection kit for CD89-IgA-IC in example 1 of the present invention;
FIG. 2 is a graph of ROC results corresponding to a single indicator of two IgA complexes according to the detection example of the present invention;
FIG. 3 is a graph of ROC results corresponding to a linear regression model of two indicators according to the test example of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The specific conditions are not specified in the specific embodiments and are carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a specific embodiment, the calibrator is a CD89-IgA complex formed by the binding of recombinant CD89 (rCD 9) to recombinant multimeric IgA (rPolyIgA), specifically designated rCD89-rPolyIgA. The preparation method of the recombinant poly IgA refers to a patent of a recombinant poly IgA protein, a preparation method and application thereof (publication number: CN 116789806A), wherein the preparation of the rCD89-rPolyIgA complex is as follows:
1. preparing a mixed solution of rCD89 and rPolyIgA by using 0.01M PBS (pH 7.4) according to a molar ratio of 1:1-1:100; preferably, the rCD89 concentration is 10nM and the rPolyIgA concentration is in the range of 10nM to 10000nM. The preferable proportion is that the mol ratio of rCD89 to rPolyIgA is 1:20;
2. placing the rCD89-rPolyIgA mixed solution at 18-25 ℃ for standing for 3 hours, and fully and uniformly mixing;
3. after the reaction is finished, the materials are packaged and stored in a refrigerator at the temperature of minus 20 ℃ to avoid repeated freezing and thawing.
In a specific embodiment, the recombinant CD89 can also be commercial CD89, and the poly IgA can also be commercial human serum IgA, and the CD89-IgA compound is obtained after the binding reaction of the CD89 and the commercial human serum IgA.
In a specific embodiment, the other calibrator is a CD89-IgA complex that is native to commercial human serum IgA. Commercial human serum IgA calibrator was purchased from Shanghai ludol organisms under the trade designation LY210-1mg with a calibration curve initial gradient of 1600ng/mL, and was sequentially diluted by 1:2 fold to obtain calibrator titer curves containing 1600, 800, 400, 200, 100, 50, 25 ng/mL.
In a specific embodiment, the rabbit anti-human CD89 antibody is purchased from Sino Biological, inc. of Beijing Yiqiao Shenzhou technologies, inc., cat# 10414-R002; commercial human serum IgA calibrator was purchased from Shanghai ludol organisms under the designation LY210-1mg; HRP-labeled mouse anti-human IgA antibodies were purchased from the industrial bioengineering ltd of basjohnsony (Boster Biological Technology); streptavidin modified magnetic particles were purchased from: JSR corporation, cat No. MS300, strepitavidin; color development liquid A and color development liquid B were purchased from Sino Biological Co., ltd., beijing Yiqiao Shenzhou technology Co., ltd., product number: SEKCR01; CD89 protein standards were purchased from beijing, samphire sciences inc.
The biotinylated rabbit anti-human CD89 antibodies used in the examples were self-produced by the following methods,
1. materials:
biotin-N-succinimidyl ester, rabbit anti-human CD89 antibody, buffer of 0.1mol/L sodium bicarbonate buffer (pH 8.0) or 0.5mol/L boric acid buffer (pH 8.6), 1mol/L NH 4 Cl, organic solvent DMSO, glycerol.
2. The steps are as follows:
A. the protein to be biotinylated is diluted to 1mg/mL with 0.1mol/L sodium bicarbonate buffer (pH 8.0) or 0.5mol/L boric acid buffer (pH 8.6), and the biotinylation volume applied in a common laboratory is 1-2.5 mL;
B. the protein was extensively dialyzed against either 0.1mol/L sodium bicarbonate buffer (pH 8.0) or 0.5mol/L boric acid buffer (pH 8.6) alternately;
C. 1mg of biotin succinimidyl ester (NHSB) was dissolved in 1mL of DMSO;
D. to 1mL of the protein solution (i.e., containing 1mg of protein), 120. Mu.l of NHSB solution (i.e., containing 120. Mu.g of NHSB) was added;
E. stirring continuously at room temperature, and preserving heat for 0.5-4 hours;
F. 9.6 mu L of 1mol/L NH was added 4 Cl (1. Mu.l per 25. Mu.g NHSB) was stirred at room temperature for 10 to 60 minutesA clock;
G. thoroughly dialyzing the PBS at 4deg.C to remove free biotin;
H. slowly eluting a sample on a 1mL molecular sieve column with PBS, collecting 1 mL/tube, and washing out protein between 1 and 3 mL;
I. finally, the samples were added with sodium azide or PC300 preservative 0.05%. The combined product is preserved at 4deg.C for a short period of time and in dark place, or 50% glycerol can be added, and preserved at-20deg.C for a long period of time.
In particular embodiments, the term "antibody" includes polyclonal antibodies and monoclonal antibodies; the rabbit anti-human CD89 antibody can also be selected from anti-human CD89 specific antibodies of other animals; the mouse anti-human IgA antibody can also be selected from anti-human IgA antibodies of other animals; the goat anti-chicken IgY antibody can also be selected from anti-chicken IgY antibodies of other animals.
Example 1
The embodiment provides an ELISA detection kit for human serum or plasma CD89-IgA-IC and a detection method thereof:
1. ELISA detection kit of CD89-IgA-IC, the kit comprises the following components:
ELISA plate: 8X 12 well standard plate;
molecular probe: rabbit anti-human CD89 antibody;
coating buffer solution: a 0.05M carbonate buffer at pH 9.6;
calibration material: commercialized human serum IgA;
and (2) secondary antibody: HRP-labeled mouse anti-human IgA antibody;
color development liquid A: fructus Ziziphi Spinosae + citric acid + TMB + H 2 O 2 ；
Color development liquid B: fructus Ziziphi Spinosae + citric acid + ABTS + H 2 O 2 ；
Stop solution: sulfuric acid with the mass concentration of 10%;
washing liquid: 0.15M phosphate buffer containing Tween-20 at a mass concentration of 0.05%;
sample dilution: a solution containing BSA with a mass concentration of 1%;
blocking buffer: a solution containing 5% by mass of BSA prepared using a washing solution;
sealing film and self-sealing bag.
2. The detection method of the ELISA detection kit of the CD89-IgA-IC specifically comprises the following steps:
(1) Sample preparation: collecting peripheral blood by using a disposable test tube, and extracting serum (blood plasma can be selected as well) to serve as a sample to be detected for a subsequent detection process (the sample can be selected to be used in time after sub-packaging and freezing storage);
(2) And (3) detection: the schematic diagram of the detection flow is shown in fig. 1, and specifically includes the following steps:
a) Coating a molecular probe: adding 100 mu L of coating buffer solution of rabbit anti-human CD89 antibody with the concentration of 2ng/mL into each hole of an ELISA plate, sealing the film, preserving at 4 ℃ for 16h (overnight coating), throwing away the solution in the holes, gently washing the plate for 3 times by using a washing solution, standing for 60s each time, pouring out the washing solution, drying the ELISA plate on clean absorbent paper after washing, adding 200 mu L of sealing solution into each hole, sealing the film, and preserving for 2h;
b) Sample adding: pouring off the sealing liquid, washing once again by using a washing liquid, diluting the sample to be detected by using a sample diluent, diluting a calibrator, a negative control (sample diluent) and a positive control (CD 89 protein standard, 1 mg/mL), adding 100 mu L of each of the diluted sample, the calibrator, the negative control and the positive control into different enzyme-labeled plate holes, and preserving the sealing film for 2 hours;
c) Adding a secondary antibody: washing 3 times by using a washing solution, adding 100 mu L of HRP-labeled mouse anti-human IgA antibody with the concentration of 2ng/mL, and preserving for 60 minutes by sealing a membrane;
d) Color development: washing 3 times by using a washing solution, taking 50 mu L of each of the color development liquid A and the color development liquid B from each hole, uniformly mixing, adding the mixture into an ELISA plate, and developing the color at room temperature in a dark place for 10min;
e) And (3) terminating: 50. Mu.L of stop solution was added to each well,
f) Measuring luminosity by using an enzyme-labeled instrument: after the ELISA plate is gently oscillated, the detection result is measured on an ELISA apparatus, the OD value is measured by using two wavelengths, the first wavelength is 450nm, and the second wavelength is 630nm;
g) And (3) data processing: the CD89-IgA-IC concentration of the sample is calculated using a four-parameter model (alternatively, a quadratic polynomial model or a log-log linear model may be used to fit the calibration curve) and using the calibration curve.
Example 2
The embodiment provides an ELISA detection kit for human urine CD89-IgA-IC and a detection method thereof: the difference between this example and example 1 is that "collect peripheral blood", extract serum (plasma may be selected) and "collect urine" in the test method of example 1.
Example 3
The embodiment provides a chemiluminescence detection kit for human serum or plasma CD89-IgA-IC and a detection method thereof:
1. the chemiluminescent method detection kit for the CD89-IgA-IC comprises the following components:
m reagent: magnetic particles with concentration of 0.2mg/mL, diameter of 300nm and modified streptavidin, preservative PC300;
r1 reagent: molecular probe: 1.0mg/mL biotinylated rabbit anti-human CD89 antibody;
r1 buffer: phosphate buffer with pH value of 7.5 and concentration of 0.05M, preservative PC300;
r2 reagent: an acridinium ester labeled mouse anti-human IgA antibody;
r2 buffer: phosphate buffer with pH value of 7.5 and concentration of 0.05M;
pre-excitation liquid a: containing HNO with a molar concentration of 0.1M 3 A solution of hydrogen peroxide at a mass concentration of 1%;
excitation liquid B: a solution containing sodium hydroxide at a molar concentration of 0.5mM and CTAC at a molar concentration of 7 mM;
calibration material: commercialized human serum IgA;
washing liquid: 0.15M phosphate buffer containing Tween-20 at a mass concentration of 0.05%;
sample dilution: a solution containing BSA with a mass concentration of 1%;
2. the detection method of the chemiluminescent method detection kit of the CD89-IgA-IC specifically comprises the following steps:
(1) Sample preparation: collecting peripheral blood by using a disposable test tube, extracting serum (blood plasma can be selected), collecting by using a standard or vacuum tube with separating gel (split charging and freezing and timely and quickly thawing at 37 ℃ below zero can be selected), and diluting 50 times by using a sample diluent to obtain a diluted sample;
(2) And (3) detection: the method specifically comprises the following steps:
the components of the kit and the diluted sample are added to the corresponding reagent positions of the full-automatic chemiluminescence immunoassay analyzer, and the analyzer automatically performs the following steps:
a) 20. Mu.L of calibrator and diluted sample were added to the reaction cup, respectively;
b) Adding 50 mu L of M reagent and 50 mu L of R1 reagent into each reaction cup, incubating and reacting for 10min at 37 ℃, and cleaning the instrument for 3 times;
c) Adding 100 mu L of R2 reagent into each reaction cup, incubating at 37 ℃ for 10min, and cleaning the instrument for 3 times;
d) Sequentially adding the pre-excitation liquid A and the excitation liquid B to generate chemiluminescence reaction;
e) Outputting a detection result, wherein a proportional relation exists between the total amount of CD89-IgA-IC in the sample and the value of a Relative Light Unit (RLUs) detected by the system, a four-parameter model (a quadratic polynomial model or a logarithmic-logarithmic linear model can be selected to fit a calibration curve) is used, and then the CD89-IgA-IC concentration of the sample is calculated by using the calibration curve.
The full-automatic chemiluminescence immunoassay analyzer used in the embodiment can be a Shine i900 or Shine i2000 full-automatic chemiluminescence immunoassay analyzer.
Example 4
The embodiment provides a chemiluminescent detection kit of human urine CD89-IgA-IC and a detection method thereof: the difference between this example and example 3 is that "collect peripheral blood", extract serum (plasma may be selected) and "collect urine" in the test method of example 3.
Example 5
The embodiment provides a time-resolved immunochromatography (TRFIA) detection kit for human serum or plasma CD89-IgA-IC and a detection method thereof:
1. the time-resolved immunochromatography detection kit for the CD89-IgA-IC comprises the following components:
and (3) detecting card: the detection card is provided with two parallel detection lines, namely a detection line T line and a quality control line C line, wherein the T line is coated with rabbit anti-human CD89 antibody, the C line is coated with chicken IgY, the front end of the detection card is provided with a sample hole and an antibody binding pad, the antibody binding pad is positioned between the sample hole and the detection card, and the antibody binding pad contains fluorescent microsphere marked mouse anti-human IgA antibody and fluorescent microsphere coated goat anti-chicken IgY antibody;
sample dilution: PBS buffer containing 1% BSA;
IC card (standard curve card): providing a standard curve;
quality control product: PBS buffer containing different CD89-IgA-IC concentrations;
2. the detection method of the POCT fluorescent immunochromatography detection kit for the CD89-IgA-IC specifically comprises the following steps:
(1) Sample preparation: collecting venous blood by using a disposable test tube, extracting serum (blood plasma can be selected), diluting 50 times by using a sample diluent to obtain a diluted sample, and storing the rest sample at-80 ℃;
(2) And (3) detection: the method specifically comprises the following steps:
a) Taking 100 mu L of diluted sample and adding the diluted sample into a sample hole;
b) Reacting for 15min at normal temperature;
c) Placing the detection card in a fluorescence detector, detecting the detection card by using the fluorescence detector, respectively detecting fluorescent values of a T line and a C line, and calculating the ratio of a T line signal to a C signal;
d) The concentration of CD89-IgA-IC in the sample was calculated based on the standard curve provided by the IC card.
Example 6
The embodiment provides a chemiluminescent detection kit of human urine CD89-IgA-IC and a detection method thereof: the difference between this example and example 5 is that "collect peripheral blood", extract serum (plasma may be selected) and "collect urine" in the test method of example 5.
Example 7
The present embodiment provides a detection method for detecting all IgA immune complexes of a sample:
two IgA immune complexes, a CD89-IgA-IC immune complex was detected based on a method in which CD89 was the capture molecule by using the protocol of the patent "application of a reagent for detecting IgA immune complexes" (patent number: 2022102944568), and a CD89-IgA-IC immune complex was detected based on an antibody specific to human CD89 as the capture molecule by using the protocol of example 1 (example 2, example 3, example 4, example 5, or example 6 may be also selected).
Through the above detection, the samples are divided into four types according to the detection results:
non-CD 89-binding IgA complex (+) & CD89-IgA-IC (+);
non-CD 89-binding IgA complex (+) & CD89-IgA-IC (-);
non-CD 89-binding IgA complex (-) & CD89-IgA-IC (+);
non-CD 89-binding IgA complex (-) & CD89-IgA-IC (-);
wherein, + represents positive, -represents negative.
Through the detection of all IgA immune complexes in the embodiment, the indexes of the two detection schemes can be utilized to carry out linear regression to establish the comprehensive indexes of the IgA immune complexes, and the method has important significance for carrying out auxiliary diagnosis, disease tracking, medication evaluation and the like on IgA nephropathy and purpura. In addition, the statistical characteristics, clinical characteristics, treatment effects and the like of different crowds can be established, so that the personal accurate medical treatment and disease management of IgA nephropathy can be performed.
Test case
The detection example uses the kit and the detection method disclosed in the patent application of the reagent for detecting IgA immune complexes (patent number: 2022102944568) to detect the IgA immune complexes which are not bound with CD89, and simultaneously uses the ELISA detection kit and the detection method of the CD89-IgA-IC of the scheme of the invention to detect the CD89-IgA-IC, thereby detecting all IgA immune complexes in a sample, improving the clinical effect of single detection of the IgA immune complexes and further being more accurately used for assisting in diagnosing diseases such as IgA nephropathy (IgAN) or purpura (HSP).
(1) The detection method comprises the following steps:
the test example used 9 positive samples, including 7 IgA nephropathy diagnosed by renal puncture and 2 purpura patients plasma samples; in addition, 18 negative samples were used, including urine samples from 4 Diabetic Nephropathy (DN), 5 Membranous Nephropathy (MN) and 6 micro-pathologically altered nephropathy (MCD) patients. The samples were captured using CD89 and anti-human CD89 antibody capture, respectively, and after the corresponding subsequent detection steps, non-CD 89-bound IgA complex titers and CD89-IgA-IC Optical Density (OD) were obtained, and since the calibrator for the CD89-IgA-IC ELISA detection kit of the present invention was also well developed, the analysis was directly performed using the measured luminescence value, i.e., OD value, and in general, the model for conversion of OD values to titers by the calibrator was made by a log-linear model, so here two linear regression methods were used: one is to directly perform linear regression using two indices; alternatively, the titers of non-CD 89-bound IgA immune complexes are first log and then linearly regressed with the OD values of the latter.
(2) Analysis of detection results:
the ROC results of two IgA immune complexes and their linear combinations for diagnosis of IgA nephropathy and purpura using subject operating characteristics (ROC) for analytical comparison are shown in table 1, wherein auc represents the area under the curve; fpr represents false positive rate; tpr represents true positive rate; cutoff represents a positive judgment value; acc represents accuracy.
TABLE 1 ROC results of two IgA immune complexes and their linear combinations for diagnosis of IgA nephropathy and purpura
| Index (I) | auc | fpr | tpr | cutoff | acc |
| CD89-IgA-IC (OD value) | 0.8 | 0.22 | 0.78 | 0.5 | 0.78 |
| IgA immune activity index (non-CD 89 linked) | 0.81 | 0.33 | 0.78 | 7.51 | 0.7 |
| Linear model: OD+IgA immune Activity index (non-CD 89 ligation) | 0.83 | 0.28 | 0.89 | 0.24 | 0.78 |
| Linear model: OD+log (IgA immune Activity index (non-CD 89 ligation)) | 0.82 | 0.33 | 1 | 0.24 | 0.74 |
Two bivariate linear regression model parameters are shown in Table 2, wherein Intercept represents the model constant term; OD_avg represents the CD89-IgA-IC sample repeat OD mean; igAN_ImmunoIndex_avg represents the detection mean of CD89 captured IgA complex sample retest; igAN_ImmunoIndex_log represents the log (log 2) of detection of CD89 captured IgA complex sample retests. The linear regression model parameters are: estime represents the weight of the linear model, stderr is the standard deviation, tv is the t-statistic (t-statistics), pv is the corresponding p-value, and rsq is the R-square of the model.
Table 2 two bivariate linear regression model parameters
The ROC for a single indicator plotted for both IgA complexes is shown in figure 2. The ROC corresponding to the two bivariate linear regression models is shown in fig. 3, and the od+iga immune activity index linear model is: score=0.22×cd89_iga_od+0.04×noccd89_iga_ic-0.15 (left panel in fig. 3), od+log 2 The (IgA immune activity index) linear model is: score=0.33×cd89_iga_od+0.22×log 2 (NoCD 89_iga_ic) -0.75 (right panel in fig. 3), wherein NoCD89_iga_ic represents non-CD 89 bound IgA immune complex titers.
From the above results, it is clear that CD89-IgA-IC has similar specificity (1-fpr) than IgA immune activity index that is not CD89 binding: 78% vs 67%, where CD89-IgA-IC is relatively good, probably due to bias in test sample selection; two linear models combining CD89-IgA-IC and non-CD 89-bound IgA immune activity indicators were better sensitive than each single indicator, two linear models: an od+iga immune activity index, the true positive rate, i.e., sensitivity, was 89%, and an od+log (IgA immune activity index), the latter was 100%, whereas the sensitivity of the single index was only 78%.
For the detection method of CD89-IgA-IC in this embodiment, the OD value of CD89-IgA-IC of the IgA nephropathy group and the other group not having IgA nephropathy group was shown by ROC comparison (first line of Table 1), auc was 0.8, specificity (1-false positive rate) was 78%, sensitivity was 78%, and accuracy was 78%. In contrast, detection of non-CD 89 bound IgA complex indicators using the kit and detection method disclosed in the patent application of reagents for detecting IgA immune complexes (patent number 2022102944568) showed that the ROC results (second line of Table 1) showed auc of 0.81, specificity (1-false positive rate) of 67%, sensitivity of 78% and accuracy of 70%. Therefore, the detection of CD89-IgA-IC according to the present embodiment has similar clinical utility and detection effect as the detection of IgA complexes other than CD89 binding by the kit and detection method disclosed in the patent application of reagent for detecting IgA immune complexes (patent number: 2022102944568). Furthermore, the linear combination of the two increased the sensitivity to 89% when the specificity was guaranteed (72%), while the linear combination of the former OD with the latter logarithm increased the sensitivity to 100% when the specificity was guaranteed (63%) (fourth row of table 1). This shows that the method for detecting CD89-IgA-IC has independent and similar detection effect and clinical value on one hand, and on the other hand, can be used as a kit and a complementary method for detecting non-CD 89-combined IgA complex disclosed in the patent application of reagent for detecting IgA immune complex (patent number: 2022102944568) to cover the cases of immune complex pathogenesis of CD89-IgA-IC as main IgA nephropathy, thereby improving sensitivity and detection comprehensiveness.
Since CD89-IgA-IC and non-CD 89 conjugated IgA immune complexes may be complementary to the diseased population, they may exhibit different levels of expression of the two pathogenic IgA complexes for different IgA kidney patients, or for the same patient at different stages of the disease, if a single test is performed, another test method may result in false negatives for patients in which only one of the IgA complexes is highly expressed, and thus, the simultaneous detection of both pathogenic IgA complexes may increase the sensitivity of the test.
In conclusion, the CD89-IgA-IC detection kit and the detection method have the advantages of high specificity and high sensitivity for detecting CD89-IgA-IC, and the detection of the IgA immune complex combined with non-CD 89 can enable the detection of the whole IgA immune complex to be perfect, so that the sensitivity of using the serological immune complex as a biomarker in the auxiliary diagnosis process of diseases such as IgA nephropathy and purpura is improved, and the detection kit and the detection method have high application value.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A reagent for detecting a CD89-IgA immune complex, wherein the reagent comprises a capture antibody and a detection secondary antibody;
wherein the capture antibody comprises an anti-CD 89 antibody and the detection secondary antibody comprises an anti-IgA antibody.
2. The reagent for detecting CD89-IgA immune complex according to claim 1, wherein the detection secondary antibody is labeled with a detectable label.
3. The detection reagent for CD89-IgA immune complex according to claim 2, wherein the detectable label is selected from the group consisting of an enzyme that catalyzes the development of a substrate, a chemiluminescent reagent, a fluorescent microsphere, a radioisotope or a nanoparticle-based label;
preferably, the enzyme catalyzing the development of a substrate is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase or glucose-6-phosphate deoxygenase;
preferably, the chemiluminescent agent is selected from the group consisting of acridinium esters and derivatives thereof, luminol and derivatives thereof, ruthenium bipyridine and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, dioxane and derivatives thereof, lomustine and derivatives thereof, or peroxyoxalate and derivatives thereof;
preferably, the fluorescent substance on the fluorescent microsphere is selected from fluorescein dye and its derivative, rhodamine dye and its derivative, cy dye and its derivative, alexa dye and its derivative or protein dye and its derivative.
4. The detection reagent for a CD89-IgA immune complex of claim 1, wherein the detection reagent further comprises a calibrator;
preferably, the calibrator comprises at least one of a CD89-IgA complex formed by binding of recombinant CD89 to recombinant multimeric IgA or multimeric IgA purified from commercial human serum IgA, a CD89-IgA complex formed by directional coupling of CD89 to multimeric IgA, a CD89-IgA complex native to commercial human serum IgA.
5. A diagnostic product of IgA nephropathy, allergic purpura and/or IgA immune complex related diseases, characterized in that the product comprises a detection reagent of CD89-IgA immune complex according to any one of claims 1 to 4.
6. A method for detecting CD89-IgA immune complexes of non-diagnostic interest, characterized in that the method uses the detection reagent of CD89-IgA immune complexes according to any one of claims 1 to 4 or the diagnostic product according to claim 5.
7. The method of detecting a CD89-IgA immune complex of claim 6 comprising the steps of:
s1, capturing a CD89-IgA immune complex by using a capture antibody;
s2, detecting the secondary antibody specific binding of the CD89-IgA immune complex captured in the step S1;
s3, detecting a luminescence value or a fluorescence value after processing the detectable marker for detecting the secondary antibody mark;
s4, calculating the content of the CD89-IgA immune complex in the sample according to a standard curve.
8. Use of a detection reagent for a CD89-IgA immune complex according to any one of claims 1 to 4 for the preparation of a diagnostic product for IgA nephropathy, allergic purpura and/or diseases associated with the IgA immune complex.
9. The use of claim 8, wherein the diagnostic product comprises any one of a kit, a chip or a test strip.
10. The use of claim 8, wherein the diagnostic product further comprises non-CD 89-bound IgA complex detection reagents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311703460.6A CN117890576A (en) | 2023-12-12 | 2023-12-12 | Reagent and method for detecting CD89-IgA immune complex and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311703460.6A CN117890576A (en) | 2023-12-12 | 2023-12-12 | Reagent and method for detecting CD89-IgA immune complex and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117890576A true CN117890576A (en) | 2024-04-16 |
Family
ID=90649950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311703460.6A Pending CN117890576A (en) | 2023-12-12 | 2023-12-12 | Reagent and method for detecting CD89-IgA immune complex and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117890576A (en) |
-
2023
- 2023-12-12 CN CN202311703460.6A patent/CN117890576A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11959912B2 (en) | Fluorescence immunochromatographic detection card and a preparation method therefor and use thereof | |
| KR101702117B1 (en) | Lateral Flow Assay Strip Senor for Measurement of Hihg Concentration of Biomolecule | |
| JP3551678B2 (en) | Method and kit for measuring hemoglobin A1c | |
| CN108414748A (en) | A kind of test strip and detection method of THSD7A antibody | |
| US20140322724A1 (en) | Homogeneous competitive lateral flow assay | |
| KR20190049898A (en) | Signal amplification in lateral flow and related immunoassays | |
| CN110361547B (en) | Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health | |
| CN106066399A (en) | The preparation method of the colloidal gold strip of detection early diabetic nephropathy | |
| CN115097129B (en) | Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1 | |
| CN107918022A (en) | A kind of cTnI detection kits and its application method | |
| JP2002530651A (en) | Method for detecting the presence of an analyte in a biological sample | |
| CN109521200A (en) | It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma | |
| KR20160120675A (en) | Rapid Quantitative Diagnostic Kit | |
| CN206038688U (en) | Immunity chromatography detection test strip's fluorescent quantitation spectral detection system | |
| CN115184620B (en) | Quantum dot fluorescence detection test strip and kit for PLA2R antibody and application of quantum dot fluorescence detection test strip and kit | |
| CN101963618B (en) | Method for identifying heterophilic antibody interference in antibody microarray system and antibody microarray chip using same for detecting target antigen | |
| CN117890576A (en) | Reagent and method for detecting CD89-IgA immune complex and application thereof | |
| US20230055382A1 (en) | Detecting gut barrier dysfunction and/or cirrhosis | |
| JP3998245B2 (en) | Method and kit for measuring oxidized apolipoprotein AI and oxidized lipoprotein containing the same | |
| CN102959398B (en) | Marker containing HPaR as active ingredient for diagnosing lung cancer | |
| KR101251222B1 (en) | Composition comprising APE1/Ref-1 for diagnosis of bladder cancer, and diagnosis kit of bladder cancer using the same | |
| US20180196042A1 (en) | Homogeneous competitive lateral flow assay | |
| JP3032891B2 (en) | Reagent kit for measuring free hemoglobin and method for measuring free hemoglobin using the same | |
| JP4588053B2 (en) | Method and kit for measuring oxidized apolipoprotein AI and oxidized lipoprotein containing the same | |
| CN109997043B (en) | point of care assay |
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 |