CN113009151A - Glycosylated CD59 detection kit based on chemiluminescence method and application thereof - Google Patents
Glycosylated CD59 detection kit based on chemiluminescence method and application thereof Download PDFInfo
- Publication number
- CN113009151A CN113009151A CN202110196369.4A CN202110196369A CN113009151A CN 113009151 A CN113009151 A CN 113009151A CN 202110196369 A CN202110196369 A CN 202110196369A CN 113009151 A CN113009151 A CN 113009151A
- Authority
- CN
- China
- Prior art keywords
- sample
- solution
- hole
- plate
- liquid
- 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.)
- Granted
Links
- 102100022002 CD59 glycoprotein Human genes 0.000 title claims abstract description 46
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000000523 sample Substances 0.000 claims abstract description 71
- 239000007788 liquid Substances 0.000 claims abstract description 55
- 238000007789 sealing Methods 0.000 claims abstract description 42
- 102000004190 Enzymes Human genes 0.000 claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 claims abstract description 40
- 239000000126 substance Substances 0.000 claims abstract description 37
- 238000005406 washing Methods 0.000 claims abstract description 31
- 239000003085 diluting agent Substances 0.000 claims abstract description 19
- 230000013595 glycosylation Effects 0.000 claims abstract description 18
- 238000006206 glycosylation reaction Methods 0.000 claims abstract description 18
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 9
- 239000012472 biological sample Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 52
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 238000000576 coating method Methods 0.000 claims description 17
- 239000011248 coating agent Substances 0.000 claims description 16
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- 101100476210 Caenorhabditis elegans rnt-1 gene Proteins 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 239000002250 absorbent Substances 0.000 claims description 9
- 230000002745 absorbent Effects 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 8
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 8
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 8
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 8
- 102000013415 peroxidase activity proteins Human genes 0.000 claims description 7
- 235000018102 proteins Nutrition 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 238000011049 filling Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000005018 casein Substances 0.000 claims description 5
- -1 casein sodium salt Chemical class 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 4
- 238000002965 ELISA Methods 0.000 claims description 4
- 238000010009 beating Methods 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000011888 foil Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 3
- 238000004020 luminiscence type Methods 0.000 claims description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 3
- VSMDINRNYYEDRN-UHFFFAOYSA-N 4-iodophenol Chemical compound OC1=CC=C(I)C=C1 VSMDINRNYYEDRN-UHFFFAOYSA-N 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000007547 defect Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000011002 quantification Methods 0.000 abstract description 2
- 102000003992 Peroxidases Human genes 0.000 abstract 2
- 206010012601 diabetes mellitus Diseases 0.000 description 14
- 238000011084 recovery Methods 0.000 description 8
- 238000012216 screening Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 241001529936 Murinae Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000101 novel biomarker Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- KQDJTBPASNJQFQ-UHFFFAOYSA-N 2-iodophenol Chemical compound OC1=CC=CC=C1I KQDJTBPASNJQFQ-UHFFFAOYSA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 108010055167 CD59 Antigens Proteins 0.000 description 1
- 102000001324 CD59 Antigens Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 1
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000009693 chronic damage Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108091005996 glycated proteins Proteins 0.000 description 1
- 230000036252 glycation Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000009461 vacuum packaging Methods 0.000 description 1
- 238000012795 verification 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a glycosylation CD59 detection kit based on a chemiluminescence method, which comprises a luminescent plate coated with an anti-gCD 59 antibody, a gCD59 standard substance gradient solution, a sample diluent, a peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, a 20 multiplied concentrated washing liquid, a luminescent liquid sealing film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the 20 multiplied concentrated washing liquid, the luminescent liquid sealing film and the sealing bag. The invention also discloses application of the kit in detecting gCD 59-containing biological samples. Experiments prove that the kit disclosed by the invention is high in stability, strong in selectivity, high in detection speed, low in cost, easy to operate, and capable of overcoming the defects of low sensitivity, long operation time and the like in gCD59 detection in the prior art, the operation time is shortened from 400 minutes to 40 minutes, the quantification limit is improved from 18.75pg/mL to 5pg/mL, and the kit has a very good clinical application prospect.
Description
Technical Field
The invention relates to detection of glycosylated CD59(gCD59), in particular to a glycosylated CD59 detection kit based on a chemiluminescence method and application thereof, and belongs to the technical field of clinical examination.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia. The chronic hyperglycemia results in chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves. In 70% of diabetic patients, proliferative lesions of small blood vessels and capillaries appear in whole body, and the pathogenesis of the proliferative lesions is related to long-term blood sugar rise, increase of glycated protein end products, expression of intercellular adhesion molecules, increase of certain cytokines and thickening of blood vessel basement membrane caused by intravascular subcutaneous tissue and polysaccharide substance deposition.
Glycosylated CD59(gCD59) is a novel biomarker. CD59 is a complement regulatory protein that protects "self" cells from complement-mediated damage (Davies CS et al. Glycation of CD59 antigens regulation on extracellular cells from diabatic subjects. in diabetes, CD59 is inactivated by non-enzymatic glycosylation to form gCD59. plasma gCD59 is a soluble form of CD59 that is shed from the cell membrane. CD59 is a widely distributed membrane-bound inhibitor of the complement cytolytic Membrane Attack Complex (MAC). CD59 acts by binding to C8 and/or C9 in nascent MAC and interferes with the insertion and polymerization of C9 membrane. this protein present in all cells is anchored to the outer surface of the membrane by a lipid tail. thus, it is exposed to extracellular and extracellular fluid glucose levels. soluble CD59 shed from the cell membrane is present in circulating and urine patients, gCD59 is present in diabetic patients in elevated blood glucose levels that are therefore evident in normal blood, the detection of the gCD59 content in the human body fluid has guiding significance for clinical detection and screening of diabetics.
In the prior art, a detection method of related glycosylated CD59 is reported, but no registration character number of the gCD59 detection kit is searched for both a domestic reagent and an imported reagent when a website of the national food and drug administration is searched. The related enzyme-linked immunoassay methods reported in the literature are all as follows: the monoclonal antibody Of anti CD59 is a capture antibody, the monoclonal antibody Of anti gCD59 is a detection antibody, the secondary antibody is labeled by an enzyme And is a goat anti-rabbit IgG-Horse Radish Peroxidase (HRP) marker or SA-HRP, the sensitivity Of the kit is not high, the operation time is as long as 200 minutes (a product Of G Biosciences, which is not registered at home) to 400 minutes (Pamela Ghosh, A Specific And Sensitive Assay For Blood Levels Of Cd < Glycated 59: A Novel Biomarker For Diabetes). There is no report on a detection method using a monoclonal antibody against gCD59 as a capture antibody and a monoclonal antibody against CD59 as a labeled antibody based on a chemiluminescence method, and a detection kit prepared by a chemiluminescence immunoadsorption method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a glycosylated CD59 detection kit based on a chemiluminescence method and application thereof.
The invention relates to a glycosylation CD59 detection kit based on a chemiluminescence method, which is composed of a luminescent plate coated with an anti-gCD 59 antibody, a gCD59 standard substance gradient solution, a sample diluent, a peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, a 20 multiplied concentrated washing solution, a luminescent liquid, a sealing plate film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the luminescent liquid, the sealing plate film, the sealing bag;
the method is characterized in that:
the luminescent plate is a transparent polystyrene 96-hole luminescent plate, and each hole of the luminescent plate is coated with a mouse monoclonal antibody Mab1 with the concentration of 5 mug/mL and resisting gCD 59;
the gCD59 standard gradient solutions were: 7 standard solutions with concentration gradients of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, SDS 0.1g, casein sodium salt 5g, TritonX-1000.1 mL, Proclin 3001 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab 2; the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate has a working titer of 1:10000, and the diluent is an enzyme conjugate diluent;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
In the above chemiluminescence-based glycosylation CD59 detection kit, the preparation method of the luminescent plate comprises the following steps:
1.1) preparing a coating liquid: namely, preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) diluting a mouse monoclonal antibody Mab1 of anti-gCD 59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking the marked enzyme label plate, using 8-pore channel gun array to spot the plate, leading the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01 percent CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
1.7) taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid into each hole for each time, washing for 2 times, and beating the absorbent paper to be dry;
1.8) adding 250 mu L of sealing liquid into each hole, and sealing for 16 hours at 2-8 ℃ overnight;
1.9) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.10) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
In the above-mentioned glycosylation CD59 detection kit based on chemiluminescence method: the mouse monoclonal antibody Mab1 resisting gCD59 and the mouse monoclonal antibody Mab2 resisting CD59 are preferably products of Shandongbao Biotechnology GmbH; wherein the amino acid sequence of antibody Mab1 directed against an epitope is: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
In the above glycosylation CD59 detection kit based on chemiluminescence method, the preparation method of the gCD59 standard substance gradient solution is: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; and preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution.
In the above-mentioned glycosylation CD59 detection kit based on chemiluminescence method, the formula of the enzyme conjugate diluent is: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
The precision and accuracy of the kit are verified by testing a median gCD59 standard product in parallel; the accuracy of the kit is verified from the aspect of sample recovery rate of the kit; the sensitivity of the kit is determined by detecting different amounts gCD59 with gCD59 concentration gradient.
The invention discloses application of a glycosylated CD59 detection kit based on a chemiluminescence method in detection of a gCD 59-containing biological sample.
Wherein the method for detecting the gCD 59-containing sample comprises the following steps:
1) sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
The glycosylation CD59 detection kit based on the chemiluminescence method is prepared based on the chemiluminescence immunoadsorption method, and tests on clinical samples show that the glycosylation CD59 detection kit (chemiluminescence method) can effectively detect the gCD59 content in a human body and is consistent with the judgment result of clinical diabetic patients, so that the glycosylation CD59 detection kit has better clinical application value. It has the obvious advantages that:
1. since gCD59 and CD59 have only a very small difference in protein structure, it is important to prepare a specific monoclonal antibody that can specifically recognize gCD59 but not CD 59. Through extensive and repeated screening and verification, the gCD59 detection antibody in the invention is a monoclonal antibody which specifically recognizes gCD59 and does not bind CD59, and therefore, the gCD59 detection antibody provides a reliable biological raw material for the specific detection gCD59 in the invention.
2. In the detection method, the gCD59 monoclonal antibody is used as the capture antibody, and the first step, namely the specificity capture gCD59 protein, is adopted in the methodology, so that the non-specific adsorption phenomenon is greatly reduced, the detection method is more direct, and the result is more accurate. This overcomes the disadvantage of non-specific binding in the first step of the reported detection method.
3. The kit provided by the invention is simple to operate, the marker is stable, the sensitivity is high, and the defects of complicated and unstable operation steps of the conventional enzyme-linked immunoassay method are completely overcome.
4. CTAB is added in the coating process, so that decontamination and impurity removal are performed, and the coating effect is improved, thereby increasing the sensitivity of the kit.
The glycosylation CD59 detection kit based on the chemiluminescence method provides a more convenient detection method based on an ELISA method, and the method has the advantages of high stability, high sensitivity, strong selectivity, high detection speed, low cost, easy operation and the like. The method overcomes the defects of low sensitivity, long operation time and the like in gCD59 detection in the prior art, shortens the operation time from 400 minutes to 40 minutes, improves the limit of quantification from 18.75pg/mL to 5pg/mL, and has excellent clinical application prospect.
Drawings
FIG. 1: the glycosylation CD59 detection kit based on the chemiluminescence method quantitatively analyzes a chart.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.
Example 1: gCD59 preparation of protein
The published gCD59 amino acid sequence (DACLITKAGLQVYNKCWKFEHC) and CD59 amino acid sequence (LQCYNCPNPTADCKTAVNCSS) are used to obtain gCD59 and CD59 corresponding nucleotide sequences by protein expression technology known to those skilled in the biological field.
Constructing a plasmid for expressing gCD59, namely pcDNA3.1-gCD59 expression vector, transfecting CHO cells, screening positive clones, culturing and purifying to obtain gCD59 protein.
The CD59 protein can be prepared by similar method.
Example 2: preparation of anti-gCD 59 murine monoclonal antibody Mab1 and anti-CD 59 murine monoclonal antibody Mab2
The obtained gCD59 and CD59 proteins were immunized to healthy BALB/c mice (8 weeks old), respectively, and after the mice produced antibodies, anti-gCD 59 murine monoclonal antibody Mab1 and anti-CD 59 murine monoclonal antibody Mab2 were prepared by conventional myeloma fusion cell technology, respectively.
Experiments prove that the amino acid sequence of the prepared antibody Mab1 for the antigen epitope is as follows: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
Wherein, the obtained Mab1 was verified not to cross-react with CD59, and Mab2 was verified not to cross-react with gCD 59. The specific process adopts hybridoma antibody preparation technology commonly known by those in the biological field.
Further, the antibody Mab2 was used to prepare enzyme conjugates by the sodium periodate-labeled HRP method.
Example 3: preparation of glycosylation CD59 detection kit based on chemiluminescence method
1. Preparing the luminescent plate:
preparing a coating solution: preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
filtering for sterilization, and storing at 4 deg.C;
the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
diluting a mouse monoclonal antibody Mab1 of gCD59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
taking a marked enzyme label plate, using an 8-pore row gun to spot the plate, enabling the coated antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
placing in a refrigerator at 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01% CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, drying absorbent paper, adding 300 μ L of washing solution into each hole, washing for 2 times, and drying absorbent paper;
adding 250 mu L of sealing liquid into each hole, sealing for 16 hours at 2-8 ℃ overnight;
taking out the coated plate, balancing at room temperature for 30min, throwing off confining liquid, and patting dry absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
vacuum packaging with aluminum foil bag, marking, and storing at 2-8 deg.C;
2. preparation of a kit solution:
preparation of the base solution
The sample diluent formula is:
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
the formula of 20 × concentrated washing solution is: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
gCD 59A standard gradient solution was prepared as follows: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution;
the formulation of the enzyme conjugate diluent was: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL;
the luminous liquid comprises a liquid A and a liquid B, and the formula of the luminous liquid is as follows: the solution A is a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L iodophenol solution in equal volume ratio; the solution B is hydrogen peroxide with the concentration of 7.5 mmol/L; the solution A and solution B were mixed at a volume ratio of 1: 1.
Example 4: method for detecting gCD 59-containing sample by using kit of the invention
1) Sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
Example 5: the glycosylation CD59 detection kit based on the chemiluminescence method has the advantages of detection of linearity, accuracy, recovery rate and precision
1) Linearity of the kit
The standard solution is adopted to have 7 concentration gradients which are respectively as follows: 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, RLU value readings of standard solutions were performed using the standard protocol of the present invention, and the linear assay curves of the kits of the present invention were plotted against the recorded test values, see FIG. 1.
As can be seen from FIG. 1, the glycosylation CD59 detection kit based on the chemiluminescence method has good linearity.
2) The accuracy of the kit was verified by sample recovery.
gCD59 was added to the standard buffer to give gCD59 final concentrations of 20ng/mL, 210ng/mL, 1050ng/mL, respectively, and each sample was tested in3 replicates and the recovery statistics calculated are shown in Table 1.
Table 1: recovery rate
The result shows that the recovery rate of gCD59 samples with high, medium and low concentrations is between 85.8 and 108.9 percent, the average recovery rate is between 88.6 and 106 percent, and the overall recovery rate is 98.9 percent.
3) Precision test:
the standard substance with 100ng/mL is tested 10 times in parallel, and the statistics of the results are shown in Table 2.
Table 2: results of precision test
Precision: CV% ═ 5.4%
Example 6: application of glycosylation CD59 detection kit based on chemiluminescence method in diabetes clinical sample screening
gCD59 is detected by using the kit, and the index is simultaneously applied to screening in the diabetic patients.
Through cooperation with a cooperative hospital, the hospital collects 100 diabetic clinical serum samples and 200 non-diabetic clinical serum samples. The result of using the kit to detect gCD59 in the serum sample shows that gCD59 is obviously higher in individuals with diabetes than in individuals without diabetes, and is independently related to glycosylated hemoglobin, so that the diabetic patients are determined to have high specificity and sensitivity. See table 3.
Table 3: diabetic patient screening
Claims (7)
1. A glycosylation CD59 detection kit based on chemiluminescence method comprises a luminescent plate coated with anti-gCD 59 antibody, gCD59 standard substance gradient solution, sample diluent, peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, 20 Xconcentrated washing solution, luminescent solution, a sealing plate film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the 20 Xconcentrated washing solution, the luminescent solution, the sealing plate film, the sealing bag and the instruction book are arranged;
the method is characterized in that:
the luminescent plate is a transparent polystyrene 96-hole luminescent plate, and each hole of the luminescent plate is coated with a mouse monoclonal antibody Mab1 with the concentration of 5 mug/mL and resisting gCD 59;
the gCD59 standard gradient solutions were: 7 standard solutions with concentration gradients of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, SDS 0.1g, casein sodium salt 5g, TritonX-1000.1 mL, Proclin 3001 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab 2; the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate has a working titer of 1:10000, and the diluent is an enzyme conjugate diluent;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double steamIn water, NaCl 8.0g and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
2. The chemiluminescence-based glycosylation CD59 assay kit according to claim 1, wherein the luminescent plate is prepared by:
1.1) preparing a coating liquid: namely, preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) diluting a mouse monoclonal antibody Mab1 of anti-gCD 59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking the marked enzyme label plate, using 8-pore channel gun array to spot the plate, leading the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01 percent CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
1.7) taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid into each hole for each time, washing for 2 times, and beating the absorbent paper to be dry;
1.8) adding 250 mu L of sealing liquid into each hole, and sealing for 16 hours at 2-8 ℃ overnight;
1.9) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.10) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
3. The chemiluminescence-based glycosylated CD59 detection kit according to claim 1, wherein: the mouse monoclonal antibody Mab1 resisting gCD59 and the mouse monoclonal antibody Mab2 resisting CD59 are products of Torilebo Biotech, Inc., Shandong; wherein the amino acid sequence of antibody Mab1 directed against an epitope is: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
4. The chemiluminescence-based glycosylated CD59 detection kit according to claim 1, wherein: the preparation method of the gCD59 standard substance gradient solution comprises the following steps: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; and preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution.
5. The glycosylated CD59 enzyme-linked immunoassay kit according to claim 1, wherein the enzyme conjugate diluent is formulated as: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
6. Use of the chemiluminescence-based glycosylated CD59 detection kit of claim 1 for detecting gCD 59-containing biological samples.
7. The use of claim 6, wherein the gCD 59-containing sample is detected by:
1) sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110188962 | 2021-02-19 | ||
| CN2021101889624 | 2021-02-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113009151A true CN113009151A (en) | 2021-06-22 |
| CN113009151B CN113009151B (en) | 2024-04-02 |
Family
ID=76405535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110196369.4A Active CN113009151B (en) | 2021-02-19 | 2021-02-22 | Glycosylated CD59 detection kit based on chemiluminescence method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113009151B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2612303A1 (en) * | 2004-06-17 | 2006-01-26 | Jose Halperin | Anti-glycated cd59 antibodies and uses thereof |
| US20180265589A1 (en) * | 2010-08-25 | 2018-09-20 | President And Fellows Of Harvard College | Glycated cd59 peptides, their preparation, and uses thereof |
| CN110045123A (en) * | 2018-01-13 | 2019-07-23 | 博奥赛斯（天津）生物科技有限公司 | A kind of carbohydrate antigen 242 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN110907649A (en) * | 2019-12-23 | 2020-03-24 | 山东莱博生物科技有限公司 | Kit for detecting short-chain fatty acid and application thereof |
-
2021
- 2021-02-22 CN CN202110196369.4A patent/CN113009151B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2612303A1 (en) * | 2004-06-17 | 2006-01-26 | Jose Halperin | Anti-glycated cd59 antibodies and uses thereof |
| US20180265589A1 (en) * | 2010-08-25 | 2018-09-20 | President And Fellows Of Harvard College | Glycated cd59 peptides, their preparation, and uses thereof |
| CN110045123A (en) * | 2018-01-13 | 2019-07-23 | 博奥赛斯（天津）生物科技有限公司 | A kind of carbohydrate antigen 242 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN110907649A (en) * | 2019-12-23 | 2020-03-24 | 山东莱博生物科技有限公司 | Kit for detecting short-chain fatty acid and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 聊菲 等: "CD59锚定蛋白对CD55介导的T细胞信号转导的增强效应", 细胞与分子免疫学杂志, vol. 27, no. 2, 31 December 2011 (2011-12-31), pages 128 - 130 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113009151B (en) | 2024-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5290678A (en) | Diagnostic kit for diagnosing and distinguishing chest pain in early onset thereof | |
| CN111057150B (en) | Latex microsphere, application thereof and glycosylated hemoglobin detection kit | |
| CN108570104A (en) | Recombinating adiponectin antigen, antibody and adiponectin nano rubber latex enhances immunoturbidimetry kit | |
| CN112904009B (en) | Magnetic microsphere detection kit for detecting glycosylated CD59 and application thereof | |
| EP3640644B1 (en) | Target marker gp73 for detecting steatohepatitis and detection application method | |
| CN109613280A (en) | A kind of serum iron determination kit and its preparation method and application | |
| CN113009152B (en) | Glycosylated CD59 enzyme-linked immunoassay kit and preparation method and application thereof | |
| CN113009151A (en) | Glycosylated CD59 detection kit based on chemiluminescence method and application thereof | |
| CN112285359A (en) | Saliva liquefaction sugar chain antigen determination kit and detection method thereof | |
| CN115097138B (en) | Application of buffer solution in PGP9.5 detection kit | |
| CN114966055B (en) | Application of buffer solution in colloidal fiber acidic protein detection kit | |
| CN111398600B (en) | ELISA detection kit for human soluble BCMA protein, and use method and application thereof | |
| CN111856037B (en) | Application of ratio of UNC45A to HSP10 in early warning, diagnosis and prognosis evaluation of POP (point of sale) and product | |
| CN111596068B (en) | Application of Utrophin in early warning, diagnosis and prognosis evaluation of POP (Point of Presence) and product | |
| CN108226520A (en) | A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology | |
| CN110068687B (en) | Quantitative measurement method for non-diagnostic purpose of myocardial troponin I complex in serum | |
| CN112485441A (en) | Anti-streptolysin O detection kit | |
| CN109444431A (en) | A kind of quantitative detecting method and detection kit of cardiac myosin binding protein C | |
| CN108226529A (en) | A kind of NT-proBNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology | |
| CN108107222A (en) | A kind of Soluble growth stimulates 2 protein immunization detection reagent of expressing gene and its preparation and detection method | |
| CN113156130B (en) | ELISA kit for detecting CD47 and preparation method and application thereof | |
| EP1256802A1 (en) | Method for examination of feces occult blood | |
| CN115290892B (en) | Application of buffer solution in brain-specific protein product 9.5 detection kit | |
| CN112903998B (en) | EB virus NA1-IgA antibody enzyme-linked immunoassay kit and detection method thereof | |
| CN111596067B (en) | Application of ZC3H8 in early warning, diagnosis and prognosis evaluation of POP (Point of Presence) |
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 |