CN113721032A - Novel quantitative detection kit for coronavirus neutralizing antibody and application thereof - Google Patents
Novel quantitative detection kit for coronavirus neutralizing antibody and application thereof Download PDFInfo
- Publication number
- CN113721032A CN113721032A CN202111025551.XA CN202111025551A CN113721032A CN 113721032 A CN113721032 A CN 113721032A CN 202111025551 A CN202111025551 A CN 202111025551A CN 113721032 A CN113721032 A CN 113721032A
- Authority
- CN
- China
- Prior art keywords
- neutralizing antibody
- microporous plate
- solution
- protein
- concentration
- 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
- 230000003472 neutralizing effect Effects 0.000 title claims abstract description 158
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 57
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 239000000523 sample Substances 0.000 claims abstract description 52
- 238000005406 washing Methods 0.000 claims abstract description 50
- 239000000243 solution Substances 0.000 claims abstract description 37
- 239000003085 diluting agent Substances 0.000 claims abstract description 34
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 claims abstract description 25
- 102100037236 Tyrosine-protein kinase receptor UFO Human genes 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 21
- 101000807561 Homo sapiens Tyrosine-protein kinase receptor UFO Proteins 0.000 claims abstract description 20
- 239000012086 standard solution Substances 0.000 claims abstract description 20
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 claims abstract description 19
- 108091005804 Peptidases Proteins 0.000 claims abstract description 18
- 239000004365 Protease Substances 0.000 claims abstract description 18
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 17
- 239000012089 stop solution Substances 0.000 claims abstract description 14
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 claims abstract description 13
- 239000013642 negative control Substances 0.000 claims abstract description 11
- 239000013641 positive control Substances 0.000 claims abstract description 11
- 239000012472 biological sample Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 61
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 102000035195 Peptidases Human genes 0.000 claims description 17
- 238000000576 coating method Methods 0.000 claims description 15
- 238000011161 development Methods 0.000 claims description 14
- 238000007789 sealing Methods 0.000 claims description 14
- 238000010009 beating Methods 0.000 claims description 13
- 239000011248 coating agent Substances 0.000 claims description 13
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 13
- 229920000936 Agarose Polymers 0.000 claims description 12
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 12
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 12
- 239000004005 microsphere Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 12
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000002372 labelling Methods 0.000 claims description 9
- 230000031700 light absorption Effects 0.000 claims description 9
- 238000002791 soaking Methods 0.000 claims description 9
- 239000004793 Polystyrene Substances 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 229920002223 polystyrene Polymers 0.000 claims description 8
- 238000004364 calculation method Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 claims description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- 230000009977 dual effect Effects 0.000 claims description 6
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 101710192735 Tyrosine-protein kinase receptor UFO Proteins 0.000 claims description 5
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 5
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical group OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 108091005634 SARS-CoV-2 receptor-binding domains Proteins 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 3
- 238000003287 bathing Methods 0.000 claims description 3
- 229940078916 carbamide peroxide Drugs 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 229960005486 vaccine Drugs 0.000 abstract description 20
- 238000012360 testing method Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 7
- 238000006386 neutralization reaction Methods 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- 238000002255 vaccination Methods 0.000 description 6
- 241000700605 Viruses Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000006957 competitive inhibition Effects 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010998 test method 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
- G01N33/6854—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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit, which consists of a microporous plate 1 coated with a new coronavirus S1 protein, a microporous plate 2 coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book, wherein the microporous plate 1 is arranged in a box body. The invention also discloses application of the kit in detecting biological samples containing the novel coronavirus neutralizing antibody. The neutralizing antibody titer in a trace cell neutralizing test can be effectively estimated according to the concentration of the neutralizing antibody determined by the kit, and the method is used for evaluating the generation of the neutralizing antibody after the novel coronavirus vaccine is inoculated. The kit has good sensitivity and specificity and has important value for accurately evaluating the vaccine effect clinically.
Description
Technical Field
The invention relates to detection of a coronavirus neutralizing antibody, in particular to a novel quantitative detection kit of a coronavirus (SARS-CoV-2) neutralizing antibody and application thereof, belonging to the technical field of clinical examination.
Background
Since the new coronavirus (SARS-CoV-2, hereinafter referred to as new crown) was reported in 2019, the new crown epidemic spread continuously all over the world and the number of infection cases has been increasing. To effectively cope with epidemic situations, the development of new corona vaccines is being accelerated in all countries of the world, and up to now, several vaccines have been approved successively in each country and region for population vaccination. By 8 months in 2021, china has completed 19 hundred million doses of new crown vaccination.
In the evaluation of the clinical effectiveness of vaccines, the protective efficacy of the vaccines (i.e., the percentage of the incidence of disease prevented after vaccination) is the most critical evaluation index. Meanwhile, the detection of the neutralizing antibody also plays an important role in evaluating the immune response level of a human body after vaccination and the like.
The laboratory gold standard for detection of neutralizing antibodies is an infection inhibition assay, which mainly includes a Plaque Reduction Neutralization Test (PRNT) using live virus and a microcytotic neutralization test (CPE) that is analyzed by detecting the amount of cytopathic effect. In both methods, a certain amount of live virus is mixed with equivalent serum of different dilutions, and then a monolayer of cells prepared in advance is inoculated, and the degree of pathological changes of the cells is evaluated through different indexes to evaluate the titer of neutralizing antibodies. Neutralization assays with live viruses cultured in vitro reflect the titer of total neutralizing antibodies in human blood samples that block viral infection. However, the test method needs a professional cell culture process, has high requirements on the experimental grade, long period and complex operation, and is not suitable for high-throughput screening of common vaccination population.
At present, many scientific research institutions and enterprises at home and abroad take the neutralizing antibody detection reagent as a research and development direction and conduct much exploration. Generally, the detection of neutralizing antibodies is carried out by adopting an enzyme-linked immunosorbent assay based on the competitive inhibition principle based on single epitope or multiple epitopes. The proposed intended use of the product is related to the evaluation of the effectiveness after vaccination. However, the above products or technologies are all neutralizing antibody in vitro diagnostic and qualitative products, and the correlation between the concentration value of the neutralizing antibody and the protective efficacy of the vaccine cannot be effectively evaluated. Through retrieval, the detection kit which can accurately and quantitatively determine the novel coronavirus neutralizing antibody in the body of a new corona vaccine injection population, determine the affinity of the neutralizing antibody and facilitate the evaluation of the vaccine protection efficacy, and the preparation method and the application thereof are not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel coronavirus (SARS-CoV-2) neutralizing antibody quantitative detection kit and application thereof.
The invention relates to a novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit, which consists of a microporous plate 1 which is arranged in a box body and is coated with a new coronavirus S1 protein, a microporous plate 2 which is coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book;
the method is characterized in that:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate is coated with a new coronavirus S1 protein with the concentration of 1-5 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate is coated with an AXL protein (tyrosine protein kinase receptor UFO) with the concentration of 1-5 mug/ml and an ACE2 protein (angiotensin converting enzyme 2) with the concentration of 2-5 mug/ml, wherein the coating proportion of the AXL protein and the ACE2 protein in each micropore is 1: 2-1: 4;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL,pH 7.4;
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is an enzyme conjugate diluent, and the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL;
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 display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
In the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit: each hole of the micro-porous plate 1 is preferably coated with the new coronavirus S1 protein with the concentration of 3 mug/ml; each well of the microplate 2 is preferably coated with an AXL protein, i.e. tyrosine protein kinase receptor UFO, at a concentration of 2 μ g/ml and an ACE2 protein, i.e. angiotensin converting enzyme 2, at a concentration of 4 μ g/ml, wherein the AXL protein and the ACE2 protein are preferably coated in a ratio of 1:2 per well.
In the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit: the RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells; the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, measuring an NTD neutralizing antibody variable region sequence, fusing with humanized Fc, and performing recombinant expression through CHO cells.
The invention relates to the application of the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit in the detection of biological samples containing the novel coronavirus neutralizing antibody.
Further, the detection of the novel coronavirus neutralizing antibody comprises a content determination of the neutralizing antibody and an affinity determination of the neutralizing antibody; wherein,
the content determination method of the neutralizing antibody comprises the following steps:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 mu L of color development liquid A into the microporous plate 1 and 50 mu L of color development liquid B into the microporous plate 2, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of a standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample;
the method for measuring the affinity of the neutralizing antibody comprises the following steps:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: and calculating the inhibition rate according to the light absorption value, and judging the result.
And (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
Where S is an abbreviation for sample and CO is an abbreviation for cut off.
The invention relates to application of a novel coronavirus neutralizing antibody quantitative detection kit and application of an index in neutralizing antibody evaluation of a new corona vaccine inoculated population and a new corona infected rehabilitation population.
The novel quantitative detection kit for the coronavirus neutralizing antibody provided by the invention has the advantages that the concentration of the new corona neutralizing antibody and the affinity of the new corona neutralizing antibody in the human body injected with the new corona vaccine can be intuitively and accurately reflected; the concentration of the new corona neutralizing antibody in the new corona vaccine injection population is directly measured through the detection result of the microporous plate 1, and the high and low affinity of the new corona neutralizing antibody is directly reflected through the detection result of the microporous plate 2; the two indexes complement each other, and are beneficial to epidemiological investigation of epidemic diseases, immune antibody monitoring and vaccine immunity efficacy evaluation.
The detection principle of the novel quantitative detection kit for the coronavirus neutralizing antibody is as follows: coating a polystyrene microporous plate with new crown S1 protein, adding a sample diluent and a sample, combining a neutralizing antibody in the sample with the new crown S1 protein to form a complex, washing, combining with horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate (HRP-RBD-NTD) enzyme, forming an 'S1 protein-neutralizing antibody-RBD-NTD' complex if the sample contains a novel coronavirus neutralizing antibody, and not developing if the sample does not contain the novel coronavirus neutralizing antibody, and finally adding an enzyme substrate for developing, and reading OD values at 450nm and 630nm on an enzyme labeling instrument. The OD value is in direct proportion to the concentration of RBD neutralizing antibodies in the sample; coating a polystyrene microporous plate with AXL protein and ACE2 protein, adding sample diluent, a sample and a recombinant RBD + NTD protease conjugate (HRP-RBD-NTD), wherein a neutralizing antibody in the sample competes with the HRP-RBD-NTD to bind with the AXL protein and the ACE2 protein on the microporous plate, forming a 'neutralizing antibody-RBD-NTD' complex if the sample contains a novel coronavirus neutralizing antibody, forming an 'HRP-ACE 2-RBD' complex and an 'HRP-AXL-RBD' complex if the sample does not contain the novel coronavirus neutralizing antibody, finally adding an enzyme substrate for color development, and reading OD values at 450nm and 630nm on an enzyme labeling instrument. The OD value is inversely proportional to the activity of the neutralizing antibodies in the sample.
The invention has the beneficial effects that:
(1) detecting a new crown neutralizing antibody by using the S1 protein and the recombinant RBD + NTD fusion protein in a double-antigen sandwich form, tracing a neutralizing antibody standard product according to a WHO neutralizing antibody standard disc, and obtaining a detection result which is an international unit and has international standardization;
(2) there are two main sites for neocorona neutralizing antibodies: RBD zone and NTD zone. The published or developed novel in vitro detection mode of the crown neutralizing antibody mainly aims at the RBD region for detection, and a kit for the combined detection of the RBD region neutralizing antibody and the NTD region neutralizing antibody is rarely reported. The method utilizes the AXL protein and the ACE2 protein as capture proteins to carry out activity identification of neutralizing antibodies by adopting a competitive inhibition method, and is an effective supplement and improvement to the prior art.
(3) The kit for detecting the novel coronavirus neutralizing antibody based on the enzyme-linked immunosorbent assay disclosed by the invention provides a more convenient detection method, and has the effects of intuitively and accurately reflecting the concentration of the novel corona neutralizing antibody and the affinity of the novel corona neutralizing antibody in a novel corona vaccine injection population.
Drawings
FIG. 1 is a graph showing the correlation between the concentrations of neutralizing antibodies in the standard.
FIG. 2 is a linear analysis of the novel quantitative detection kit for coronavirus neutralizing antibodies of the invention.
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: preparation of novel coronavirus neutralizing antibody quantitative detection kit
1. Coating of the micro-porous plate:
the coating method of the micro-porous plate 1 comprises the following steps:
1.1) preparing a coating liquid: preparing carbonate buffer solution and adjusting the pH value to 9.6;
Na2CO3 1.59g
NaHCO3 2.94g
the purified water is fixed to the volume of 1000mL
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 the new coronavirus S1 protein to the working concentration of 1-5 mug/mL by using a coating solution, uniformly mixing, and standing for 10 min;
1.5) taking a marked microporous plate, using an 8-pore row gun to spot the plate, enabling the protein solution of the new coronavirus S1 to reach 150 mu L/pore, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-16 hours;
1.7) taking out the coated plate, balancing for 30min at room temperature, throwing off protein liquid, beating the water-absorbing paper to be dry, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating the water-absorbing 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.
The coating method of the micro-porous plate 2 comprises the following steps:
1.11) preparing a coating liquid: preparing carbonate buffer solution and adjusting the pH value to 9.6;
Na2CO3 1.59g
NaHCO3 2.94g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.12) 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;
1.13) 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;
1.14) respectively diluting the AXL protein and the ACE2 protein by using coating liquid to working concentrations of 1-5 mu g/ml AXL protein and 2-5 mu g/ml ACE2, calculating and controlling the coating amount of the AXL protein and the ACE2 protein per micropore to be 1: 2-1: 4, uniformly mixing, and standing for 10 min;
1.15) taking the marked microporous plate, using an 8-pore row gun to spot the plate, enabling the protein solution to reach 150 mu L/pore, and covering a cover plate film;
1.16) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-16 hours;
1.17) taking out the coated plate, balancing for 30min at room temperature, throwing off protein liquid, beating the water-absorbing paper to be dry, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating the water-absorbing paper to be dry;
1.18) adding 250 mu L of sealing liquid into each hole, sealing for 16 hours at 2-8 ℃ overnight;
1.19) 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;
1.20) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
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.
The RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells;
the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, fusing a determined NTD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO (Chinese hamster ovary) cells;
the neutralizing antibody standard substance gradient solution is respectively as follows: 7 standard solutions with concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, wherein 0AU/mL is standard buffer solution; the standard buffer solution is as follows: the pH of the phosphate buffer solution is 7.4.
The negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody.
The positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
in the above kit for quantitatively detecting a neutralizing antibody against a novel coronavirus, the formula of the diluent of the enzyme conjugate is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL。
The preparation method of the recombinant RBD + NTD enzyme conjugate comprises the following steps: the conjugate of the recombinant RBD + NDT protein marked by a sodium periodate method and horseradish peroxidase (HRP) is utilized. The preparation method is not limited to this method, and other protein labeling methods are also possible.
Optimization of the use amount of the recombinant RBD + NTD enzyme conjugate: and (3) selecting according to the sensitivity and the detection range as indexes by adopting a chessboard method. Preferred use titer concentrations are 1: 7000.
the formulation of the enzyme conjugate diluent was: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL;
The color developing agent is Tetramethylbenzidine (TMB) solution with the concentration of 0.3mg/mL and prepared by taking dimethyl sulfoxide (DMSO) as a solvent, named as solution A, and urea peroxide solution with the concentration of 0.74mg/mL and prepared by taking disodium hydrogen phosphate with the pH value of 5.5 and 0.2mol/L and citric acid buffer solution with the concentration of 0.1mol/L as a solvent, named as solution B, and when the color developing agent is used, the solution A and the solution B are mixed according to the volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
Example 2: the invention discloses application of a novel coronavirus neutralizing antibody quantitative detection kit in detection of a biological sample containing the novel coronavirus neutralizing antibody.
The novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit comprises a microporous plate 1 coated with a new coronavirus S1 protein, a microporous plate 2 coated with an AXL protein and an ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, a 20 multiplied concentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction book, wherein the microporous plate 1 is arranged in a box body;
specifically, the method comprises the following steps:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate 1 is coated with a new coronavirus S1 protein with the concentration of 3 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate 2 is coated with 2 mug/ml of AXL protein (tyrosine protein kinase receptor UFO) and 4 mug/ml of ACE2 protein (angiotensin converting enzyme 2), wherein the coating proportion of the AXL protein and the ACE2 protein in each hole is 1: 2;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL,pH 7.4;
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is an enzyme conjugate diluent, and the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL;
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 display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
The preferred method for detecting samples containing novel coronavirus neutralizing antibodies is:
a, content determination of neutralizing antibodies:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 muL of color development liquid A into the microporous plate 1 and the microporous plate 2, then adding 50 muL of color development liquid B, and carrying out dark warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
B neutralizing antibody affinity assay:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: and calculating the inhibition rate according to the light absorption value, and judging the result.
And (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
Example 3: the invention discloses a detection method for linearity, accuracy, recovery rate and precision of a novel coronavirus neutralizing antibody quantitative detection kit
1) Tracing and calibrating a neutralizing antibody standard product:
the neutralizing antibody standard solution is as follows: 7 standard solutions with a concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, comprising RBD neutralizing antibody and NTD neutralizing antibody at a molar ratio of 2: 1;
the first international standard (NIBSC code: 20/136) of the human anti-SARS-CoV-2 immunoglobulin of the world health organization is diluted according to the conversion relation of the neutralizing antibody (the international standard is the concentration of the neutralizing antibody/0.00798), and the corresponding international standard concentrations are respectively: 0AU/mL, 19.586AU/mL, 39.160AU/mL, 78.321AU/mL, 156.642AU/mL, 313.283AU/mL, 626.566AU/mL, the detection results of the international standard samples at each concentration are shown in Table 1, respectively, by performing the detection according to step A of example 2:
TABLE 1
Concentration correlation analysis: the correlation analysis of the concentration of neutralizing antibody in the standard is shown in FIG. 1.
2) Linearity of the kit
1) Preparing a standard neutralizing antibody: and 7 standard solutions with concentration gradient of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, wherein the RBD neutralizing antibody and the NTD neutralizing antibody are contained at a molar ratio of 2: 1.
2) And (3) testing a calibration sample: the OD value was obtained by testing the prepared calibrator by the method described in step a of example 2.
3) Establishment of a standard curve: and generating a calibration curve of the batch of reagents by adopting a proper fitting mode according to the prepared concentration and the OD value of the calibrator. See table 2 and figure 2.
Table 2: reagent kit linearity
Concentration of | 0AU/mL | 0.1563AU/mL | 0.3125AU/mL | 0.625AU/mL | 1.25AU/mL | 2.5AU/mL | 5AU/mL |
OD value | 0.012 | 0.150 | 0. | 0.412 | 0.441 | 1.089 | 2.357 |
Example 4 application of the novel coronavirus neutralizing antibody quantitative detection kit in screening clinical samples of injection vaccines
The immune samples of clinically injected new corona vaccines of 200 cases and the immune samples of not injected new corona vaccines of 400 cases are compared with the trace cell neutralization experiment, the statistical index of the coincidence or difference degree of the determination result of the kit of the invention and the trace cell neutralization experiment result by using the microporous plate 2 is calculated, and the evaluation method is shown in the table 3.
Table 3: the kit of the invention is compared with a trace cell neutralization test
And (3) sensitivity calculation: 194/194 × 100% ═ 100%.
And (3) calculating the specificity: 400/406X 100% ═ 98.52%
The total coincidence rate is as follows: (194+ 400)/600X 100% ═ 99%
The above tests show that the kit for detecting the novel coronavirus neutralizing antibody and the detection method thereof provided by the invention have good consistency with the gold standard for detecting the neutralizing antibody.
The results of the experiments of neutralizing the trace cells of the immune samples after completing the clinical injection of the new corona vaccine in 200 cases and the results of the assay of the kit of the invention by using the microplate 1 are counted, and the relationship of the antibody titer is shown in Table 4:
TABLE 4
Neutralization assays with live viruses cultured in vitro reflect the titer of total neutralizing antibodies in human blood samples that block viral infection. Due to the source of the live virus strain and the limitation of operation in a high-grade biosafety laboratory, the method has certain limitations on popularization and operability. The invention provides a method for quantitatively detecting a novel coronavirus neutralizing antibody and application thereof, which can effectively presume the neutralizing antibody titer in a trace cell neutralizing test according to the concentration of the detected neutralizing antibody, can be used for evaluating the generation of the neutralizing antibody after the inoculation of a novel coronavirus vaccine, has good sensitivity and specificity, and is favorable for accurately evaluating the vaccine effect clinically.
Claims (5)
1. A novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit comprises a microporous plate 1 coated with new coronavirus S1 protein, a microporous plate 2 coated with AXL protein and ACE2 protein, a neutralizing antibody standard solution, a positive control, a negative control, a sample diluent, a recombinant RBD + NTD protease conjugate, 20 Xconcentrated washing solution, a display solution A, a display solution B, a stop solution and an instruction, which are arranged in a box body;
the method is characterized in that:
the microporous plate 1 is a polystyrene microporous plate, and each hole of the microporous plate is coated with a new coronavirus S1 protein with the concentration of 1-5 mug/ml;
the microporous plate 2 is a polystyrene microporous plate, and each hole of the microporous plate is coated with an AXL protein (tyrosine protein kinase receptor UFO) with the concentration of 1-5 mug/ml and an ACE2 protein (angiotensin converting enzyme 2) with the concentration of 2-5 mug/ml, wherein the coating proportion of the AXL protein and the ACE2 protein in each micropore is 1: 2-1: 4;
the neutralizing antibody standard solution is a mixed solution containing an RBD neutralizing antibody and an NTD neutralizing antibody, wherein the molar ratio of the RBD neutralizing antibody to the NTD neutralizing antibody is 2: 1; the standard solution is 7 standard solutions with concentration gradients of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL and 5AU/mL respectively, wherein 0AU/mL is standard buffer solution: phosphate buffer, pH 7.4;
the positive control is a mixed solution of 3ug/ml RBD neutralizing antibody and 1ug/ml NTD neutralizing antibody;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、CTAB 1g、BSA 5g、TritonX-100 0.1mL、Proclin300 1mL,pH 7.4;
The RBD + NTD protease conjugate is a horseradish peroxidase-labeled recombinant RBD + NTD protease conjugate; the enzyme-labeled enzyme conjugate has a working titer of 1:7000, the diluent is enzyme conjugate diluentThe formula is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、BSA 3.5g、Proclin300 1mL;
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 display liquid A is a Tetramethylbenzidine (TMB) solution with the concentration of 0.3 mg/mL; the display liquid B is a carbamide peroxide solution with the concentration of 0.74 mg/mL; mixing the display liquid A and the display liquid B at a volume ratio of 1: 1;
the stop solution is a sulfuric acid solution with the concentration of 2 mol/L.
2. The kit for quantitatively detecting a neutralizing antibody against SARS-CoV-2 as a coronavirus according to claim 1, wherein: each hole of the micropore plate 1 is coated with a new coronavirus S1 protein with the concentration of 3 mug/ml; the microporous plate 2 is coated with 2 mug/ml AXL protein (tyrosine protein kinase receptor UFO) and 4 mug/ml ACE2 protein (angiotensin converting enzyme 2) in each hole, wherein the ratio of the AXL protein to the ACE2 protein in each hole is 1: 2.
3. The kit for quantitatively detecting a neutralizing antibody against SARS-CoV-2 as a coronavirus according to claim 1, wherein: the RBD neutralizing antibody is obtained by filtering inactivated novel coronavirus infector serum through an agarose microsphere chromatographic column provided with recombinant RBD protein coupling, wherein the RBD neutralizing antibody can be combined to an agarose microsphere, obtaining a purified RBD neutralizing antibody through impurity washing and elution, detecting and screening titer, fusing a determined RBD neutralizing antibody variable region sequence with humanized Fc, and performing recombinant expression through CHO cells; the NTD neutralizing antibody is obtained by filtering inactivated serum of a novel coronavirus infected person through an agarose microsphere chromatographic column provided with recombinant NTD protein coupling, combining the NTD neutralizing antibody on an agarose microsphere, washing, eluting to obtain a purified NTD neutralizing antibody, performing titer detection and screening, measuring an NTD neutralizing antibody variable region sequence, fusing with humanized Fc, and performing recombinant expression through CHO cells.
4. Use of the novel coronavirus SARS-CoV-2 neutralizing antibody quantitative detection kit as claimed in claim 1 in the detection of biological samples containing the novel coronavirus neutralizing antibody.
5. Use according to claim 4, characterized in that: the detection of the novel coronavirus neutralizing antibody comprises the content determination of the neutralizing antibody and the affinity determination of the neutralizing antibody; wherein,
the content determination method of the neutralizing antibody comprises the following steps:
(1) respectively adding 25uL of sample diluent into each hole in the microporous plate 1 and the microporous plate 2, and then respectively adding 25uL of samples to be detected into the holes of the microporous plate containing the sample diluent; in the microplate 1, 7 standard solutions of 0AU/mL, 0.1563AU/mL, 0.3125AU/mL, 0.625AU/mL, 1.25AU/mL, 2.5AU/mL, 5AU/mL, respectively, were added to each well containing the sample diluent at 25 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100uL of RBD + NTD protease conjugate into each of the microporous plate 1 and the microporous plate 2, mixing, sealing the reaction plate with a self-adhesive sealing sheet, and bathing at 37 deg.C for 20 min;
(4) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(5) respectively adding 50 mu L of color development liquid A into the microporous plate 1 and 50 mu L of color development liquid B into the microporous plate 2, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(6) respectively adding 50 mu L of stop solution into the microporous plate 1 and the microporous plate 2, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(7) the calculation method of the microplate 1 is as follows: drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the OD value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured OD value; or calculating a linear regression equation of a standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample to be measured into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample;
the method for measuring the affinity of the neutralizing antibody comprises the following steps:
(1) adding 25uL of sample diluent into each hole in the microporous plate 2, respectively adding 25uL of the sample to be detected, the positive control and the negative control into the hole of the microporous plate containing the sample diluent, adding 100uL of RBD + NTD protease conjugate, and carrying out warm bath at 37 ℃ for 30 minutes;
(2) after the reaction is finished, washing the plate 5 +/-1 times by using washing liquid, soaking for 20-40 s each time, adding liquid in each hole of not less than 350 mu L, and drying by beating after washing;
(3) adding 100 mu L of color development liquid A into a microporous plate, then adding 100 mu L of color development liquid B, and carrying out light-proof warm bath at 37 ℃ for 10 minutes;
(4) adding 50 mu L of stop solution into the microporous plate, stopping the reaction, and detecting the light absorption value of each hole at the dual wavelengths of 450nm and 630nm by using an enzyme-labeling instrument;
(5) the calculation result method of the micro-porous plate 2 is as follows: calculating the inhibition rate according to the light absorption value, and judging the result;
and (4) result judgment standard: S/CO is more than or equal to 30%: novel coronavirus neutralizing antibody positive, S/CO < 30%: the novel coronavirus neutralizing antibody is negative.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111025551.XA CN113721032B (en) | 2021-09-02 | 2021-09-02 | Novel quantitative detection kit for neutralizing antibodies of coronaviruses and application of kit |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111025551.XA CN113721032B (en) | 2021-09-02 | 2021-09-02 | Novel quantitative detection kit for neutralizing antibodies of coronaviruses and application of kit |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113721032A true CN113721032A (en) | 2021-11-30 |
CN113721032B CN113721032B (en) | 2024-03-19 |
Family
ID=78680923
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111025551.XA Active CN113721032B (en) | 2021-09-02 | 2021-09-02 | Novel quantitative detection kit for neutralizing antibodies of coronaviruses and application of kit |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113721032B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
CN112098660A (en) * | 2020-11-03 | 2020-12-18 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus neutralizing antibody detection kit |
CN112748243A (en) * | 2020-12-23 | 2021-05-04 | 北京美康基因科学股份有限公司 | Novel coronavirus neutralizing antibody detection kit and preparation method thereof |
CN113009154A (en) * | 2021-02-25 | 2021-06-22 | 山东莱博生物科技有限公司 | One-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody and application thereof |
-
2021
- 2021-09-02 CN CN202111025551.XA patent/CN113721032B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
CN112098660A (en) * | 2020-11-03 | 2020-12-18 | 北京百普赛斯生物科技股份有限公司 | Novel coronavirus neutralizing antibody detection kit |
CN112748243A (en) * | 2020-12-23 | 2021-05-04 | 北京美康基因科学股份有限公司 | Novel coronavirus neutralizing antibody detection kit and preparation method thereof |
CN113009154A (en) * | 2021-02-25 | 2021-06-22 | 山东莱博生物科技有限公司 | One-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody and application thereof |
Non-Patent Citations (1)
Title |
---|
张赛;向乐;李林海;李辉军;王刚;钱纯亘;: "新型冠状病毒(2019-nCoV)IgM/IgG抗体检测试剂的研制及性能评价", 中国生物工程杂志, no. 08, 15 August 2020 (2020-08-15) * |
Also Published As
Publication number | Publication date |
---|---|
CN113721032B (en) | 2024-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113030483B (en) | Novel coronavirus neutralizing antibody ELISA detection kit and application thereof | |
CN111024954A (en) | Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof | |
AU2002248996B2 (en) | Detection of candida | |
CN113009154B (en) | Novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof | |
CN113009153B (en) | New coronavirus neutralizing antibody detection kit based on magnetic particle chemiluminescence and application thereof | |
CN111337673B (en) | Synthetic polypeptide composition for novel coronavirus immunodetection and application | |
CN109765383B (en) | Cat distemper virus antibody fluorescence detection test strip and preparation method and application thereof | |
CN111521786A (en) | Respiratory tract pathogen IgM antibody joint detection kit and preparation method thereof | |
CN111273017B (en) | Fluorescent immunochromatography kit for rapidly detecting novel coronaviruses | |
CN101735319A (en) | Monoclonal antibody against GP73 protein, preparation method and application thereof | |
CN112305218A (en) | Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof | |
CN111423496B (en) | Polypeptide or combination thereof for detecting novel coronavirus | |
CN110261623A (en) | A kind of detection sheep echinococcosis granulosa antibody indirect ELISA detection kit and its application | |
CN113321715A (en) | Novel coronavirus antigen and detection use thereof | |
CN113985037A (en) | Novel detection method, detection test strip and kit for coronavirus neutralizing antibody | |
JP2003279577A (en) | Composition for flow through type inspection, and kit using the same, and inspection method | |
CN115078737B (en) | Kit for detecting immune plasma titer of rabies, and preparation method and application thereof | |
Vrublevskaya et al. | Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera | |
CN113721032B (en) | Novel quantitative detection kit for neutralizing antibodies of coronaviruses and application of kit | |
CN113702634A (en) | Novel coronavirus high-affinity neutralizing antibody detection kit and preparation method and application thereof | |
Yang et al. | Development of immunochromatographic test strips for rapid, quantitative detection of H9AIV antibodies | |
Maya et al. | Evaluation of diagnostic accuracy of developed rapid SARS-COV-2 IgG antibody test kit using novel diluent system | |
CN113075405A (en) | Hepatitis B virus surface antigen detection kit and preparation method thereof | |
Feng et al. | Serological diagnosis of infectious mononucleosis by chemiluminescent immunoassay using capsid antigen p18 of Epstein-Barr virus | |
CN113607957A (en) | Specific neutralizing antibody competition method ELISA kit aiming at SARS-CoV-2 RBD structural domain |
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 |