CN113267624A - Method and product for detecting RBD and NTD neutralizing antibody - Google Patents
Method and product for detecting RBD and NTD neutralizing antibody Download PDFInfo
- Publication number
- CN113267624A CN113267624A CN202110477341.8A CN202110477341A CN113267624A CN 113267624 A CN113267624 A CN 113267624A CN 202110477341 A CN202110477341 A CN 202110477341A CN 113267624 A CN113267624 A CN 113267624A
- Authority
- CN
- China
- Prior art keywords
- reagent
- ntd
- sample
- protein
- rbm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000003472 neutralizing effect Effects 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims description 16
- 238000001514 detection method Methods 0.000 claims abstract description 49
- 241000711573 Coronaviridae Species 0.000 claims abstract description 16
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 90
- 108090000623 proteins and genes Proteins 0.000 claims description 42
- 102000004169 proteins and genes Human genes 0.000 claims description 40
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 claims description 27
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 claims description 27
- 239000012528 membrane Substances 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 11
- 102100031673 Corneodesmosin Human genes 0.000 claims description 10
- 101710139375 Corneodesmosin Proteins 0.000 claims description 10
- 239000003550 marker Substances 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 239000011859 microparticle Substances 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 4
- 210000003722 extracellular fluid Anatomy 0.000 claims description 4
- 210000004880 lymph fluid Anatomy 0.000 claims description 4
- 210000004698 lymphocyte Anatomy 0.000 claims description 4
- 210000003296 saliva Anatomy 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 claims description 4
- 210000002700 urine Anatomy 0.000 claims description 4
- 241000315672 SARS coronavirus Species 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000000243 solution Substances 0.000 description 16
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 15
- 238000002372 labelling Methods 0.000 description 12
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 10
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 10
- 230000000903 blocking effect Effects 0.000 description 8
- 239000006249 magnetic particle Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000003018 immunoassay Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 description 5
- 239000007987 MES buffer Substances 0.000 description 5
- 108091005609 SARS-CoV-2 Spike Subunit S1 Proteins 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 102000048657 human ACE2 Human genes 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 3
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000003365 glass fiber Substances 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- -1 particulate Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229940096437 Protein S Drugs 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710198474 Spike protein Proteins 0.000 description 2
- 230000010530 Virus Neutralization Effects 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 238000000643 oven drying Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000010618 wire wrap Methods 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282375 Herpestidae Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000711467 Human coronavirus 229E Species 0.000 description 1
- 241001109669 Human coronavirus HKU1 Species 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 241001428935 Human coronavirus OC43 Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 241000283966 Pholidota <mammal> Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 101150054399 ace2 gene Proteins 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000008876 conformational transition Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 239000006148 magnetic separator Substances 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000007860 single-cell PCR Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
Abstract
The invention relates to the field of antibody detection, in particular to a double competition detection method, a detection component and application of related antibodies, which can be applied to a detection method and a product of a new coronavirus (SARS-CoV-2) neutralizing antibody and can simultaneously detect RBD and NTD neutralizing antibodies.
Description
Technical Field
The invention relates to the field of antibody detection, in particular to a detection method and a product.
Background
The frequent sporadic accidents in China and the quick approval of the vaccine on the market are particularly urgent. Some vaccines are currently marketed globally with authorization for emergency use or additional conditions.
However, in the face of brand new viruses, under the background that more relevant basic researches and more mechanism researches are not thorough, a large number of vaccine designs rapidly enter the clinic, and more contents still need to be further researched on the effectiveness, the protection period, the quality control and the accessibility of the vaccines.
The first vaccine in China is mainly an inactivated vaccine, however, BPL (BPL-based protein) inactivation adopted by the inactivated vaccine is adopted, and Spike protein has high proportion of post-fusion conformation and RBD (reduced beta-amyloid) conformation, so that the evidence that the total antibody level response is relatively low and the neutralizing antibody titer is not high is found in the primary immune population analysis.
In addition, from the current basic research of new crowns, not all individuals will produce sufficient titers of neutralizing antibodies after infection with new crowns, with the risk of secondary infection. The neutralizing antibody titer gradually decreases after reaching the peak value in 1 month, and a small part of low-titer convalescent people can decrease below the detection limit. With reference to other coronaviruses, new corona antibodies may exist for about 1-2 years and do not form long-lasting protection.
Neutralizing antibodies to SARS-CoV-2 can effectively control infection by blocking or inhibiting the interaction between SARS-CoV-2 and the host cell. The best studied mechanism is the interaction between the Receptor Binding Domain (RBD) on the S1 subunit of the SARS-CoV-2 Spike protein and the host cell Receptor ACE2, followed by conformational transition and membrane fusion. ACE2, also known as achh and known as angiotensin converting enzyme 2, is a metalloprotease with 805 amino acids in total length and is a type I transmembrane glycoprotein with a single extracellular catalytic domain. A Receptor Binding Motif (RBM), which is part of the Receptor Binding Domain (RBD) that is in contact with ACE 2.
Many new neutralizing antibodies have also been isolated in current research, such as those that bind to the NTD region of the S1 subunit.
Partial vaccines currently show evidence of low positive conversion and titers of neutralizing antibodies after immunization. In addition, SARS-CoV-2 as RNA virus has very high mutation frequency, and the neutralizing effect of serum and vaccine in convalescent period shows lower neutralizing capacity to partial mutation, and the accumulation of the partial mutation may result in immunological escape. Thus, higher demands are made on the sensitivity and sensitivity of detection of neutralizing antibodies.
The existing product mainly detects a neutralizing antibody blocking the interaction of RBD-ACE2 by an RBD-ACE2 competition method, and cannot detect an NTD neutralizing antibody.
The invention is therefore proposed.
Disclosure of Invention
The invention provides at least one of the following embodiments:
in some embodiments, the invention relates to a method for detecting a coronavirus neutralizing antibody, comprising the steps of:
(1) contacting the sample with a fragment containing RBD, a reagent 1 and a reagent 2;
reagent 1: ACE2 or ACE2 fragment;
reagent 2: neutralizing antibodies that bind NTD;
wherein one of the reagent 1 or the reagent 2 is connected with a marker, and the other is fixed on a solid phase carrier;
(2) and detecting the signal.
In some embodiments, the contacting comprises any one of:
(a) simultaneously contacting the sample with protein containing RBM and NTD structural domains, a reagent 1 and a reagent 2;
(b) the sample is firstly contacted with protein containing RBM and NTD structural domains, a reagent 1 and then contacted with a reagent 2;
(c) firstly, contacting a sample with protein containing RBM and NTD structural domains, a reagent 2 and then a reagent 1;
(d) firstly, contacting a sample with protein containing RBM and NTD structural domains, and then contacting with a reagent 1 and a reagent 2;
(e) the sample is firstly contacted with protein containing RBM and NTD structural domains, then contacted with a reagent 1, and then contacted with a reagent 2;
(f) the sample is contacted with the protein containing the RBM and NTD domains, then with reagent 2, and then with reagent 1.
In some embodiments, the coronavirus is selected from SARS-CoV-2 or SARS-CoV.
In some embodiments, the sample is selected from any sample that may contain antibodies.
In some embodiments, the sample is selected from serum, plasma, whole blood, lymph fluid, cerebrospinal fluid, interstitial fluid, saliva, urine, or lymphocytes.
In some embodiments, the protein comprising the RBM and NTD domains is optionally S1 protein or S protein.
In some embodiments, a detection assembly for coronavirus neutralizing antibodies, comprising:
(a) an RBD-containing fragment according to any one of the embodiments above;
(b) the reagent 1 according to any of the above embodiments;
(c) the reagent 2 according to any of the above embodiments.
In some embodiments, the solid support is selected from a magnetic microparticle or an Elisa plate.
In some embodiments, a detection assembly for coronavirus neutralizing antibodies, comprising: protein with RBM and NTD structural domains and test paper; there are sample pad, conjugate pad, chromatographic carrier, absorbent pad and backing plate on the test paper, the test paper still includes: reagents 1 and 2 as described in any of the above embodiments.
Wherein, the reagent 1 is connected with a marker and arranged on the bonding pad, and the reagent 2 is fixed on the chromatographic membrane; or; the reagent 2 is connected with a marker and is arranged on the bonding pad, and the reagent 1 is fixed on the chromatographic membrane.
In some embodiments, the protein comprising the RBM and NTD domains is disposed on a sample pad.
In some embodiments, the detection method of any one of the above embodiments, or the detection module of any one of the above embodiments, is used for antibody detection or for preparing an antibody detection reagent.
Drawings
FIG. 1 shows the principle of the methodology of the dual competition assay neutralizing antibody, in which M represents a solid phase carrier, NTD-NAb represents an NTD neutralizing antibody, and RBD-NAb represents an RBD neutralizing antibody.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Herein, "neutralizing antibodies are certain antibodies produced by B lymphocytes that are capable of binding to antigens on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to the target cell receptor and invading the cell.
Herein, a "sample" is understood to be any sample that may contain antibodies, in some embodiments, the sample is from a sample after infection or active immunization; in some embodiments, the sample is selected from the group consisting of bodily fluids, excreta, cells; such as, but not limited to, serum, plasma, whole blood, lymph fluid, cerebrospinal fluid, interstitial fluid, saliva, urine, lymphocytes, etc.
Herein, "label" is understood to be capable of directly generating a signal; or directly or indirectly trigger the specific substance to generate a signal, and the label may be directly or indirectly linked to the labeled substance. For example, labels commonly used for immunodetection include, but are not limited to, metal particles, fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, electrochemiluminescent labels, radioactive labels, nucleic acid labels, polypeptide labels, or enzymes. In some embodiments, the label can be colloidal gold, fluorescein, fluorescent microspheres, acridinium ester, horseradish peroxidase, alkaline phosphatase, latex microspheres, ruthenium triad, luminols, Eu chelates.
As used herein, "solid support" is understood to mean a solid support capable of being immobilized to an object to be immobilized (e.g., a protein, a polypeptide), such as is commonly used in immunoassays, including plastic, particulate, or membrane supports. The plastic may be, for example, polystyrene; the particles may be, for example, magnetic particles; the membrane support may be, for example, a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane.
Herein, "detection signal" is understood to mean the acquisition or identification of the intensity or level of a detection signal in a manner that enables identification of the signal substance.
Herein, "contacting" is understood to allow binding thereof to occur. The contact time is not particularly limited, and may vary from embodiment to embodiment and from platform to platform, but is within the purview of one skilled in the art.
Herein, "protein containing RBM and NTD structural domain" is understood to mean a recombinant protein composed of only RBM and NTD amino acids constructed by recombinant technology, and may be a natural protein on coronavirus or a recombinant natural protein, wherein the amino acid sequence of RBM and NTD is included, such as S1 protein or S protein.
The invention can detect RBD neutralizing antibody and NTD neutralizing antibody simultaneously in antibody detection, especially in neutralizing antibody detection, the detected neutralizing antibody is more comprehensive and has higher sensitivity and/or detection rate, and the more comprehensive neutralizing antibody can be covered. The invention has universality, is not limited to a specific immunoassay platform and is not limited to a specific species.
In some embodiments, the present invention provides a method of detection comprising the steps of:
(1) contacting the sample with protein containing RBM and NTD structural domains, reagent 1 and reagent 2;
reagent 1: ACE2 or ACE2 fragment;
reagent 2: neutralizing antibodies that bind NTD;
one of the reagent 1 or the reagent 2 is connected with a marker, and the other is fixed on a solid phase carrier;
(2) and detecting the signal.
In some embodiments, step (1) is selected from any one of the following:
(a) simultaneously contacting the sample with protein containing RBM and NTD structural domains, a reagent 1 and a reagent 2;
(b) the sample is firstly contacted with protein containing RBM and NTD structural domains, a reagent 1 and then contacted with a reagent 2;
(c) firstly, contacting a sample with protein containing RBM and NTD structural domains, a reagent 2 and then a reagent 1;
(d) firstly, contacting a sample with protein containing RBM and NTD structural domains, and then contacting with a reagent 1 and a reagent 2;
(e) the sample is firstly contacted with protein containing RBM and NTD structural domains, then contacted with a reagent 1, and then contacted with a reagent 2;
(f) the sample is contacted with the protein containing the RBM and NTD domains, then with reagent 2, and then with reagent 1.
In some embodiments, the pathogen is a coronavirus. Among them, common human-infecting coronaviruses include HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. The SARS-CoV-2 includes its wild type and variant.
In some embodiments, the sample is selected from the group consisting of bodily fluids, excreta, cells; such as, but not limited to, serum, plasma, whole blood, lymph fluid, cerebrospinal fluid, interstitial fluid, saliva, urine, lymphocytes, etc.
In some embodiments, the protein containing the RBM and NTD domains may be a recombinant protein containing at least RBM and NTD sequences, or may be the SI or S protein (including R BM and NTD sequences) of the virus used directly. Wherein RBD is Receptor Binding Domain (Receptor Binding Domain), and NTD is N-terminal Domain (N-terminal Domain). In some embodiments, the ligand is selected from the group consisting of RBD of SARS-CoV-2S protein, NTD of SARS-CoV-2S protein. In some embodiments, the fragment containing the RBD of the SARS-CoV-2S protein can be the S protein and fragments thereof that encompass the RBD, e.g., the S protein reference Gene ID: 43740568. In some embodiments, the sequence of the R BD of the SARS-CoV-2S protein can be found in the Wrapp Daniel, Wang Nianshuan, Corbett Kizmekia S, et al Cryo-EM structure of the 2019-nCoV spike in the fusion formation n, and amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence thereto, and which still have the function of ligand-binding receptors and/or antibodies. In some embodiments, the sequence of NT D of the SARS-CoV-2S protein can be found in A neutral human antibody binding to the N-terminal domain of the spike protein of SARS-CoV-2, and amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence thereto, and which still have ligand binding receptor and/or antibody function. An "RBM" is a receptor binding domain in an "RBD" and has an amino acid sequence of SNNLDSKVGGNYNYLYRLFRKS NLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to its sequence. At least the "RBM" fragment is required for the antigen to be used to detect RBD neutralizing antibodies.
In some embodiments, "ACE 2" refers to ACE2 of any species and includes ACE2 subtypes, fragments, variants (including mutants), or homologs from any species. In some embodiments, ACE2 is mammalian (e.g., subclass veterinaria, subclass mammalia, subclass veterinaria, class protozoo, order grand, order primates (monkey, non-human primate, or human)) ACE 2. in some embodiments, ACE2 is human, bat, pangolin, civet, or pig ace2. in some embodiments, ACE2 is human ACE2, the sequence being referenced to Gene ID:59272, and amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence thereto, and which still function as receptor binding ligands.
In some embodiments, the invention also relates to a kit for the detection of coronavirus neutralizing antibodies, said kit comprising:
(1) a protein comprising an RBM and an NTD domain according to any one of the embodiments above;
(2) the reagent 1 according to any of the above embodiments;
(3) the reagent 2 according to any of the above embodiments;
in some embodiments, the solid support is selected from a magnetic microparticle or an Elisa plate.
In some embodiments, the invention also relates to a detection assembly for coronavirus neutralizing antibodies, comprising: protein and test paper containing RBM and NTD structural domains; the test paper is provided with a sample pad, a combination pad, a chromatographic membrane, a water absorption pad and a backing plate, and further comprises a reagent 1 and a reagent 2 in any one of the above embodiments; wherein, the reagent 1 is connected with a marker and arranged on the bonding pad, and the reagent 2 is fixed on the chromatographic membrane; or; the reagent 2 is connected with a marker and is arranged on the bonding pad, and the reagent 1 is fixed on the chromatographic membrane.
In some embodiments, the protein comprising the RBM and NTD domains is disposed on a sample pad.
By "kit" is herein understood a kit, e.g. a conventional kit, chromatographic strip, reagent card, etc. in the form conventional for immunological products.
The invention also relates to the detection method of any one of the above embodiments, or the application of the detection assembly of any one of the above embodiments in antibody detection or preparation of an antibody detection reagent.
The principle of the invention is illustrated by detecting the neutralizing antibody of SARS-COV-2:
(1) contacting the S protein with the positive sample, the S protein binding to total antibodies to the S protein (including total and neutralizing antibodies to the S protein NTD, total and neutralizing antibodies to the RBD, and other total and neutralizing antibodies)
(2) Contacting the immune complex with the NTD neutralizing antibody in the reagent in (1) to generate equivalent competition relationship with the level of the NTD neutralizing antibody in the sample,
(3) contacting the immunocomplex produced in (2) with ACE2 in the reagent, producing an equivalent competition relationship with the level of RBD neutralizing antibodies in the sample, producing a signal, thereby effecting an additive competition for the double neutralizing epitope, recognizing the sum of the level of NTD neutralizing antibodies and the level of RBD neutralizing antibodies, and effecting more complete detection of neutralizing antibodies than conventional methods.
Aspects and embodiments of the present application will be discussed with reference to the figures and the following examples. Other aspects and embodiments will be apparent to those skilled in the art. Although the present application has been described in conjunction with exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art in light of the present application. Accordingly, the exemplary embodiments of the present application are intended to be illustrative, not limiting. Various changes may be made to the embodiments described without departing from the spirit and scope of the present application.
Example 1 NTD neutralizing antibody immobilized on magnetic microparticles
1.1 mixing 1um magnetic particles with 0.1M MES buffer solution with pH of 5.0, uniformly mixing, placing on a magnetic separator until the supernatant is not turbid, removing the supernatant, and reserving magnetic particles; the magnetic beads were then fully suspended by adding MES buffer to the magnetic particles, re-isolated and resuspended as described above.
1.2 taking the washed magnetic particles, adding 10mg/ml carbodiimide (EDAC) to react for 30min at 25 ℃, placing on a blood mixing machine, and mixing uniformly at a medium speed.
1.3 adding NTD neutralizing antibody (obtained by screening peripheral blood after recovery of SARS-CoV-2 infected person by single cell PCR technology and purchased from Guangdong Fengpeng biological Co., Ltd.), and reacting at 25 deg.C in dark for 3h to obtain coated product; the blocking reaction was terminated by adding a quenching buffer (10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4) and reacting at 25 ℃ in the dark for 1h, and the antibody or antigen immobilized on the magnetic microparticles was collected by centrifugation. NTD neutralizing antibodies may also be used with the sequences of GenBank: MT712309.1(L chain) and GenBank: MT712296.1(H chain).
Example 2 ACE2 ligation tags
Taking human ACE2, ultrafiltering, centrifuging, replacing with 0.1M MES buffer solution with pH of 5.0, centrifuging, adding acridine ester, mixing, and labeling (labeling temperature is 37 deg.C, time is 4 hr); after the labeling reaction is finished, adding a blocking buffer (10% BSA buffer with pH 8.0 is used as a blocking buffer) for blocking (1 h); and (3) performing ultrafiltration and centrifugation after sealing to remove the unconjugated acridinium ester, and after the ultrafiltration is finished, determining the protein concentration, and storing the protein concentration as an intermediate product for reagent preparation.
EXAMPLE 3 preparation of dilution containing SARS-COV-2S1 protein
The S1 protein was purchased from Guangdong Fengpo Bio Inc. and diluted to a concentration of 1.0ug/mL for SARS-COV-2S1 protein using 100mmol/L PB, 0.1% 150mmol/L NaCl, 0.1% proclin-300, 2mmol/L EDTA x 2Na, 0.1% BSA, pH 6.0.
Comparative example 1 existing alternative virus neutralization test method reagent preparation
Reagent 1: ACE2 coated magnetic bead reagent was prepared as described in example 1 above.
Reagent 2: the labeling reagent for RBD-labeling of acridinium ester was prepared as described in example 2 above.
Test example 1
Scheme 1: samples were assigned using a pseudovirus Neutralization assay, as described in Protocol and Reagents for pseudoviral Particles with SARS-CoV-2 Spike Protein for neutrallization Assays.
Scheme 2: existing alternative virus neutralization tests: a semi-automatic chemiluminescence immunoassay (Thermo) is used as a detection tool, a methodological mode is a competition method, the sample size is 50uL, 50uL of ACE2(0.25mg/ml) coated by magnetic beads and 40uL of acridinium ester labeled RBD labeling liquid (1ug/ml) are adopted. 50ul of the sample was added to the reaction cuvette, 50ul of ACE2(0.25mg/ml) coated with magnetic beads and 40ul of acridinium ester-labeled RBD labeling solution (1ug/ml) were added thereto, and the reaction was carried out at 37 ℃ for 15min to detect the luminescence value.
Scheme 3: the test of the invention comprises the following steps: a semi-automatic chemiluminescence immunoassay analyzer (Thermo) is used as a detection tool, and a methodology mode is a double competition method. 50ul of sample was added to the reaction cuvette, 50ul of a dilution containing SARS-COV-2S1 protein (example 3) was added thereto, and the mixture was incubated at 37 ℃ for 30min, 50ul of an NTD neutralizing antibody (0.25mg/ml) coated with magnetic beads and 40ul of ACE2(1ug/ml) labeled with acridinium ester were added thereto, and the mixture was incubated at 37 ℃ for 15min to detect the luminescence value.
Test results
The feasibility of the invention was tested using 4 samples, sample 1: PBS (negative quality control), sample 2: sample of NTD neutralizing antibody, sample 3: sample of G3 antibody (G3 is RBD neutralizing antibody), specimen 4: mixed samples of sample 2 and sample 3.
The results are shown in the following table:
note: inhibition rate is 1-sample detection signal/negative quality control detection signal.
Conclusion analysis: as can be seen from the data in the above table, scheme 2 can detect only neutralizing antibodies that block RBD, but not neutralizing antibodies to NTD, and scheme 3 of the present invention can detect not only RBD neutralizing antibodies but also neutralizing antibodies to NTD. In addition, compared with the prior art, the detection inhibition rate of the scheme of the invention on the RBD neutralizing antibody (G3) is improved from 25.9% to 46.9%, and the detection inhibition rate on the mixed sample is improved from 27% to 69.9%, which indicates higher detection sensitivity.
Example 4 protocol testing of coated ACE2 and labeled NTD neutralizing antibodies
4.1 magnetic particle immobilization
Washing the magnetic particles, suspending the magnetic particles in MES buffer solution, adding carbodiimide (EDAC) for reaction, and placing the mixture on a blood mixing instrument for medium-speed mixing; adding ACE2 (purchased from Guangdong Fengcong biological Co., Ltd.), and reacting at room temperature in dark to obtain a coated product; the blocking reaction was terminated by adding a quenching buffer at room temperature away from light, and ACE2 immobilized on magnetic microparticles was collected.
4.2 linking acridine labels
Suspending the NTD neutralizing antibody in MES buffer solution, adding acridinium ester, mixing, labeling, and adding 10% BSA for blocking after the labeling reaction; and centrifuging to remove unconjugated acridinium ester, and collecting an NTD neutralizing antibody acridinium marker.
4.3 detection
A semi-automatic chemiluminescence immunoassay analyzer (Thermo) is used as a detection tool, a sample and a diluent containing SARS-COV-2S1 protein (example 3) are added into a reaction cup, incubation is carried out at 37 ℃ for sufficient reaction, an acridinium ester labeled NTD neutralizing antibody is added, incubation is carried out at 37 ℃ for sufficient reaction, ACE2 coated by magnetic beads is added, incubation is carried out at 37 ℃ for sufficient reaction, and the luminescence value is detected.
This example is equivalent to the effect of scheme 3, and can achieve the above-mentioned effects of simultaneously detecting neutralizing antibodies against NTD and RBD and improving sensitivity.
Example 5
A semi-automatic chemiluminescence immunoassay analyzer (Thermo) is used as a detection tool, a sample, a diluent containing SARS-COV-2S1 protein (example 3), an NTD neutralizing antibody coated by magnetic beads are added into a reaction cup, incubation at 37 ℃ is carried out for complete reaction, then ACE2 marked by acridinium ester is added, incubation at 37 ℃ is carried out for complete reaction, and the luminescence value is detected.
This example is equivalent to the effect of scheme 3, and can achieve the above-mentioned effects of simultaneously detecting neutralizing antibodies against NTD and RBD and improving sensitivity.
Example 6 preparation of colloidal gold chromatography kit based on the method of the present invention
6.1. Preparation of colloidal gold
The colloidal gold solution was prepared by the following method: adding 1% chloroauric acid solution with final concentration of four ten-thousandths into boiled purified water, boiling for 3min, adding 0.1M sodium citrate (580 μ l per 100ml chloroauric acid solution), stirring and heating for 10min, cooling to room temperature to obtain colloidal gold solution, and storing at 2-8 deg.C.
6.2. Colloidal gold labeling
The method adopts human ACE2 protein, and comprises the following steps of: adding K2CO3 into the colloidal gold solution, adding human ACE2 into the solution with the adjusted pH value, coupling for 5min, and adding 10% BSA to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.
6.3. Preparation of the conjugate pad
The ACE2 solution with the colloidal gold attached was diluted by 10% and spread evenly on glass fiber and dried overnight in a drying room at 37 deg.C to obtain a conjugate pad.
6.4. Preparation of chromatographic membranes
T-shaped wire wrapping: diluting NTD neutralizing antibody to 1.0mg/ml, coating on nitrocellulose membrane with gold spraying synoviograph, and oven drying at 37 deg.C for more than 2h to obtain chromatographic membrane.
6.5 sample pad treatment
SARS-COV-2S protein (purchased from Guangdong Fengcong biological Co., Ltd.) was diluted to 1ug/ml with 1XPBST, spread on a sample pad, and dried at 37 ℃ for 1 h.
6.6 Assembly
And sequentially overlapping and assembling the sample pad, the combination pad, the chromatographic membrane and the water absorption pad on a lining plate, cutting into 2.7mm, and manufacturing a corresponding detection assembly.
The detection method comprises the following steps: the sample to be tested is loaded onto the sample pad.
Example 7
The method of example 6 was repeated except that the SARS-COV-2S protein was diluted with a diluent. And during detection, the diluent is contacted with the sample and simultaneously loaded on the sample pad.
Comparative example 2 ACE2 competitive assay for neutralizing antibodies
1. Colloidal gold labeling
Colloidal gold was prepared as in example 6. Gold labeling of RBD antigen (purchased from Guangdong Fengcong biological Co., Ltd.), addition of K to the colloidal gold solution2CO3Adding RBD antigen into the solution to perform coupling reaction, and adding 10% BSA to stop the coupling reaction; centrifuging, removing supernatant, dissolving precipitate again, and storing at 2-8 deg.C.
2. Preparation of the conjugate pad
And diluting the RBD solution connected with the colloidal gold according to a dilution ratio of 10%, uniformly spreading the solution on glass fibers, and placing the glass fibers in a drying room at 37 ℃ for drying overnight to obtain the bonding pad.
3. Preparation of chromatographic membranes
T-shaped wire wrapping: diluting human ACE2 protein to 0.8mg/mL, coating on nitrocellulose membrane with gold spraying synoviograph, and oven drying at 37 deg.C for more than 2h to obtain chromatographic membrane.
4. Assembly
And sequentially overlapping and assembling the sample pad, the combination pad, the chromatographic membrane and the water absorption pad on a lining plate, cutting into 2.7mm, and manufacturing a corresponding detection assembly.
5. The detection method comprises the following steps: the sample to be tested is loaded onto the sample pad.
As a result: both example 6 and example 7 had better neutralization inhibition efficiency than comparative example 2, and could detect RBD and NTD neutralizing antibodies, whereas comparative example 2 failed to detect NTD neutralizing antibodies.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
It will be clear to those skilled in the art from the understanding of the present invention that the principle of the present invention for detecting NTD-neutralizing antibodies can be supplemented is achieved on the basis of the reaction principle of the present invention, which is improved, and RBD-neutralizing antibodies can be detected by the first competitive reaction of the RBD region in the sample and the protein containing the RBM and NTD domains with ACE 2; the NTD neutralizing antibody can be detected by the second competition reaction of the NTD neutralizing antibody and the NTD region in the protein containing the RBM and the NTD domain in the sample (as shown in FIG. 1), although the first and second competition reactions are not limited in order for easy understanding, and may be interchanged.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for detecting neutralizing antibodies to coronaviruses, comprising the steps of:
(1) contacting the sample with protein containing RBM and NTD structural domains, reagent 1 and reagent 2;
reagent 1: ACE2 or ACE2 fragment;
reagent 2: neutralizing antibodies that bind NTD;
one of the reagent 1 or the reagent 2 is connected with a marker, and the other is fixed on a solid phase carrier;
(2) and detecting the signal.
2. The detection method according to claim 1, wherein the step (1) is selected from any one of the following modes:
(a) simultaneously contacting the sample with protein containing RBM and NTD structural domains, a reagent 1 and a reagent 2;
(b) the sample is firstly contacted with protein containing RBM and NTD structural domains, a reagent 1 and then contacted with a reagent 2;
(c) firstly, contacting a sample with protein containing RBM and NTD structural domains, a reagent 2 and then a reagent 1;
(d) firstly, contacting a sample with protein containing RBM and NTD structural domains, and then contacting with a reagent 1 and a reagent 2;
(e) the sample is firstly contacted with protein containing RBM and NTD structural domains, then contacted with a reagent 1, and then contacted with a reagent 2;
(f) the sample is contacted with the protein containing the RBM and NTD domains, then with reagent 2, and then with reagent 1.
3. The method of claim 1, wherein the coronavirus is selected from the group consisting of SARS-CoV-2 and SARS-CoV.
4. The detection method according to claim 1, wherein the sample is selected from any sample that may contain an antibody;
optionally, the sample is selected from serum, plasma, whole blood, lymph fluid, cerebrospinal fluid, interstitial fluid, saliva, urine, or lymphocytes.
5. The method according to claim 1, wherein the protein comprising the RBM and NTD domains is optionally S1 protein or S protein.
6. A detection assembly for coronavirus neutralizing antibodies, the assembly comprising:
(1) the protein according to any one of claims 1 to 5, which comprises RBM and NTD domains;
(2) the reagent 1 of any one of claims 1 to 5;
(3) the reagent 2 according to any one of claims 1 to 5.
7. The detection assembly of claim 6, wherein the solid support is selected from the group consisting of magnetic microparticles and Elisa plates.
8. A detection assembly for coronavirus neutralizing antibodies, comprising: protein and test paper containing RBM and NTD structural domains; the test paper is provided with a sample pad, a combination pad, a chromatographic membrane, a water absorption pad and a backing plate, and further comprises a reagent 1 and a reagent 2 according to any one of claims 1 to 5; wherein, the reagent 1 is connected with a marker and arranged on the bonding pad, and the reagent 2 is fixed on the chromatographic membrane; or; the reagent 2 is connected with a marker and is arranged on the bonding pad, and the reagent 1 is fixed on the chromatographic membrane.
9. The test kit of claim 8, wherein the protein comprising the RBM and NTD domains is disposed on a sample pad.
10. Use of the assay of any one of claims 1-5 or the test module of any one of claims 6-9 for antibody detection or for the preparation of an antibody detection reagent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021100450997 | 2021-01-13 | ||
| CN202110045099 | 2021-01-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113267624A true CN113267624A (en) | 2021-08-17 |
Family
ID=77230026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110477341.8A Pending CN113267624A (en) | 2021-01-13 | 2021-04-29 | Method and product for detecting RBD and NTD neutralizing antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113267624A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2029011A (en) * | 1978-09-01 | 1980-03-12 | Coulson W | The use of a synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes |
| US4508830A (en) * | 1982-02-10 | 1985-04-02 | Baker Terence S | Assay |
| US4904583A (en) * | 1987-05-26 | 1990-02-27 | Becton, Dickinson And Company | Cascade immunoassay by multiple binding reactions |
| US5641639A (en) * | 1987-04-29 | 1997-06-24 | Celltech Therapeutics Limited | Binding assay device |
| CN103983767A (en) * | 2014-05-06 | 2014-08-13 | 中国食品药品检定研究院 | Kit for detecting alpha-1,3Gal antigen and application thereof |
| CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN112098644A (en) * | 2020-09-11 | 2020-12-18 | 江苏美克医学技术有限公司 | Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof |
-
2021
- 2021-04-29 CN CN202110477341.8A patent/CN113267624A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2029011A (en) * | 1978-09-01 | 1980-03-12 | Coulson W | The use of a synthetic bifunctional ligand for the immunometric determination of the concentration ratio of two solutes |
| US4508830A (en) * | 1982-02-10 | 1985-04-02 | Baker Terence S | Assay |
| US5641639A (en) * | 1987-04-29 | 1997-06-24 | Celltech Therapeutics Limited | Binding assay device |
| US4904583A (en) * | 1987-05-26 | 1990-02-27 | Becton, Dickinson And Company | Cascade immunoassay by multiple binding reactions |
| CN103983767A (en) * | 2014-05-06 | 2014-08-13 | 中国食品药品检定研究院 | Kit for detecting alpha-1,3Gal antigen and application thereof |
| CN111562369A (en) * | 2020-06-18 | 2020-08-21 | 威海威高生物科技有限公司 | SARS-CoV-2 neutralizing antibody detection kit |
| CN112098644A (en) * | 2020-09-11 | 2020-12-18 | 江苏美克医学技术有限公司 | Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| CONSTANT GILLOT, ET AL.: "An Original ELISA-Based Multiplex Method for the Simultaneous Detection of 5 SARS-CoV-2 IgG Antibodies Directed against Different Antigens", 《J. CLIN. MED.》 * |
| V.V.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.", 《JOURNAL OF VIROLOGICAL METHODS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113156129B (en) | High-sensitivity detection method and product of neutralizing antibody | |
| RU2730897C1 (en) | Method of using recombinant proteins sars-cov-2 as part of a test system for elisa test with determining igm, igg, iga class antibody levels in blood serum/plasma of covid-19 patients | |
| JP3958797B2 (en) | Antigen-specific IgM detection | |
| CN111233985A (en) | Preparation method of novel coronavirus IgA antibody rapid detection test strip | |
| Ernst et al. | Technical considerations to development of serological tests for SARS-CoV-2 | |
| JP2015007651A (en) | Methods and compositions for detection of complement fixing antibodies | |
| AU5347000A (en) | Flow-through assay for visually detecting the presence of influenza A and B | |
| WO2022151562A1 (en) | Method and product for simultaneously detecting total antibody and neutralizing antibody | |
| CA2225078A1 (en) | Immunodissociation for improving the immunochemical determination of an analyte | |
| US20230072761A1 (en) | Immunoassay for Detecting Zika Virus Infection | |
| US20210364515A1 (en) | Optimizing diagnostics for galactofuranose containing antigens | |
| Ferris et al. | Utility of recombinant integrin αvβ6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease | |
| JP2008275511A (en) | Method for measuring immunity of influenza virus antigen and device used for the same | |
| CA2874688C (en) | Method for diagnosing and differentiating hiv-2 infections | |
| CN113267624A (en) | Method and product for detecting RBD and NTD neutralizing antibody | |
| JP6675165B2 (en) | Test substance detection method, detection reagent kit and detection reagent | |
| JPH03502248A (en) | Aqueous washing solution for assay, diagnostic test kit and method for measuring herpes simplex virus | |
| CN113203855A (en) | RBD (receptor binding domain) neutralizing antibody detection method and product | |
| CN112881710B (en) | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof | |
| JP2022174540A (en) | Immunological detection method and reagent for sars-cov-2 | |
| CN113219167A (en) | Dual competition detection method and product | |
| CN114910643B (en) | Method and reagent for identifying antibody combined with mutant antigen | |
| CN112904018B (en) | Kit and method for detecting virus neutralizing antibody and application | |
| KR102605065B1 (en) | Monoclonal antibody specific to highly pathogenic avian influenza virus H5N1 and rapid diagnostic kit using the same | |
| NZ516587A (en) | Assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210817 |
|
| RJ01 | Rejection of invention patent application after publication |