CN116047065A - Novel coronavirus antigen rapid detection kit capable of identifying omacron strain - Google Patents
Novel coronavirus antigen rapid detection kit capable of identifying omacron strain Download PDFInfo
- Publication number
- CN116047065A CN116047065A CN202210408994.5A CN202210408994A CN116047065A CN 116047065 A CN116047065 A CN 116047065A CN 202210408994 A CN202210408994 A CN 202210408994A CN 116047065 A CN116047065 A CN 116047065A
- Authority
- CN
- China
- Prior art keywords
- protein
- novel coronavirus
- strain
- detection
- novel
- 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
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 135
- 238000001514 detection method Methods 0.000 title claims abstract description 112
- 239000000427 antigen Substances 0.000 title claims abstract description 64
- 102000036639 antigens Human genes 0.000 title claims abstract description 64
- 108091007433 antigens Proteins 0.000 title claims abstract description 64
- 238000012360 testing method Methods 0.000 claims abstract description 74
- 238000000034 method Methods 0.000 claims abstract description 44
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 claims abstract description 43
- 101710141454 Nucleoprotein Proteins 0.000 claims abstract description 42
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 claims abstract description 34
- 241000352457 Shivajiella indica Species 0.000 claims abstract description 15
- 102100034583 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 Human genes 0.000 claims abstract description 8
- 101000848781 Homo sapiens Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 Proteins 0.000 claims abstract description 8
- 101150013771 olfml3 gene Proteins 0.000 claims abstract description 8
- 238000002965 ELISA Methods 0.000 claims description 36
- 210000004027 cell Anatomy 0.000 claims description 28
- 241001465754 Metazoa Species 0.000 claims description 27
- 239000000020 Nitrocellulose Substances 0.000 claims description 27
- 239000012528 membrane Substances 0.000 claims description 27
- 229920001220 nitrocellulos Polymers 0.000 claims description 27
- 229920001184 polypeptide Polymers 0.000 claims description 21
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 21
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 230000003053 immunization Effects 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 17
- 235000018102 proteins Nutrition 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 210000002966 serum Anatomy 0.000 claims description 15
- 238000012216 screening Methods 0.000 claims description 14
- 230000002745 absorbent Effects 0.000 claims description 12
- 239000002250 absorbent Substances 0.000 claims description 12
- 238000005516 engineering process Methods 0.000 claims description 12
- 102100031673 Corneodesmosin Human genes 0.000 claims description 11
- 101710139375 Corneodesmosin Proteins 0.000 claims description 11
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 8
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 8
- 235000018417 cysteine Nutrition 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 210000004408 hybridoma Anatomy 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 241000283707 Capra Species 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 241000270722 Crocodylidae Species 0.000 claims description 4
- 241000287828 Gallus gallus Species 0.000 claims description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 4
- 241000271567 Struthioniformes Species 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 235000013330 chicken meat Nutrition 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 239000004816 latex Substances 0.000 claims description 4
- 229920000126 latex Polymers 0.000 claims description 4
- 239000004005 microsphere Substances 0.000 claims description 4
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 4
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 4
- 150000003573 thiols Chemical class 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 claims description 3
- 230000028327 secretion Effects 0.000 claims description 3
- 210000001138 tear Anatomy 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 210000003608 fece Anatomy 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims 2
- 239000003550 marker Substances 0.000 claims 1
- 230000000241 respiratory effect Effects 0.000 claims 1
- 238000011841 epidemiological investigation Methods 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 241000700605 Viruses Species 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 230000035772 mutation Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 101100478237 Caenorhabditis elegans ost-1 gene Proteins 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 206010002653 Anosmia Diseases 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000006083 Hypokinesia Diseases 0.000 description 1
- 206010050515 Hyposmia Diseases 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 206010028748 Nasal obstruction Diseases 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 208000036071 Rhinorrhea Diseases 0.000 description 1
- 206010039101 Rhinorrhoea Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 208000017574 dry cough Diseases 0.000 description 1
- 235000019559 hyposmia Nutrition 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 208000013465 muscle pain Diseases 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
- 244000052769 pathogen Species 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000008786 sensory perception of smell Effects 0.000 description 1
- 230000014860 sensory perception of taste Effects 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229960005486 vaccine Drugs 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a novel rapid detection kit for coronavirus antigens, which can identify Omicron strains, and comprises two independent detection strips, namely a test strip N and a test strip S, wherein the two independent detection strips are respectively marked as a test strip S, and 2 detection lines are respectively arranged; the ONT1 detection line of the test strip N is coated with an anti-novel coronavirus Omicron strain N protein specific antibody (detection type), and the NT2 detection line is coated with an anti-novel coronavirus N protein universal antibody (detection type) capable of recognizing wild type and various mutant strains; OST1 detection line of test strip S is coated with specific antibody (detection type) of anti-novel coronavirus Omicron strain S1 protein, ST2 detection line is coated with general antibody (detection type) of anti-novel coronavirus S1 protein which can recognize wild type and various mutant strains. The kit can detect various strains of novel coronaviruses by a rapid (3-15 minutes), simple and low-cost method, can identify the Omicron strain, and has important value for epidemiological investigation of the Omicron strain.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel rapid detection kit for coronavirus antigens, which can identify Omicron strains.
Background
There are currently 5 "variants of the novel coronavirus" that are Alpha (Alpha), beta (Beta), gamma (Gamma), delta (Delta), and omicon (omicon), respectively. The symptoms of patients are mainly fever, dry cough and hypodynamia. Some patients also have nasal obstruction, runny nose, sore throat, hyposmia or loss of sense of smell and taste, conjunctivitis, muscle pain, diarrhea, etc. as the main manifestations. Most patients are well prognosis and some severe cases may develop acute respiratory distress syndrome or septic shock, even death.
The novel coronavirus can continuously adapt to a host to generate mutation in the replication process, and particularly, the main epidemic strain Omicron generates a large number of area mutations, so that the effect of the previously developed vaccine is greatly reduced. The Omicron strain has the characteristics of large infectivity and rapid transmission, and is still in further mutation evolution. At present, various commonly used novel coronavirus antigen detection kits are basically targets of relatively conserved N proteins, and can not be identified by adopting a general N protein antibody although the Omicron strain N protein can be non-specifically identified. Therefore, it is very significant to develop a novel coronavirus antigen detection kit capable of rapidly identifying omacron strains.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention aims to provide a novel rapid detection kit for coronavirus antigens, which can identify Omicron strains, and is based on detection and identification of Omicron strains by developing specific antibodies of novel Omicron strain N protein and S protein, and simultaneously, the aim of detecting novel coronavirus universality is fulfilled by applying universal antibodies of novel coronavirus N protein and S protein.
The novel rapid detection kit for the coronavirus antigen capable of identifying the omacron strain comprises two independent detection strips which are respectively used for detecting novel coronavirus N protein antigen and S1 protein antigen, and are respectively marked as a test strip N and a test strip S, and each detection strip is provided with 2 detection lines. Wherein, the detection line T1 (ONT 1) of the test strip N is coated with an anti-novel coronavirus Omicron strain N protein specific antibody (detection type), and the detection line T2 (NT 2) is coated with an anti-novel coronavirus N protein universal antibody (detection type) capable of recognizing wild type and various mutant strains. The detection line T1 (OST 1) of the test strip S is coated with an anti-novel coronavirus Omicron strain S1 protein specific antibody (detection type), and the detection line T2 (ST 2) is coated with an anti-novel coronavirus S protein universal antibody (detection type) capable of recognizing wild type and various mutant strains; the anti-novel coronavirus antibody (capture type) capable of identifying wild type and various mutant strains is marked by colloidal gold, colloidal carbon, color latex or fluorescent microsphere, and is embedded on a release pad of a detection strip together with marked IgG for capturing novel coronavirus in a sample, and can be combined with a corresponding detection area to form a visible strip in the chromatography process, wherein the marked antibody of the detection strip N is an anti-novel coronavirus N protein universal capture antibody; the labeled antibody of the test strip S is a general capture antibody for resisting novel coronavirus S1 protein.
The preparation method of various anti-novel coronavirus antibodies comprises the following steps:
1) The specific polypeptide sequence near the N end of the N protein of the novel coronavirus Omicron strain is synthesized to be used as an epitope, and the synthesized specific polypeptide sequence used as the epitope is respectively coupled with proteins such as KLH and BSA or OVA and BGG to prepare the immune antigen.
2) Immunizing an animal by using the immune antigen prepared in the step 1), and separating B lymphocytes of the immunized animal after measuring high-titer immune serum to prepare a monoclonal antibody cell strain; screening out monoclonal antibodies specific to N proteins of novel coronavirus Omicron strains by taking N proteins of novel coronaviruses of wild type and various mutant strains as reaction antigens.
3) And (3) amplifying a large amount of the monoclonal antibody cell strain with the specificity of the protein N of the anti-novel coronavirus Omicron strain screened in the step (2) to prepare an antibody for later use.
4) Polypeptide sequences of 6 conserved regions of novel coronavirus N protein are synthesized to serve as antigen epitopes, and the synthesized polypeptide sequences are respectively coupled with proteins such as KLH and BSA or OVA and BGG to prepare immune antigens, so that 6 immune antigens are prepared.
5) Respectively immunizing animals with the 6 immune antigens prepared in the step 4), and separating B lymphocytes of the immunized animals after high-titer immune serum is measured to prepare monoclonal antibody cell strains; the novel coronavirus N protein universal monoclonal antibody of 2 or more different antigen epitopes is screened by taking the novel coronavirus N protein of wild type and various mutant strains as reaction antigens.
6) Immunizing an animal with a novel coronavirus Omicron strain S1 protein, and separating B lymphocytes of the immunized animal after high-titer immune serum is measured to prepare a monoclonal antibody cell strain; screening of novel coronavirus Omicron strain (B.1.1.529) S1 protein specific monoclonal antibody and novel coronavirus S1 protein universal monoclonal antibody by using wild type and novel coronavirus S1 proteins of various mutant strains as reaction antigens.
7) And (3) amplifying the novel coronavirus Omicron strain S1 protein specific monoclonal antibody cell strain and the universal monoclonal antibody cell strain screened in the step (6) in a large quantity to prepare antibodies for later use.
Further, in the step 1), the antigen epitope specific polypeptide sequence of the N near N end of the novel coronavirus Omicron strain N protein is NALRITFGGPSDSTGSNQNGGAR; during synthesis, cysteine (C) is added at the C end, and the synthesized polypeptide is coupled with proteins such as KLH and BSA or OVA and BGG through thiol-dimercapto exchange reaction by utilizing thiol (-SH) of the cysteine to prepare immune antigen;
further, in step 2), the serum titer is determined by ELISA, and the direct gradient dilution titer is at least 100 parts per million, which can be used to immunize animals; the method for preparing the monoclonal antibody cell strain comprises the steps of preparing B lymphocyte and mouse myeloma fusion cells, namely hybridomas, by using a mouse monoclonal antibody technology by taking a mouse as an immune animal; preparing monoclonal nanometer antibody cell strain by nanometer antibody preparation technology with animals such as ostriches, crocodiles, etc. as immune hosts; the monoclonal antibody cell strain can be prepared by using the genetic engineering antibody technology when other animals such as rabbits, chickens and the like are used as immune hosts; the various mutants include all novel coronavirus mutants that have been identified and published by the world organization so far: mu (B.1.621), omacron (BA.1/BA.2), delta variant B.1.617.2, lambda variant C.37, delta/Deltaplus, alpha/Beta (B.1.1.7/B.1.351/A.2.2), beta (B.1.351), gamma (P.1/P.1.1/P.1.2), epsilon (B.1.427/429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), B.1.617, B.1.617.3, B.1.618, B.1.620, A.23.1, P.2, P.3; the screening method adopts an ELISA method, namely, N proteins of wild type coronaviruses and various mutant strains are coated on a 96-well enzyme-linked immunoassay (ELISA) plate, and specific monoclonal antibodies which only react with the N proteins of novel coronaviruses Omicron (B.1.1.529) are analyzed and screened.
Further, in step 4), the polypeptide sequences of the antigen epitopes in the 6 conserved regions of the novel coronavirus N protein are respectively selected as follows by analyzing mutation sites of the N proteins of each strain type of the novel coronavirus as shown in Table 1 and simultaneously combining with antigen epitope determinant predictive analysis
ARSKQRRPQGLPNNTASWFTALTQHGK, identified as N-1;
RRIRGGDGKMKDLSPRWYFYYLGTGPE, identified as N-2;
KGFYAEGSRGGSQASSRSSSRSRNSSR, identified as N-3;
GQQQQGQTVTKKSAAEASKKPRQKRTA, identified as N-4;
GAIKLDDKDPNFKDQVILLNKHIDAYK, identified as N-5;
QQTVTLLPAADLDDFSKQLQQSMSSAD, identified as N-6;
during synthesis, cysteine (C) is added at the C terminal of the 6 polypeptide sequences, and the synthesized polypeptide is coupled with proteins such as KLH and BSA or OVA, BGG and the like through thiol-dimercapto exchange reaction by utilizing the thiol (-SH) of the cysteine to prepare the immune antigen.
TABLE 1 novel N protein mutation sites of coronavirus strains
Further, in step 5), the serum titer is determined by ELISA, and the direct gradient dilution titer is at least 100 parts per million, which can be used to immunize an animal; the method for preparing the monoclonal antibody cell strain comprises the steps of preparing B lymphocyte and mouse myeloma fusion cells, namely hybridomas, by using a mouse monoclonal antibody technology by taking a mouse as an immune animal; preparing monoclonal nanometer antibody cell strain by nanometer antibody preparation technology with animals such as ostriches, crocodiles, etc. as immune hosts; the monoclonal antibody cell strain can be prepared by using the genetic engineering antibody technology when other animals such as rabbits, chickens and the like are used as immune hosts; the various mutants include all novel coronavirus mutants that have been identified and published by the world organization so far: mu (B.1.621), omacron (BA.1/BA.2), delta variant B.1.617.2, lambda variant C.37, delta/Deltaplus, alpha/Beta (B.1.1.7/B.1.351/A.2.2), beta (B.1.351), gamma (P.1/P.1.1/P.1.2), epsilon (B.1.427/429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), B.1.617, B.1.617.3, B.1.618, B.1.620, A.23.1, P.2, P.3; the screening method adopts an ELISA method, namely, N proteins of wild type and various mutant novel coronaviruses are coated on a 96-hole enzyme-linked immunoassay (ELISA) plate, and at least 2 or more than 2 universal monoclonal antibodies with high affinity which can react with the N proteins of all the novel coronaviruses are screened out through analysis.
Further, in step 6), the serum titer is determined by ELISA, and the direct gradient dilution titer is at least 100 parts per million, which can be used to immunize an animal; the method for preparing the monoclonal antibody cell strain comprises the steps of preparing B lymphocyte and mouse myeloma fusion cells, namely hybridomas, by using a mouse monoclonal antibody technology by taking a mouse as an immune animal; preparing monoclonal nanometer antibody cell strain by nanometer antibody preparation technology with animals such as ostriches, crocodiles, etc. as immune hosts; the monoclonal antibody cell strain can be prepared by using the genetic engineering antibody technology when other animals such as rabbits, chickens and the like are used as immune hosts; the various mutants include all novel coronavirus mutants that have been identified and published by the world organization so far: mu (B.1.621), omacron (BA.1/BA.2), delta variant B.1.617.2, lambda variant C.37, delta/Deltaplus, alpha/Beta (B.1.1.7/B.1.351/A.2.2), beta (B.1.351), gamma (P.1/P.1.1/P.1.2), epsilon (B.1.427/429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), B.1.617, B.1.617.3, B.1.618, B.1.620, A.23.1, P.2, P.3; the screening method adopts an ELISA method, namely, S1 proteins of wild type coronaviruses and various mutant coronaviruses are coated on a 96-well enzyme-linked immunoassay (ELISA) plate, and specific monoclonal antibodies which only react with the Omacron (B.1.1.529) S1 proteins and high-affinity general monoclonal antibodies which can react with S1 proteins of all novel strain coronaviruses are screened out by analysis, wherein at least 2 or more general monoclonal antibodies are selected.
Further, the kit further comprises a label of a capture antibody; the capture antibody adopts a general type, comprising anti-novel coronavirus N protein and S1 protein general type capture antibody, and the antibody source can be commercially proven antibodies or can be prepared by the method of the invention; the labeling medium may be colloidal gold, colloidal carbon, colored latex or fluorescent microspheres, but is not limited thereto; rabbit IgG was labeled with the same medium at the same time for use in a quality control system.
Furthermore, a novel rapid detection kit for coronavirus antigens, which can identify Omicron strains, comprises a test strip N and a test strip S of the kit, wherein the immunochromatographic test strip comprises a substrate, a nitrocellulose membrane, a sample pad, a release pad and absorbent paper; the sample pad, the release pad, the nitrocellulose membrane and the absorbent paper are lapped and assembled on the bottom lining, the release pad and the absorbent paper are respectively overlapped at two ends of the nitrocellulose membrane, a detection area is formed on the surface of the nitrocellulose membrane, and the sample pad is overlapped on the release pad; a detection area T1 line, a detection area T2 line and a control area C line are sequentially arranged on the nitrocellulose membrane of the detection area from the release pad to the absorbent paper;
the preparation of the test strip N comprises the following steps: mixing a marked general capture antibody for resisting novel coronavirus N protein and marked IgG, spraying on a release pad of an immunochromatographic test strip, and coating a detection zone T1 line on a nitrocellulose membrane with an anti-novel coronavirus Omicron strain N protein specific monoclonal antibody, which is marked as ONT1; t2 line is coated with a general monoclonal antibody (detection type) resisting novel coronavirus N protein, and is marked as NT2; the nitrocellulose membrane of the C line area is coated with goat anti-rabbit IgG polyclonal antibody (GAR), and the mark is C1;
the preparation of the test strip S comprises the following steps: mixing a marked general capture antibody for resisting novel coronavirus S protein and marked IgG, spraying on a release pad of an immunochromatographic test strip, and coating an anti-novel coronavirus Omicron strain S1 protein specific monoclonal antibody on a T1 line of a detection area on a nitrocellulose membrane, wherein the mark is OST1; t2 line is coated with a general monoclonal antibody (detection type) resisting novel coronavirus S1 protein, and is marked as ST2; the nitrocellulose membrane of the C line area is coated with goat anti-rabbit IgG polyclonal antibody (GAR), and the mark is C2;
the test strip N and the test strip S are assembled in parallel on the duplex clamping plate for simultaneous detection of novel coronavirus N protein and S protein, and the Omicron mutant strain can be rapidly identified.
Furthermore, the kit is suitable for detecting respiratory tract secretions, feces, urine, tears, environmental samples and the like, and can be applied to rapid identification detection of novel coronaviruses and Omicron mutant strains.
Furthermore, the technical scheme of the kit is not only suitable for the research and development of the novel coronavirus Omicron mutant strain identification detection kit, but also suitable for the research and development of the novel coronavirus future novel mutant strain identification detection kit.
The beneficial effects obtained by the invention are as follows:
the invention adopts a method of immunochromatography test paper strips, and the kit comprises two independent test strips, wherein 2 detection lines are respectively arranged on a nitrocellulose membrane of each test strip, a detection line 1 (T1) is coated with an anti-novel coronavirus Omicron strain specific antibody (detection type), and a detection line 2 (T2) is coated with an anti-novel coronavirus antibody (detection type) capable of identifying wild type and various mutant strains; the anti-novel coronavirus antibodies (capture type) which can identify wild type and various mutant strains are marked by colloidal gold, colloidal carbon, colored latex or fluorescent microspheres, and are embedded on a release pad of a detection strip together with marked IgG for capturing novel coronaviruses in a sample, and can be combined with a corresponding detection area to form a visible strip in the chromatography process. Since the omicon strain-specific antibody of the detection line 1 (T1) can only specifically bind to the novel coronavirus omicon strain, if the T1 line is colored, it is indicated that the novel coronavirus omicon strain is present in the detection sample; if T1 does not develop, no Omacron strain is present. If the T2 line is developed, the novel coronavirus in the sample is detected, and any strain can be used.
The kit of the invention can detect various strains of novel coronaviruses by a rapid (3-15 minutes), simple and low-cost method, can identify the Omicron strain, and has important value for epidemiological investigation of the Omicron strain. The Omicron strain is a novel coronavirus highly mutant strain and a strain type which is mainly popular at present, and has the characteristic of strong transmission capacity. The method has the advantages of being rapid, simple, convenient, low in cost, stable, good in detection, capable of realizing home detection and the like in the aspect of novel coronavirus antigen detection, also can directly identify the Omicron mutant strain, has important value for rapidly and widely grasping the epidemic situation of the Omicron mutant strain, and can be used for further mutation and evolution monitoring of novel coronaviruses.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the kit of the present invention
Detailed Description
The invention will be further illustrated with reference to specific examples, but the scope of the invention is not limited thereto.
In the following examples, the synthesis of the novel coronavirus N protein epitope polypeptide sequence and its coupling to proteins were prepared by Shanghai blaze biotechnology limited; recombinant novel coronavirus Omicron strain N protein and spinous process protein S1 are provided by meierde biotechnology limited, hangzhou; the paired universal monoclonal antibodies for detecting novel coronavirus N protein and for detecting novel coronavirus S1 protein are provided by Hangzhou Michael biosciences.
EXAMPLE 1 preparation of novel coronavirus Omicron strain N protein-specific monoclonal antibody
1) The novel antigen epitope specific polypeptide sequence NALRITFGGPSDSTGSNQNGGAR with the purity of more than or equal to 95% near N end of N protein of coronavirus Omicron strain is synthesized by Shanghai blaze biotechnology limited company, and is respectively coupled with KLH and BSA, and the protein is named ONN-KLH and ONN-BSA respectively.
2) ONN-KLH was prepared into a 0.5mg/mL solution with 0.01M PBS (pH 7.4), and split into 5 pieces with 1.5mLEP tube, 250. Mu.L/piece; frozen stock was used as the immunizing antigen for later use.
3) Taking 5 male healthy BALB/C mice of 6-8 weeks old; one (250. Mu.L) of the frozen ONN-KLH is taken, after natural thawing, 250. Mu.L of 501 adjuvant is added, and after complete mixing, mice are immunized by the method of calf intramuscular injection, and 100. Mu.L/mouse is immunized.
4) The same method was used to boost the immunity once after 12 days; the immunization was then boosted 3 times every 10 days; tail tips of mice were bled 7 days after the last booster immunization and titers were determined; titer determination ELISA was performed using ONN-BSA coated ELISA plates to obtain serum direct gradient dilution titers of more than 100 parts per million for 2 mice.
5) Directly injecting the high-titer immunized mice into the abdominal cavity by using ONN-KLH of 0.5mg/mL, and performing impact immunization by 200 mu L of each mouse; after 3 days, spleen was taken to prepare monoclonal antibody hybridomas.
6) The screening method adopts ELISA method, namely, the N protein of the novel coronaviruses of the wild type and various mutant strains is coated on a 96-well enzyme-linked immunoassay (ELISA) plate, and the specific monoclonal antibody which only reacts with the N protein of the novel coronavirus Omicron (B.1.1.529) is analyzed and screened, and is marked as nCovON1.
EXAMPLE 2 preparation of novel coronavirus N protein Universal monoclonal antibody
1) A polypeptide sequence KGFYAEGSRGGSQASSRSSSRSRNSSR of a novel coronavirus N protein conserved region epitope with the purity of more than or equal to 95 percent is synthesized by Shanghai blaze biotechnology limited company and is marked as N-3; and GQQQQGQTVTKKSAAEASKKPRQKRTA with purity more than or equal to 95 percent, which is marked as N-4; and coupled to KLH and BSA, respectively, designated N-3-KLH, N-3-BSA, and N-4-KLH, N-4-BSA, respectively.
2) N-3-KLH was prepared into a 0.5mg/mL solution with 0.01M PBS (pH 7.4), and split into 5 branches, 250. Mu.L/branch, with 1.5mLEP tube; frozen stock was used as the immunizing antigen for later use.
3) Taking 5 male healthy BALB/C mice of 6-8 weeks old; one frozen N-3-KLH branch (250 mu L) is taken, after natural thawing, 250 mu L of 501 adjuvant is added, and after complete mixing, mice are immunized by a method of calf intramuscular injection, and 100 mu L/mouse is immunized.
4) The same method was used to boost the immunity once after 12 days; the immunization was then boosted 3 times every 10 days; tail tips of mice were bled 7 days after the last booster immunization and titers were determined; titer determination ELISA was performed using N-3-BSA coated ELISA plates to obtain serum direct gradient dilution titers of more than 100 parts per million for 2 mice.
5) Directly injecting the high-titer immunized mice into the abdominal cavity by using 0.5mg/mL of N-3-KLH, and performing impact immunization by 200 mu L of each mouse; after 3 days, spleen was taken to prepare monoclonal antibody hybridomas.
6) The screening method adopts ELISA method, namely, the N protein of the novel coronaviruses of the wild type and various mutant strains is coated on a 96-well enzyme-linked immunoassay (ELISA) plate, and the universal monoclonal antibody which can react with the N protein of the novel coronaviruses of the wild type and various mutant strains is analyzed and screened, wherein the identification epitope is KGFYAEGSRGGSQASSRSSSRSRNSSR, and the identification epitope is nCovN3.
7) A universal monoclonal antibody which can react with N protein of novel coronaviruses of wild type and various mutant strains is prepared by immunizing mice with N-4-KLH according to the same method as in 2) to 6), and the recognition epitope is GQQQQGQTVTKKSAAEASKKPRQKRTA and is marked as nCovN4.
EXAMPLE 3 preparation of novel coronavirus S1 protein Universal and Omicron Strain-specific monoclonal antibodies
1) The immune antigen is novel coronavirus Omicron strain S1 protein with purity more than or equal to 95%, and is provided by Hangzhou Michael biotechnology limited company; s1 protein was prepared in 0.01M PBS (pH 7.4) to a solution of 0.5mg/mL, and split-packed into 5 pieces with 1.5mLEP tubes, 250. Mu.L/piece; frozen stock was used as the immunizing antigen for later use.
2) Taking 5 male healthy BALB/C mice of 6-8 weeks old; taking one (250 mu L) of the frozen S1 protein, naturally thawing, adding 250 mu L of 501 adjuvant, and fully mixing to obtain 100 mu L/mouse by calf intramuscular injection.
3) The same method was used to boost the immunity once after 12 days; the immunization was then boosted 3 times every 10 days; tail tips of mice were bled 7 days after the last booster immunization and titers were determined; titer measurement ELISA was performed using ELISA plates coated with Omicron strain S1 protein to obtain 3 mice with serum direct gradient dilution titers of over 100 parts per million.
4) Directly injecting the high-titer immunized mice into the abdominal cavity with 0.5mg/mL of S1 protein, and performing impact immunization with 200 mu L of each; after 3 days, spleen was taken to prepare monoclonal antibody hybridomas.
5) The screening method adopts ELISA method, namely, the S1 protein of the novel coronaviruses of the wild type and various mutant strains is coated on a 96-well enzyme-linked immunoassay (ELISA) plate, and the specific monoclonal antibody which only reacts with the S1 protein of Omacron (B.1.1.529) is screened out by analysis, and is marked as nCovOS1, and 2 high-affinity general monoclonal antibodies which can react with the S1 protein of all the novel coronaviruses are respectively marked as nCovS2 and nCovS3.
Example 4 preparation of novel coronavirus antigen detection test strip (schematic diagram of the detection principle of the kit of the invention is shown in FIG. 1)
1. Preparation of test strip N:
1) The nCovN3 monoclonal antibodies and rabbit IgG prepared in the above example 2 were respectively marked with colloidal gold, and marked as CG-nCovN3 and CG-RIgG; and mixing CG-nCovN3 and CG-RIgG, spraying on a release pad of a test strip, and drying at 37 ℃ for 12 hours for standby.
2) The nCovON1 mab prepared in example 1, nCovN4 mab prepared in example 2 and goat anti-rabbit IgG polyclonal antibody were diluted to 0.5mg/mL with coating dilution (150 mmpb, ph 7.4), respectively, and sprayed and streaked uniformly on nitrocellulose membrane ONT1, NT2 detection line region and control line region (marked as C1 line region) with a spraying amount of 1 μl/cm, respectively, and dried at 37 ℃ for 12 hours, and sealed for later use.
3) The sample pad, the release pad, the nitrocellulose membrane and the absorbent paper are lapped and assembled on the bottom lining, the release pad and the absorbent paper are respectively overlapped at two ends of the nitrocellulose membrane, and a detection area (an ONT1 line area is close to the release pad, and a NT2 line area is close to a C1 line area) is formed on the surface of the nitrocellulose membrane; the C1 line area is close to the water absorption paper), the sample pad is overlapped on the release pad, and a novel coronavirus N protein antigen detection test paper large plate is formed after assembly, and can be cut into test paper strips with the width of 3-4mm according to the clamping case (figure 1 (A)) for standby.
2. Preparation of test strip S:
1) The nCovS2 monoclonal antibodies and rabbit IgG prepared in the above example 3 were respectively marked with colloidal gold, and marked as CG-nCovS2 and CG-RIgG; and mixing CG-nCovS2 and CG-RIgG, spraying on a release pad of a test strip, and drying at 37 ℃ for 12 hours for standby.
2) The ncovis 1 mab and ncovis 3 mab prepared in example 3 above and the goat anti-rabbit IgG polyclonal antibody were diluted to 0.5mg/mL with coating dilution (150 mmpb, ph 7.4), respectively, and uniformly sprayed and streaked on the nitrocellulose membrane OST1, ST2 detection line area and control line area (marked as C2 line area) at a spray film amount of 1 μl/cm, respectively, and dried at 37 ℃ for 12 hours, and sealed for use.
3) The sample pad, the release pad, the nitrocellulose membrane and the absorbent paper are lapped and assembled on the bottom lining, the release pad and the absorbent paper are respectively overlapped at two ends of the nitrocellulose membrane, and a detection area (an OST1 line area is close to the release pad, and an ST2 line area is close to a C2 line area) is formed on the surface of the nitrocellulose membrane; the C2 line area is close to the water absorption paper), the sample pad is overlapped on the release pad, and a novel coronavirus N protein antigen detection test paper large plate is formed after assembly, and can be cut into test paper strips with the width of 3-4mm according to the clamping case (figure 1 (B)) for standby.
3. Card-loading
The prepared test strip N and test strip S are parallelly connected into a duplex card, so that the novel coronavirus antigen rapid detection kit paper (shown in fig. 1 (C)) capable of identifying the omacron strain is prepared, can be used for simultaneous detection of novel coronavirus N protein and S protein, and can rapidly identify omacron mutant strain.
Example 5 test of novel coronavirus antigen Rapid test kit capable of identifying Omacron strain
The novel coronavirus antigen rapid detection kit capable of identifying the Omicron strain, which is developed based on the technical scheme of the invention, can be applied to simultaneous detection of N protein and S protein of all novel coronavirus strains at present, and can rapidly identify Omicron mutant strains; the method is suitable for detecting various biological samples such as excrement, urine, tears, oral mucosa liquid, respiratory tract secretion, whole blood, blood plasma, serum and the like and environmental samples.
1. Sensitivity experiment
1) Test strip S novel coronavirus detection sensitivity experiment:
recombinant expression of novel coronavirus spinous process protein S1 was obtained, and included wild-type and various mutants Mu (B.1.621), delta variant B.1.617.2, lambda variant C.37, delta/Delta plus, alpha/Beta (B.1.1.7/B.1.351/A.2.2), beta (B.1.351), gamma (P.1/P.1.2), epsilon (B.1.427/429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), B.1.617, B.1.617.3, B.1.618, B.1.620, A.23.1, P.2, P.3, respectively, prepared with virus lysate (0.01M, PBS containing 1% NP-40, 0.5% Triton X-100, 5.5, pH 7.5, 0.05, 0.01 ml, and blank concentrations of PBS, 0.01 ml. The test strip S and the test strip N of the kit are respectively spotted, 100 mu l of each hole is used for chromatography, and a T line (namely two detection lines) is observed by naked eyes after 10-15 minutes of chromatography; 5 replicates were set for each concentration. Results: the test strip N has no color development on both T lines (ONT 1 and NT 2) and is negative. The OST1 lines of the test strips S are all non-developed and negative, which indicates that the Omacron strain S1 protein detection line does not cross react with other strains. The concentration S1 proteins of the ST2 line pairs 5, 1, 0.5, 0.1, 0.05 and 0.01ng/ml of the test strip S are all colored, positive, and the change from deep to shallow is shown, so that the effective relationship is realized; the blank control and the 0.005ng/ml concentration group are not developed, and are negative, so that the kit can detect the novel coronavirus wild type and the S1 protein of each mutant strain, and the sensitivity is 10pg/ml.
The recombinant expressed novel coronaviruses OmicronBA.1 and BA.2 spinous process protein S1 are respectively prepared into concentrations of 5, 1, 0.5, 0.1, 0.05, 0.01 and 0.005ng/ml by using virus lysate, and blank is the virus lysate. The test strip S and the test strip N of the kit are respectively spotted, 100 mu l of each hole is used for chromatography, and a T line (namely two detection lines) is observed by naked eyes after 10-15 minutes of chromatography; 5 replicates were set for each concentration. Results: the test strip N has no color development on both T lines (ONT 1 and NT 2) and is negative. OST1 line pairs 5, 1, 0.5, 0.1, 0.05 and 0.01ng/ml of test strip S are colored, positive and show the change from deep to shallow, and the effective relationship; the blank control and the 0.005ng/ml concentration group are not developed, and are negative, so that the kit can identify novel strains OmicronBA.1 and BA.2 of coronaviruses, and the sensitivity is 10pg/ml of spinous process protein S1.
2) Test strip N novel coronavirus detection sensitivity experiment:
recombinant expression of novel coronavirus N proteins, including wild-type and mutant strains Mu (B.1.621), lambda variant C.37, delta/Delta plus, alpha/Beta (B.1.1.7/B.1.351/A.2.2), gamma (P.1/P.1.1/P.1.2), eta (B.1.525), iota (B.1.526), B.1.617, B.1.617.3, B.1.618, A.23.1, P.2 were prepared with virus lysates (0.01M PBS containing 1% NP-40, 0.5% Triton X-100, pH 7.4) at concentrations of 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005ng/ml, respectively, with blanks of virus lysates. The test strip S and the test strip N of the kit are respectively spotted, 100 mu l of each hole is used for chromatography, and a T line (namely two detection lines) is observed by naked eyes after 10-15 minutes of chromatography; 5 replicates were set for each concentration. Results: the test strip S has no color development on both T lines (OST 1 and ST 2), and is negative. No color development was observed on ONT1 lines of test strip N, which were negative, indicating that the Omacron strain N protein detection line did not cross-react with other strains. The concentration N proteins of NT2 line pairs 5, 1, 0.5, 0.1, 0.05 and 0.01ng/ml of the test strip N are all colored, positive, and the change from deep to shallow is shown, so that the quantitative effect relationship is realized; the blank control and the 0.005ng/ml concentration group are not developed, and are negative, so that the kit can detect the novel coronavirus wild type and the N protein of each mutant strain, and the sensitivity is 10pg/ml.
N proteins of recombinant expressed novel coronaviruses Omicron BA.1 and BA.2 are respectively prepared into concentrations of 5, 1, 0.5, 0.1, 0.05, 0.01 and 0.005ng/ml by using virus lysate, and blank is the virus lysate. The test strip S and the test strip N of the kit are respectively spotted, 100 mu l of each hole is obtained, and the T line is observed by naked eyes after chromatography for 10-15 minutes; 5 replicates were set for each concentration. Results: the test strip S has no color development on both T lines (OST 1 and ST 2), and is negative. The ONT1 line pair 5, 1, 0.5, 0.1, 0.05 and 0.01ng/ml of the test strip N are colored, positive and show the change from deep to shallow, and have an effective relationship; the blank control and the 0.005ng/ml concentration group are not developed, and are negative, so that the kit can identify novel strains of coronaviruses Omicron BA.1 and BA.2, and the sensitivity is 10pg/ml of spinous process protein.
2. Cross-reaction experiments:
using the product of the invention, cross-reaction tests were performed on other common human-epidemic coronavirus (HKU 1, OC43, NL63 and 229E) pathogens. Through data analysis, the product does not cross react with human epidemic coronaviruses (HKU 1, OC43, NL63 and 229E).
3. Clinical test:
using the product of the invention, nasal swab samples of 100 normal healthy volunteers were tested negative, indicating a specificity of 100%. The nasal swab samples of 100 clinically diagnosed patients are detected positively, which indicates that the detection rate is 100%; 67 of the samples are identified as Omicron strains, and the accuracy rate is 100% through nucleic acid detection, so that the product provided by the invention can be used for effectively detecting novel coronavirus antigens and identifying whether the novel coronavirus antigens are Omicron strains or not.
Claims (10)
1. The novel rapid detection kit for the coronavirus antigen is characterized by comprising two independent detection strips, wherein the two independent detection strips are respectively used for detecting novel coronavirus N protein antigen and novel coronavirus S protein antigen, are respectively marked as a test strip N and a test strip S, and are respectively provided with 2 detection lines;
wherein the ONT1 detection line of the test strip N is coated with an anti-novel coronavirus Omicron strain N protein specific antibody (detection type), and the NT2 detection line is coated with an anti-novel coronavirus N protein universal antibody (detection type) which can identify wild type and various mutant strains; OST1 detection line of test strip S is coated with specific antibody (detection type) of anti-novel coronavirus Omicron strain S1 protein, ST2 detection line is coated with general antibody (detection type) of anti-novel coronavirus S1 protein which can recognize wild type and various mutant strains.
2. A novel rapid detection kit for coronavirus antigens capable of identifying omacron strain as claimed in claim 1, wherein the preparation method of the antibody specific for omacron strain N protein of anti-novel coronavirus and the antibody universal for novel coronavirus N protein comprises the steps of:
1) Synthesizing a novel coronavirus Omicron strain N protein near-N-terminal specific polypeptide sequence as an antigen epitope, and coupling the synthesized specific polypeptide sequence as the antigen epitope with immune protein to prepare an immune antigen;
2) Immunizing an animal by using the immune antigen prepared in the step 1), and separating B lymphocytes of the immunized animal after measuring high-titer immune serum to prepare a monoclonal antibody cell strain; screening out a monoclonal antibody specific to the novel coronavirus Omicron strain N protein by taking the novel coronavirus N protein of the wild type mutant strain and various mutant strains as reaction antigens;
3) The monoclonal antibody cell strain with the specificity of the anti-novel coronavirus Omicron strain N protein screened in the step 2) is largely amplified to prepare antibodies for later use;
4) Synthesizing polypeptide sequences of 6 conserved regions of novel coronavirus N protein as antigen epitopes, respectively coupling the synthesized polypeptide sequences as antigen epitopes to immune proteins, and preparing 6 immune antigens;
5) Respectively immunizing animals with the 6 immune antigens prepared in the step 4), and separating B lymphocytes of the immunized animals after high-titer immune serum is measured to prepare monoclonal antibody cell strains; screening 2 or more than 2 novel coronavirus N protein universal monoclonal antibodies with novel coronavirus N proteins of wild type and various mutant strains as reaction antigens;
the preparation method of the anti-novel coronavirus Omicron strain S1 protein specific antibody and the anti-novel coronavirus S protein universal antibody comprises the following steps:
s1: immunizing an animal with a novel coronavirus Omicron strain S1 protein, and separating B lymphocytes of the immunized animal after high-titer immune serum is measured to prepare a monoclonal antibody cell strain; screening a novel coronavirus Omicron strain S1 protein specific monoclonal antibody and a novel coronavirus S1 protein general monoclonal antibody by taking novel coronavirus S1 proteins of wild type and various mutant strains as reaction antigens;
s2: and (3) amplifying the novel coronavirus Omicron strain S1 protein specific monoclonal antibody cell strain and the universal monoclonal antibody cell strain screened in the step (S1) in a large quantity to prepare antibodies for later use.
3. The rapid detection kit for novel coronavirus antigens capable of identifying omacron strain according to claim 2, wherein in step 1), the sequence of antigen epitope specific polypeptide near N-terminal of novel omacron strain N protein is NALRITFGGPSDSTGSNQNGGAR, a cysteine is added at C-terminal during synthesis, and the synthesized polypeptide is coupled to an immune protein by thiol-dimercapto exchange reaction by utilizing thiol of cysteine, wherein the immune protein is KLH, BSA, OVA or BGG.
4. The rapid detection kit for novel coronavirus antigens capable of identifying omacron strain according to claim 2, wherein in step 2), 5) or S1, the method for testing high titer immune serum is ELISA measurement, and the serum direct gradient dilution titer of immune animal is more than 100 parts per million, namely the immune animal can be adopted; the method for preparing the monoclonal antibody cell strain comprises the steps of preparing B lymphocyte and mouse myeloma fusion cells, namely hybridomas, by using a mouse monoclonal antibody technology by taking a mouse as an immune animal; preparing monoclonal nano antibody cell strain by using nano antibody preparation technology with ostriches or crocodiles animals as immune hosts; preparing a monoclonal antibody cell strain by using a genetic engineering antibody technology and taking rabbits or chickens as immune hosts;
the screening method in the step 2) adopts an ELISA method, namely, N proteins of novel coronaviruses of wild type and various mutant strains are coated on a 96-well enzyme-linked immunoassay ELISA plate, and a specific monoclonal antibody which only reacts with the N proteins of the novel coronaviruses Omicron strain is screened out by analysis.
5. A novel rapid detection kit for coronavirus antigens capable of identifying omacron strain according to claim 2, wherein in step 4), the polypeptide sequence of the 6 conserved region epitopes of said novel coronavirus N protein is selected from the group consisting of:
ARSKQRRPQGLPNNTASWFTALTQHGK, identified as N-1;
RRIRGGDGKMKDLSPRWYFYYLGTGPE, identified as N-2;
KGFYAEGSRGGSQASSRSSSRSRNSSR, identified as N-3;
GQQQQGQTVTKKSAAEASKKPRQKRTA, identified as N-4;
GAIKLDDKDPNFKDQVILLNKHIDAYK, identified as N-5;
QQTVTLLPAADLDDFSKQLQQSMSSAD, identified as N-6;
during synthesis, cysteine is added at the C end of the 6 polypeptide sequences, and the synthesized polypeptide is coupled to immune protein KLH, BSA, OVA or BGG through thiol-dimercapto exchange reaction by utilizing the thiol of the cysteine.
6. The rapid detection kit for novel coronavirus antigens capable of identifying omacron strain as claimed in claim 2, wherein in step 5), the screening method adopts ELISA method, i.e. the N proteins of wild type and various mutant novel coronaviruses are coated on 96-well ELISA plate, and the high affinity universal monoclonal antibodies capable of reacting with the N proteins of all strains of novel coronaviruses are screened out by analysis, at least 2 or more.
7. The rapid detection kit for novel coronavirus antigens capable of identifying Omicron strain according to claim 2, wherein in step S1, the screening method adopts an ELISA method, i.e. the novel coronavirus S1 proteins of wild type and various mutant strains are coated on a 96-well enzyme-linked immunoassay ELISA plate, and specific monoclonal antibodies which only react with the novel coronavirus Omicron strain S1 protein and high affinity universal monoclonal antibodies which can react with all strain type novel coronavirus S1 proteins are screened out by analysis, wherein the number of the universal monoclonal antibodies is at least 2 or more.
8. A novel rapid detection kit for coronavirus antigens capable of identifying omacron strains according to claim 1, wherein said kit further comprises a marker for a capture antibody; the capture antibody adopts a general type, and comprises anti-novel coronavirus N protein and S1 protein general type capture antibodies; meanwhile, the rabbit IgG is marked by the same medium and is used for a quality control system;
the anti-novel coronavirus antibody (capture type) capable of identifying wild type and various mutant strains is marked by colloidal gold, colloidal carbon, colored latex or fluorescent microsphere and is used for capturing novel coronaviruses in a sample, and the novel coronaviruses can be combined with a corresponding detection area to form a visible strip in the chromatographic process, wherein the capture type antibody of the test strip N is an anti-novel coronavirus N protein universal type capture antibody; the capture antibody of the test strip S is a general capture antibody for resisting novel coronavirus S1 protein.
9. The rapid detection kit for novel coronavirus antigens capable of identifying omacron strain according to claim 8, wherein the test strip N and the test strip S of the kit comprise immunochromatographic test strips, and the immunochromatographic test strip comprises a substrate, a nitrocellulose membrane, a sample pad, a release pad and absorbent paper; the sample pad, the release pad, the nitrocellulose membrane and the absorbent paper are lapped and assembled on the bottom lining, the release pad and the absorbent paper are respectively overlapped at two ends of the nitrocellulose membrane, a detection area is formed on the surface of the nitrocellulose membrane, and the sample pad is overlapped on the release pad; a detection area T1 line, a detection area T2 line and a control area C line are sequentially arranged on the nitrocellulose membrane of the detection area from the release pad to the absorbent paper;
the preparation of the test strip N comprises the following steps: mixing a marked general capture antibody for resisting novel coronavirus N protein and marked IgG, spraying on a release pad of an immunochromatographic test strip, and coating a detection zone T1 line on a nitrocellulose membrane with an anti-novel coronavirus Omicron strain N protein specific monoclonal antibody, which is marked as ONT1; t2 line is coated with a general monoclonal antibody (detection type) resisting novel coronavirus N protein, and is marked as NT2; the nitrocellulose membrane of the C line area is coated with goat anti-rabbit IgG polyclonal antibody (GAR), and the mark is C1;
the preparation of the test strip S comprises the following steps: mixing a marked general capture antibody for resisting novel coronavirus S protein and marked IgG, spraying on a release pad of an immunochromatographic test strip, and coating an anti-novel coronavirus Omicron strain S1 protein specific monoclonal antibody on a T1 line of a detection area on a nitrocellulose membrane, wherein the mark is OST1; t2 line is coated with a general monoclonal antibody (detection type) resisting novel coronavirus S1 protein, and is marked as ST2; the nitrocellulose membrane of the C line area is coated with goat anti-rabbit IgG polyclonal antibody (GAR), and the mark is C2;
the test strip N and the test strip S are assembled in parallel on the duplex clamping plate for simultaneous detection of novel coronavirus N protein and S protein, and the Omicron mutant strain can be rapidly identified.
10. The rapid detection kit for novel coronavirus antigens capable of identifying omacron strain according to claim 9, wherein the kit is suitable for detection of respiratory secretions, feces, urine, tears or environmental samples, and can be applied to rapid identification detection of novel coronaviruses and omacron mutant strains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210408994.5A CN116047065A (en) | 2022-04-19 | 2022-04-19 | Novel coronavirus antigen rapid detection kit capable of identifying omacron strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210408994.5A CN116047065A (en) | 2022-04-19 | 2022-04-19 | Novel coronavirus antigen rapid detection kit capable of identifying omacron strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116047065A true CN116047065A (en) | 2023-05-02 |
Family
ID=86126115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210408994.5A Pending CN116047065A (en) | 2022-04-19 | 2022-04-19 | Novel coronavirus antigen rapid detection kit capable of identifying omacron strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116047065A (en) |
-
2022
- 2022-04-19 CN CN202210408994.5A patent/CN116047065A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7022969B2 (en) | Methods and Reagents for Diagnosing SARS-CoV-2 Infections | |
| Walls et al. | Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses | |
| AU2008334976B2 (en) | Compositions and methods for early pregnancy diagnosis | |
| US20060188519A1 (en) | Peptides, antibodies, and methods for the diagnosis of SARS | |
| TW202200603A (en) | Epitope of antibody against structural protein of sars-cov-2, antibody that reacts with the epitope, method for detecting sars-cov-2 using the antibody, kit for detecting sars-cov-2 containing the antibody, method for detecting anti-sars-cov-2 antibody containing polypeptide of the epitope, kit for detecting anti-sars-cov-2 antibody containing polypeptide of the epitope, vaccine for sars-cov-2 containing polypeptide of the epitope, and therapeutic agent for sars-cov-2 infectious disease containing the antibody | |
| EP1894005B1 (en) | Methods and compositions for detecting herpes simplex virus type 2 | |
| CN111474345A (en) | SARS-CoV-2 antibody detection method | |
| KR101782862B1 (en) | Monoclonal antibody for diagnosing MERS virus and immunochromatographic diagnostic kit | |
| JP6616333B2 (en) | Immunological detection method and kit for Mycoplasma pneumoniae | |
| EP4113121A1 (en) | Antigen for 2019 novel coronavirus and detection use thereof | |
| CN105891491A (en) | Kit and application thereof | |
| WO2005042579A1 (en) | Anti-sars virus antibody, hybridoma producing the antibody and immunoassay reagent using the antibody | |
| JP6616332B2 (en) | Immunological detection method and kit for Mycoplasma pneumoniae | |
| KR100832870B1 (en) | Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof | |
| CN113150133B (en) | Monoclonal antibodies against SARS-CoV-2 or antigen binding fragments thereof | |
| Vidal et al. | A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of African swine fever virus antigens and antibodies | |
| US20220144912A1 (en) | Leptin immunogens, hybridoma cells, monoclonal antibodies, polyclonal antibodies and use thereof | |
| CN116047065A (en) | Novel coronavirus antigen rapid detection kit capable of identifying omacron strain | |
| KR100832867B1 (en) | Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof | |
| Potgieter et al. | Quantitation of canine distemper virus and antibodies by enzyme-linked immunosorbent assays using protein A and monoclonal antibody capture | |
| CN111704656A (en) | Duck adenovirus I type Penton protein and preparation method and application thereof | |
| JP2010276441A (en) | Detection method of streptococcus pneumoniae | |
| KR101287602B1 (en) | Antibody recognizing kidney type and respiratory type infectious bronchitis virus and use thereof | |
| EP0291479A1 (en) | Immunoassay method for the diagnosis of chlamydia infection | |
| CN113072625B (en) | Polypeptide, novel detection test paper and detection kit for coronavirus antibody |
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 |