CN112326966A - Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof - Google Patents
Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof Download PDFInfo
- Publication number
- CN112326966A CN112326966A CN202011205245.XA CN202011205245A CN112326966A CN 112326966 A CN112326966 A CN 112326966A CN 202011205245 A CN202011205245 A CN 202011205245A CN 112326966 A CN112326966 A CN 112326966A
- Authority
- CN
- China
- Prior art keywords
- pad
- monoclonal antibody
- solution
- sample
- kit
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 93
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 33
- 239000000427 antigen Substances 0.000 title claims abstract description 27
- 102000036639 antigens Human genes 0.000 title claims abstract description 27
- 108091007433 antigens Proteins 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000012528 membrane Substances 0.000 claims abstract description 56
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 42
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 42
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 36
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 33
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 32
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 32
- 238000003908 quality control method Methods 0.000 claims abstract description 31
- 241000283707 Capra Species 0.000 claims abstract description 10
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims abstract description 9
- 238000010521 absorption reaction Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 101710114810 Glycoprotein Proteins 0.000 claims abstract description 6
- 101710167605 Spike glycoprotein Proteins 0.000 claims abstract description 6
- 238000002331 protein detection Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 86
- 239000000243 solution Substances 0.000 claims description 61
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 38
- 238000001035 drying Methods 0.000 claims description 34
- 235000018102 proteins Nutrition 0.000 claims description 34
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 24
- 229940098773 bovine serum albumin Drugs 0.000 claims description 24
- 239000000337 buffer salt Substances 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 23
- 238000006748 scratching Methods 0.000 claims description 22
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 19
- 239000013504 Triton X-100 Substances 0.000 claims description 19
- 229920004890 Triton X-100 Polymers 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 19
- 239000011248 coating agent Substances 0.000 claims description 19
- 238000000576 coating method Methods 0.000 claims description 19
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 19
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 19
- 239000005018 casein Substances 0.000 claims description 18
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 18
- 235000021240 caseins Nutrition 0.000 claims description 18
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 18
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 16
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 16
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- 239000003365 glass fiber Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- 230000002393 scratching effect Effects 0.000 claims description 10
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 6
- 239000003223 protective agent Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 5
- 239000002981 blocking agent Substances 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 239000002250 absorbent Substances 0.000 claims description 4
- 230000002745 absorbent Effects 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 4
- 230000031700 light absorption Effects 0.000 claims description 4
- 239000012470 diluted sample Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 125000000647 trehalose group Chemical group 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 9
- 238000012360 testing method Methods 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 9
- 101710141454 Nucleoprotein Proteins 0.000 description 8
- 102100031673 Corneodesmosin Human genes 0.000 description 6
- 101710139375 Corneodesmosin Proteins 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 101710137302 Surface antigen S Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 241000008904 Betacoronavirus Species 0.000 description 4
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 4
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 241000700605 Viruses Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 101710204837 Envelope small membrane protein Proteins 0.000 description 2
- 101710145006 Lysis protein Proteins 0.000 description 2
- 101710085938 Matrix protein Proteins 0.000 description 2
- 101710127721 Membrane protein Proteins 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000007888 film coating Substances 0.000 description 2
- 238000009501 film coating Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 201000009240 nasopharyngitis Diseases 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000010200 validation analysis 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
- G01N33/577—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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/569—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
- 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
Abstract
The invention discloses a novel rapid detection kit for coronavirus antigens, which comprises a box body and a reagent strip placed in the box body, and is characterized in that the reagent strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped end to end along the length direction of the bottom plate; the combination pad is coated with a colloidal gold labeled anti-nucleocapsid protein monoclonal antibody and an anti-spinous process glycoprotein monoclonal antibody; the cellulose nitrate membrane is respectively coated with a nucleocapsid protein detection line N line formed by an anti-nucleocapsid protein monoclonal antibody, a spike glycoprotein detection line S line formed by an anti-spike glycoprotein monoclonal antibody and a quality control line C line formed by a goat anti-mouse IgG antibody. The invention also discloses a preparation method and application of the kit. The rapid detection kit prepared by the invention can directly detect the novel coronavirus, is simple and convenient to operate, has short detection time, and is suitable for large-scale crowd screening and field screening.
Description
Technical Field
The invention belongs to the technical field of biological immunodetection and analysis, and particularly relates to a novel rapid detection kit for coronavirus antigens, and a preparation method and application thereof.
Background
The new coronavirus (2019-nCoV) appeared at the end of 2019, which attracts extensive attention in all communities and can be transmitted through respiratory droplets, contact and the like.
The existing detection method aiming at the novel coronavirus mainly comprises two types of nucleic acid detection and antibody detection: the nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity and the like, is the 'gold standard' for determining new coronary pneumonia, but has higher requirements on detection equipment, can be carried out in a laboratory with relatively complete conditions, has the average detection time of more than 4 hours, is complex to operate and depends on a real-time quantitative PCR instrument and high-level technicians; the antibody detection method has the advantages of being convenient and fast to use, short in detection time, capable of effectively breaking through the limitation of the existing detection technology on personnel and places, and capable of shortening detection time. However, the antibody production is in a window period, which causes false negative results in antibody detection, and the IgG antibody exists for a long time and cannot be used for rehabilitation period examination, and the existing infected person cannot be effectively distinguished.
Therefore, in order to overcome the technical defects of the above detection, a new detection method for detecting a novel coronavirus is needed.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a novel rapid detection kit for coronavirus antigens and a preparation method and application thereof aiming at the defects of the prior art.
The novel coronavirus antigen detection reagent disclosed by the invention is used for selecting a detection target antigen, can be used as an antigen to induce immune response in the novel coronavirus, and is a protein combined with a specific antibody, and mainly comprises 4 structural proteins: spike glycoprotein (S protein for short), membrane glycoprotein (E protein for short), small membrane glycoprotein (M protein for short) and nucleocapsid protein (N protein for short), the corresponding antibodies are mainly N protein antibody and S protein antibody; the N protein is relatively conserved, has high homology with N proteins of other beta coronaviruses, for example, the homology with coronaviruses (Sars-COV) is more than 90%, and is infected by other common beta coronaviruses (such as HKU1, OC43, NL63 and 229E) to cause a patient with common cold, and is also easily recognized by a specific antibody marked by colloidal gold to generate 'false positive', however, the S protein has high specificity as a main action protein of the novel coronaviruses entering cells, and has homology with coronaviruses (Sars-COV) of about 56%, so that the N antigen and the S antigen of the novel coronaviruses are selected as detection targets of the antigens of the novel coronaviruses.
The technical scheme for realizing the invention is as follows:
the invention relates to a novel rapid detection kit for coronavirus antigens, which comprises a box body and a reagent strip placed in the box body, wherein the reagent strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped end to end along the length direction of the bottom plate;
the combination pad is coated with a colloidal gold labeled anti-nucleocapsid protein monoclonal antibody and an anti-spinous process glycoprotein monoclonal antibody;
the cellulose nitrate membrane is respectively coated with a nucleocapsid protein detection line N line formed by an anti-nucleocapsid protein monoclonal antibody, a spike glycoprotein detection line S line formed by an anti-spike glycoprotein monoclonal antibody and a quality control line C line formed by a goat anti-mouse IgG antibody.
Preferably, the colloidal gold-labeled anti-nucleocapsid protein monoclonal antibody and anti-spinous process glycoprotein monoclonal antibody coated on the conjugate pad are prepared by the following method:
preparing a gold-labeled antibody, spraying the prepared gold-labeled antibody on a bonding pad, drying the bonding pad by adopting an SB-08 glass fiber membrane in a drying environment at the temperature of 37 ℃ for 5 hours to solidify the gold-labeled antibody on the bonding pad, and sealing and storing.
Preferably, the preparation method of the gold-labeled antibody is as follows: taking 1mL of colloidal gold solution, wherein the wavelength range corresponding to the maximum visible light absorption value of the colloidal gold solution is 520-530 nm, adding 10 mu L of potassium carbonate with the use concentration range of 0.02-0.5M, and uniformly mixing for 2 min; adding the anti-nucleocapsid protein monoclonal antibody with the final concentration of 5-20 mug/mL and the anti-spinous process glycoprotein monoclonal antibody with the final concentration of 5-20 mug/mL into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; then adding 0.1mL of BSA as a blocking agent, wherein the use mass fraction concentration range of the BSA is 1% -10%, and uniformly mixing for 30 min; adding a BSA (bovine serum albumin) uniformly mixed solution, centrifuging for 10min at the rotation speed of 12500rpm at 4 ℃, carefully discarding the supernatant, then re-suspending and precipitating by using a Tris-HCL buffer solution with the concentration of 8.0 being 40 mu L, pH, adding sucrose serving as a protective agent into the re-suspended solution, wherein the final concentration of the sucrose in percentage by mass is 5-30%, and fully mixing to prepare the gold-labeled antibody.
Further, in the invention, the wavelength corresponding to the maximum visible light absorbance of the colloidal gold solution is 525nm, the use concentration of potassium carbonate is 0.2M, the final concentration of the anti-nucleocapsid protein monoclonal antibody is 5 mug/mL, the final concentration of the anti-spinous process glycoprotein monoclonal antibody is 5 mug/mL, the use mass fraction concentration of BSA is 10%, the use concentration range of Tris-HCL buffer solution is 0.01M, and the final mass fraction concentration of sucrose is 20%.
Preferably, the kit further comprises a membrane scratching coating solution, the anti-nucleocapsid protein monoclonal antibody and the anti-spinous-process glycoprotein monoclonal antibody are diluted to 1-2 mg/mL by the membrane scratching coating solution, then the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous-process glycoprotein monoclonal antibody are scratched on one end, close to the binding pad, of the nitrocellulose membrane in sequence to serve as a detection line, a goat anti-mouse IgG antibody is scratched on the other end of the nitrocellulose membrane to serve as a quality control line, and the scribed nitrocellulose membrane is placed in a drying environment at 37 ℃ to be dried for 48 hours to obtain the nitrocellulose membrane with the detection line and the quality control line.
Preferably, the film-scratching coating solution comprises PBS buffer salt, isopropanol, Triton X-100 and trehalose; the PBS buffer salt is PBS buffer salt with the final concentration range of 0.001M-0.05M, pH-8.2; the isopropanol is isopropanol with the volume fraction final concentration range of 1-5%; the Triton X-100 uses Triton X-100 with the volume fraction final concentration range of 0.0001-0.001%; the trehalose is used with a final concentration range of 0.1-1%.
Further, in the film-coating solution of the present invention, the final concentration of PBS buffer salt is preferably 0.01M, the final concentration of isopropanol is 2% by volume fraction, the final concentration of Triton X-100 is 0.0005% by volume fraction, the final concentration of trehalose is 0.3% by volume fraction, and the dilution concentration of the anti-SPP monoclonal antibody against the nucleocapsid protein monoclonal antibody is 2 mg/mL.
Preferably, the sample pad is soaked in the sample pad treatment solution for 30min, redundant liquid is discarded, and the sample pad is placed in a drying environment with the temperature of 37 ℃ for drying for 12h and taken out for standby.
Preferably, the sample pad treatment solution comprises PBS buffer, casein, and Tween-20; the PBS buffer solution uses PBS buffer salt with the concentration range of 0.01M-0.1M, pH-7.4; the casein is casein with the mass fraction concentration of 0.1-1%; the Tween-20 uses Tween-20 with the volume fraction concentration of 0.1-1%; preferably, the sample pad treatment solution in the present invention comprises PBS buffer solution with 0.01M, pH of 7.4, casein with mass fraction of 1%, and Tween-20 with volume fraction of 1%; the sample pad used a SB-08 glass fiber membrane.
A method for preparing the kit, which comprises the following steps:
step one, preparing a sample pad: the sample pad adopts an SB-08 glass fiber membrane, and is prepared with sample pad treatment fluid, wherein the sample pad treatment fluid comprises PBS buffer solution, casein and Tween-20; the PBS buffer solution uses PBS buffer salt with the concentration range of 0.01M-0.1M, pH-7.4; the casein is casein with the mass fraction concentration of 0.1-1%; the Tween-20 uses Tween-20 with the volume fraction concentration of 0.1-1%; in the embodiment, the preferable technical scheme is that the sample pad treatment solution comprises PBS buffer solution with 0.01M, pH being 7.4, casein with the mass fraction of 1% and Tween-20 with the volume fraction of 1%;
and (3) soaking the sample pad in the sample pad treatment solution for 30min, removing redundant liquid, drying the soaked sample pad for 12h in a drying environment at the temperature of 37 ℃, and taking out for later use.
Step two, preparing a bonding pad: the combination pad adopts an SB-08 glass fiber membrane;
firstly, preparing a gold-labeled antibody, taking 1mL of colloidal gold solution, adding 10 mu L of potassium carbonate with the use concentration range of 0.02-0.5M into the colloidal gold solution, and uniformly mixing for 2min, wherein the wavelength range corresponding to the maximum visible light absorbance value of the colloidal gold solution is 520-530 nm; adding the anti-nucleocapsid protein monoclonal antibody with the final concentration of 5-20 mug/mL and the anti-spinous process glycoprotein monoclonal antibody with the final concentration of 5-20 mug/mL into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; then adding 0.1mL of BSA as a blocking agent, wherein the use mass fraction concentration range of the BSA is 1% -10%, and uniformly mixing for 30 min; adding a BSA (bovine serum albumin) uniformly mixed solution, centrifuging for 10min at the rotation speed of 12500rpm at 4 ℃, carefully discarding the supernatant, then re-suspending and precipitating by using a Tris-HCL buffer solution with the concentration of 8.0 being 40 mu L, pH, adding sucrose serving as a protective agent into the re-suspended solution, wherein the final concentration of the sucrose in mass fraction is 5-30%, and fully mixing uniformly to prepare a gold-labeled antibody;
in the invention, preferably, the wavelength corresponding to the maximum visible light absorbance value of the colloidal gold solution is 525nm, the use concentration of potassium carbonate is 0.2M, the final concentration of the anti-nucleocapsid protein monoclonal antibody is 5 mug/mL, the final concentration of the anti-spinous-process glycoprotein monoclonal antibody is 5 mug/mL, the use mass fraction concentration of BSA is 10%, the use concentration range of a Tris-HCL buffer solution is 0.01M, and the final mass fraction concentration of sucrose is 20%;
then spraying the prepared gold-labeled antibody on a bonding pad, drying for 5h in a drying environment at the temperature of 37 ℃ to solidify the gold-labeled antibody on the bonding pad, and sealing and storing;
step three, film dispensing: preparing a film-scratching coating liquid;
the film-scratching coating solution comprises PBS buffer salt, isopropanol, Triton X-100 and trehalose; the PBS buffer salt is PBS buffer salt with the final concentration range of 0.001M-0.05M, pH-8.2; the isopropanol is isopropanol with the volume fraction final concentration range of 1-5%; the Triton X-100 uses Triton X-100 with the volume fraction final concentration range of 0.0001-0.001%; the trehalose is trehalose with a final concentration range of 0.1-1%;
in the present invention, it is preferable that the final concentration of PBS buffer salt is 0.01M, the final concentration of isopropanol is 2% by volume fraction, the final concentration of Triton X-100 is 0.0005% by volume fraction, the final concentration of trehalose is 0.3% by volume fraction, and the dilution concentration of the anti-SPP monoclonal antibody against the nucleocapsid protein monoclonal antibody is 2 mg/mL;
diluting the anti-nucleocapsid protein monoclonal antibody and the anti-spinous-process glycoprotein monoclonal antibody to 1-2 mg/mL by using a film-scratching coating solution, scratching one end of the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous-process glycoprotein monoclonal antibody, which is close to a binding pad, on a nitrocellulose membrane in sequence to serve as a detection line, scratching the other end of the nitrocellulose membrane by using a goat anti-mouse IgG antibody to serve as a quality control line, and placing the scribed nitrocellulose membrane in a drying environment at 37 ℃ for drying for 48 hours to obtain the nitrocellulose membrane with the detection line and the quality control line.
Step four, assembling the reagent card: and (3) sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad which are processed in the first step to the third step to a bottom plate, cutting the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad into reagent strips with the width of 3.00mm, and filling the reagent strips into a box body template to prepare the kit.
Preferably, a sample adding hole and an observation window are arranged on the kit body of the kit; the sample adding hole is positioned above the sample pad, and the observation window is positioned above the detection line and the quality control line.
The application of the kit prepared by the preparation method comprises the following steps:
after a sample to be detected is diluted by the sample diluent, the diluted sample to be detected is dripped on a sample pad of the reagent strip through the sample adding hole, and results of a detection line and a quality control line on the reagent strip are observed through an observation window within 15-20 minutes.
The interpretation of the results on the test strips of the present invention includes: and (4) positive result: when any one of the detection line N line 3 and the detection line S line 4 appears a red strip and the quality control line C line 5 normally appears a red strip, judging that the detection result is positive; when only one red strip appears in the detection line N line 3 and the detection line S line 4, a positive result is judged, and a nucleic acid detection reagent is recommended to be used for carrying out rechecking detection; negative result: if only the quality control line C line 5 has a red strip, and the detection line N line 3 and the detection line S line 4 do not have color development, judging that the detection result is a negative result; ③ invalid result: when the red strip does not appear on the line 5 of the quality control line C, the detection result is invalid.
By adopting the technical scheme, the invention has the following beneficial effects:
(1) the kit can directly detect the novel coronavirus by jointly detecting the N antigen and the S antigen of the novel coronavirus, has no window period, and effectively distinguishes a former infected person and an existing infected person; and the kit is simple and convenient to operate, the detection time period is short, the judgment of the detection result does not depend on large-scale equipment, and the kit is suitable for large-scale crowd screening and field screening.
(2) The method for detecting by using the kit jointly detects the N antigen and the S antigen of the new coronavirus and optimizes a reaction system, overcomes the defect of insufficient antigen detection sensitivity to a great extent, has high consistency with an RT-PCR method, and has high sensitivity and clinical use value; compared with the RT-PCR method, the detection method provided by the invention is simple to operate, detection can be performed without depending on high-level technicians, and the detection result is judged visually.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a schematic structural diagram of a reagent strip in the kit according to the present invention;
FIG. 2 is a schematic diagram illustrating the determination of the test result of the test strip according to the present invention.
In the figure, 1-sample pad, 2-combination pad, 3-detection line N line, 4-detection line S line, 5-quality control line C line, 6-water absorption pad, 7-nitrocellulose membrane and 8-bottom plate.
Detailed Description
The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the embodiments.
Example (b): the embodiment provides a rapid detection kit for a novel coronavirus antigen, the detection method of the kit comprises the steps of detecting the novel coronavirus antigen by using a double-antibody sandwich method, respectively coating and marking different specific monoclonal antibodies for the novel coronavirus on a nitrocellulose membrane and colloidal gold, and when a detected sample contains the novel coronavirus antigen, a common color strip appears in a corresponding area on a reagent strip of the kit to serve as a positive signal value; the performance of antigen detection reagents depends largely on the choice of the paired monoclonal antibody, and therefore screening for a paired monoclonal antibody with good sensitivity and specificity requires a great deal of validation.
In this embodiment, the selection of the target antigen for detection of the novel coronavirus antigen detection reagent can be used as an antigen to induce an immune response, and the protein bound to the specific antibody mainly comprises 4 structural proteins: spike glycoprotein (S protein for short), membrane glycoprotein (E protein for short), small membrane glycoprotein (M protein for short) and nucleocapsid protein (N protein for short), the corresponding antibodies are mainly N protein antibody and S protein antibody; the N protein is relatively conserved, has high homology with N proteins of other beta coronaviruses, for example, the homology with coronaviruses (Sars-COV) is more than 90%, and is infected by other common beta coronaviruses (such as HKU1, OC43, NL63 and 229E) to cause a patient with common cold, and is also easily recognized by a specific antibody marked by colloidal gold to generate 'false positive', however, the S protein has high specificity as a main action protein of the novel coronaviruses entering cells, and has homology with coronaviruses (Sars-COV) of about 56%, so that the N antigen and the S antigen of the novel coronaviruses are selected as detection targets of the antigens of the novel coronaviruses.
The kit for rapidly detecting the coronavirus antigen comprises a box body and a reagent strip placed in the box body, wherein the reagent strip comprises a bottom plate 8, and a sample pad 1, a combination pad 2, a nitrocellulose membrane 7 and a water absorption pad 6 which are sequentially overlapped end to end along the length direction of the bottom plate 8, as shown in figure 1;
the base plate 8 in this embodiment is preferably a PVC base plate; the sample pad 1 in the embodiment preferably adopts an SB-08 glass fiber membrane, the sample pad 1 is soaked in the sample pad treatment solution for 30min, the redundant liquid is discarded, the sample pad is placed in a drying box with the temperature of 37 ℃ for drying for 12h, and the sample pad is taken out for standby; the sample pad treatment solution in this example contained PBS buffer, casein, and Tween-20; PBS buffer was used at a concentration range of 0.01M to 0.1M, pH ═ 7.4 in PBS buffer salt; casein with mass fraction concentration of 0.1-1% is used; the Tween-20 is Tween-20 with volume fraction concentration of 0.1-1%; preferably, the sample pad treatment solution in this embodiment comprises PBS buffer solution with 0.01M, pH of 7.4, casein with a mass fraction of 1%, and Tween-20 with a volume fraction of 1%.
The combination pad in the embodiment is preferably an SB-08 glass fiber membrane, and the combination pad 2 is coated with a colloidal gold labeled anti-nucleocapsid protein monoclonal antibody and an anti-spinous process glycoprotein monoclonal antibody; the preparation method of the anti-nucleocapsid protein monoclonal antibody and the anti-spinous process glycoprotein monoclonal antibody marked by the colloidal gold coated on the binding pad 2 comprises the following steps:
preparing a gold-labeled antibody, wherein the preparation method of the gold-labeled antibody comprises the following steps:
taking 1mL of colloidal gold solution, wherein the wavelength range corresponding to the maximum visible light absorption value of the colloidal gold solution is 520-530 nm, adding 10 mu L of potassium carbonate with the use concentration range of 0.02-0.5M, and uniformly mixing for 2 min; adding the anti-nucleocapsid protein monoclonal antibody with the final concentration of 5-20 mug/mL and the anti-spinous process glycoprotein monoclonal antibody with the final concentration of 5-20 mug/mL into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; then adding 0.1mL of BSA serving as a blocking agent, wherein the BSA is used in a mass fraction concentration range of 1-10%, and uniformly mixing for 30 min; adding a uniformly mixed solution of BSA, centrifuging for 10min at the rotation speed of 12500rpm at 4 ℃, carefully discarding the supernatant, then re-suspending the precipitate by using a Tris-HCL buffer solution with the concentration of 8.0 mu L, pH, wherein the use concentration range of the Tris-HCL buffer solution is 0.01-0.1M, adding sucrose serving as a protective agent into the re-suspended solution, and fully mixing to prepare a gold-labeled antibody, wherein the final concentration of the sucrose in mass fraction is 5-30%; in the embodiment, the preferable technical scheme is that the wavelength corresponding to the maximum visible light absorbance value of the colloidal gold solution is 525nm, the use concentration of potassium carbonate is 0.2M, the final concentration of the anti-nucleocapsid protein monoclonal antibody is 5 mug/mL, the final concentration of the anti-spinous process glycoprotein monoclonal antibody is 5 mug/mL, the use mass fraction concentration of BSA is 10%, the use concentration range of a Tris-HCL buffer solution is 0.01M, and the final mass fraction concentration of sucrose is 20%;
spraying the prepared gold-labeled antibody on the bonding pad 2, drying the bonding pad 2 in a drying environment at 37 ℃ for 5h to solidify the gold-labeled antibody on the bonding pad 2, and sealing for storage.
As shown in fig. 1, a nucleocapsid protein detection line N line 3 formed by an anti-nucleocapsid protein monoclonal antibody, a spike glycoprotein detection line S line 4 formed by an anti-spinous glycoprotein monoclonal antibody, and a quality control line C line 5 formed by a goat anti-mouse IgG antibody are respectively coated on the nitrocellulose membrane 7 in this embodiment.
The embodiment also comprises a film-scratching coating solution, wherein the film-scratching coating solution comprises PBS buffer salt, isopropanol, Triton X-100 and trehalose; PBS buffer salts were used at final concentrations ranging from 0.001M to 0.05M, pH ═ 8.2; the isopropanol is isopropanol with the volume fraction final concentration range of 1-5%; triton X-100 uses Triton X-100 with volume fraction final concentration range of 0.0001% -0.001%; the final concentration range of the trehalose is 0.1-1%;
in the preferred film-coating solution of this example, the final concentration of PBS buffer salt is preferably 0.01M, the final concentration of isopropanol is 2% by volume fraction, the final concentration of Triton X-100 is 0.0005% by volume fraction, the final concentration of trehalose is 0.3% by volume fraction, and the dilution concentration of the anti-SPP monoclonal antibody against the nucleocapsid protein monoclonal antibody is 2 mg/mL;
diluting the anti-nucleocapsid protein monoclonal antibody and the anti-spinous-process glycoprotein monoclonal antibody to 1-2 mg/mL by using a film-scratching coating solution, scratching one end of the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous-process glycoprotein monoclonal antibody, which is close to a binding pad, on a nitrocellulose membrane in sequence to serve as a detection line, scratching the other end of the nitrocellulose membrane by using a goat anti-mouse IgG antibody to serve as a quality control line, and placing the scribed nitrocellulose membrane in a drying environment at 37 ℃ for drying for 48 hours to obtain the nitrocellulose membrane with the detection line and the quality control line.
The kit in this embodiment also relates to the assembly of the reagent card: and (3) sequentially adhering the processed sample pad 1, the combined pad 2, the nitrocellulose membrane 7 and the water absorption pad 6 to a bottom plate 8, cutting the sample pad, the combined pad, the nitrocellulose membrane and the water absorption pad into reagent strips with the width of 3.00mm by using a cutting machine, and filling the reagent strips into a box template to prepare the kit.
The kit body of the kit in the embodiment is provided with a sample adding hole and an observation window; the sample adding hole is positioned above the sample pad 1, the observation window is positioned above the detection line and the quality control line, a sample to be detected is added on the sample pad through the sample adding hole, after reaction for a period of time, the display results of the detection line and the quality control line are observed through the observation window, and then the attribute of the sample to be detected is judged.
The kit in the embodiment is used for detection, has the advantages of simple and convenient operation and quick diagnosis, does not depend on instruments and equipment and high-level technicians, and is suitable for large-scale crowd screening and field screening.
The preparation method of the kit adopts the preferred technical scheme in each step in the preparation method in the embodiment, and the preferred preparation method comprises the following steps:
step one, preparing a sample pad 1: the sample pad 1 adopts an SB-08 glass fiber membrane, and is prepared with a sample pad treatment solution, wherein the sample pad treatment solution comprises PBS buffer salt with 0.01M, pH of 7.4, casein with the mass fraction concentration of 1% and Tween-20 with the volume fraction concentration of 1%; and (3) soaking the sample pad 1 in the sample pad 1 treatment solution for 30min, removing redundant liquid, drying the soaked sample pad 1 in a drying environment at the temperature of 37 ℃ for 12h, and taking out for later use.
Step two, preparing the bonding pad 2: the combination pad 2 adopts an SB-08 glass fiber membrane, firstly, a gold-labeled antibody is prepared, 1mL of colloidal gold solution is taken, the wavelength corresponding to the maximum visible light absorption value of the colloidal gold solution is 525nm, 10 mu L of 0.2M potassium carbonate is added, and the mixture is uniformly mixed for 2 min; adding 5 mug/mL of anti-nucleocapsid protein monoclonal antibody and 5 mug/mL of anti-spinous process glycoprotein monoclonal antibody into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; adding 0.1mL of BSA with the mass fraction concentration of 10% as a sealant, and uniformly mixing for 30 min; adding a uniformly mixed solution of BSA, centrifuging at the temperature of 4 ℃ and the rotating speed of 12500rpm for 10min, carefully discarding a supernatant, then re-suspending and precipitating by using a Tris-HCL buffer solution with the concentration of 0.01M, pH of 8.0, adding sucrose with the final mass fraction concentration of 20% into a re-suspending solution as a protective agent, and fully and uniformly mixing to prepare a gold-labeled antibody; then, spraying the prepared gold-labeled antibody on the bonding pad 2, drying for 5h in a drying environment at the temperature of 37 ℃ to solidify the gold-labeled antibody on the bonding pad 2, and sealing for storage;
step three, film dispensing: preparing a film-scratching coating solution, wherein the film-scratching coating solution comprises PBS buffer salt with the final concentration of 0.01M, pH being 8.2, isopropanol with the final concentration of 2% by volume fraction, Triton X-100 with the final concentration of 0.0005% by volume fraction and trehalose with the final concentration of 0.3%; diluting the anti-nucleocapsid protein monoclonal antibody and the anti-spinous-process glycoprotein monoclonal antibody to 2mg/mL by using a film-drawing coating solution, then drawing the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous-process glycoprotein monoclonal antibody on one end of the nitrocellulose membrane 7 adjacent to the binding pad 2 in sequence to be used as a detection line, drawing a goat anti-mouse IgG antibody on the other end of the nitrocellulose membrane 7 to be used as a quality control line, and placing the drawn nitrocellulose membrane 7 in a drying environment at 37 ℃ for drying for 48h to obtain the nitrocellulose membrane 7 with the detection line and the quality control line;
step four, assembling the reagent card: sequentially adhering the sample pad 1, the combination pad 2, the nitrocellulose membrane 7 and the water absorption pad 6 which are processed in the first step to the third step to a bottom plate 8, cutting the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad into reagent strips with the width of 3.00mm by a cutting machine, and filling the reagent strips into a box template to prepare the kit; a sample adding hole and an observation window are arranged on the box body of the kit; the sample adding hole is positioned above the sample pad 1, and the observation window is positioned above the detection line and the quality control line.
By the preparation method in the embodiment, the kit can be quickly prepared, and the success rate of preparing finished products of the kit can be improved.
The application of the kit prepared by the preparation method comprises the following application steps:
after a sample to be detected is diluted by sample diluent, the diluted sample to be detected is dripped on a sample pad 1 of the reagent strip through a sample adding hole, and the results of a detection line and a quality control line on the reagent strip are observed through an observation window within 15-20 minutes; as shown in FIG. 2, interpretation of the results on the test strip includes: and (4) positive result: when any one of the detection line N line 3 and the detection line S line 4 appears a red strip and the quality control line C line 5 normally appears a red strip, judging that the detection result is positive; when only one red strip appears in the detection line N line 3 and the detection line S line 4, a positive result is judged, and a nucleic acid detection reagent is recommended to be used for carrying out rechecking detection; negative result: if only the quality control line C line 5 has a red strip, and the detection line N line 3 and the detection line S line 4 do not have color development, judging that the detection result is a negative result; ③ invalid result: when the red strip does not appear on the line 5 of the quality control line C, the detection result is invalid.
For the sensitivity evaluation of the kit prepared in this example, the evaluation procedure was as follows:
(1) diluting the titer-determined inactivated virus culture medium to 2000TCID with a sample diluent50/mL、1000TCID50/mL、500TCID50/mL、200TCID50/mL、100TCID50/mL、50TCID50/mL、20TCID50/mL、10TCID50/mL、5TCID50A total of 9 dilution gradients, such as/mL, and the detection is carried out by using the prepared kit, and the inactivated virus culture solution of the 9 dilution gradients is detected for 20 times by each gradient;
(2) sucking 100 mu L of the 9-gradient diluents to be slowly dripped onto the sample pad through the sample adding hole;
(3) observing the displayed result within 15-20 minutes; and the result judgment is carried out according to the description of the figure 2;
(4) the results are shown in table 1 below:
TABLE 1
As shown in Table 1, the sensitivity of the kit prepared in this example was 10TCID as shown by the results of 20 dilution gradients of 9 inactivated virus culture solutions50And the sensitivity is higher when the probe is used for detecting the specific surface area of the sample.
Clinical efficacy evaluation of the kit prepared in this example:
the kit prepared in the embodiment detects clinical samples, the clinical samples are contained in 166 clinical samples in total, 47 clinical samples are positive in diagnosis, 119 clinical samples are negative in diagnosis, and the consistency analysis is carried out on the detection result of the kit and the nucleic acid detection result of a commercial RT-PCR instrument; the results are shown in table 2 below:
TABLE 2
Of 166 clinical samples, 47 were positive and 119 were negative; as can be seen from the test results in table 2, the test results using the kit of this example showed 39 positive test cases and 127 negative test cases, and 8 positive test cases and negative test cases; the detection sensitivity of the kit in this example was 83%, the specificity was 100%, and the kappa identity coefficient was 0.875; therefore, the test results of the kit in this example are highly consistent with the current clinical diagnostic criteria.
The kit in the embodiment jointly detects the N antigen and the S antigen of the novel coronavirus and optimizes a reaction system, so that the defect of insufficient antigen detection sensitivity is overcome to a great extent, and the kit has high consistency with an RT-PCR method and clinical use value; moreover, the kit is simple and convenient to operate, short in detection time, independent of large-scale equipment for interpretation of detection results, and suitable for large-scale crowd screening and field screening.
As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited thereto. Various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A novel rapid detection kit for coronavirus antigens comprises a kit body and a reagent strip placed in the kit body, and is characterized in that the reagent strip comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped end to end along the length direction of the bottom plate;
the combination pad is coated with a colloidal gold labeled anti-nucleocapsid protein monoclonal antibody and an anti-spinous process glycoprotein monoclonal antibody;
the cellulose nitrate membrane is respectively coated with a nucleocapsid protein detection line N line formed by an anti-nucleocapsid protein monoclonal antibody, a spike glycoprotein detection line S line formed by an anti-spike glycoprotein monoclonal antibody and a quality control line C line formed by a goat anti-mouse IgG antibody.
2. The kit according to claim 1, wherein the gold-labeled anti-nucleocapsid protein monoclonal antibody and anti-spinous process glycoprotein monoclonal antibody coated on the conjugate pad are prepared as follows:
preparing a gold-labeled antibody, spraying the prepared gold-labeled antibody on a bonding pad, drying the bonding pad by adopting an SB-08 glass fiber membrane in a drying environment at the temperature of 37 ℃ for 5 hours to solidify the gold-labeled antibody on the bonding pad, and sealing and storing.
3. The kit according to claim 2, wherein the gold-labeled antibody is prepared by the following method: taking 1mL of colloidal gold solution, wherein the wavelength range corresponding to the maximum visible light absorption value of the colloidal gold solution is 520-530 nm, adding 10 mu L of potassium carbonate with the use concentration range of 0.02-0.5M, and uniformly mixing for 2 min; adding the anti-nucleocapsid protein monoclonal antibody with the final concentration of 5-20 mug/mL and the anti-spinous process glycoprotein monoclonal antibody with the final concentration of 5-20 mug/mL into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; then adding 0.1mL of BSA as a blocking agent, wherein the use mass fraction concentration range of the BSA is 1% -10%, and uniformly mixing for 30 min; adding a BSA (bovine serum albumin) uniformly mixed solution, centrifuging for 10min at the rotation speed of 12500rpm at 4 ℃, carefully discarding the supernatant, then re-suspending and precipitating by using a Tris-HCL buffer solution with the concentration of 8.0 being 40 mu L, pH, adding sucrose serving as a protective agent into the re-suspended solution, wherein the final concentration of the sucrose in percentage by mass is 5-30%, and fully mixing to prepare the gold-labeled antibody.
4. The kit according to claim 1, further comprising a scratching coating solution, wherein the scratching coating solution is used for diluting the anti-nucleocapsid protein monoclonal antibody and the anti-spinous process glycoprotein monoclonal antibody to 1-2 mg/mL, then the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous process glycoprotein monoclonal antibody are scratched on one end, close to the binding pad, of the nitrocellulose membrane in sequence to serve as a detection line, the goat anti-mouse IgG antibody is scratched on the other end of the nitrocellulose membrane to serve as a quality control line, and the scribed nitrocellulose membrane is placed in a drying environment at 37 ℃ for drying for 48 hours to obtain the nitrocellulose membrane with the detection line and the quality control line.
5. The kit of claim 4, wherein the streaking coating solution comprises PBS buffer salt, isopropanol, Triton X-100, and trehalose; the PBS buffer salt is PBS buffer salt with the final concentration range of 0.001M-0.05M, pH-8.2; the isopropanol is isopropanol with the volume fraction final concentration range of 1-5%; the Triton X-100 uses Triton X-100 with the volume fraction final concentration range of 0.0001-0.001%; the trehalose is used with a final concentration range of 0.1-1%.
6. The kit according to claim 1, wherein the sample pad is soaked in the sample pad treatment solution for 30min, the excess solution is discarded, and the sample pad is dried in a drying environment at 37 ℃ for 12h and taken out for standby.
7. The kit of claim 6, wherein the sample pad treatment solution comprises PBS buffer, casein, and Tween-20; the PBS buffer solution uses PBS buffer salt with the concentration range of 0.01M-0.1M, pH-7.4; the casein is casein with the mass fraction concentration of 0.1-1%; the Tween-20 uses Tween-20 with the volume fraction concentration of 0.1-1%; the sample pad used a SB-08 glass fiber membrane.
8. A method for preparing a kit according to any one of claims 1 to 7, comprising the steps of:
step one, preparing a sample pad: the sample pad adopts an SB-08 glass fiber membrane, and is provided with a sample pad treatment solution, wherein the sample pad treatment solution comprises a PBS buffer solution, casein and Tween-20; the PBS buffer solution uses PBS buffer salt with the concentration range of 0.01M-0.1M, pH-7.4; the casein is casein with the mass fraction concentration of 0.1-1%; the Tween-20 uses Tween-20 with the volume fraction concentration of 0.1-1%; and (3) soaking the sample pad in the sample pad treatment solution for 30min, removing redundant liquid, drying the soaked sample pad for 12h in a drying environment at the temperature of 37 ℃, and taking out for later use.
Step two, preparing a bonding pad: the combination pad adopts an SB-08 glass fiber membrane;
firstly, preparing a gold-labeled antibody, taking 1mL of colloidal gold solution, adding 10 mu L of potassium carbonate with the use concentration range of 0.02-0.5M into the colloidal gold solution, and uniformly mixing for 2min, wherein the wavelength range corresponding to the maximum visible light absorbance value of the colloidal gold solution is 520-530 nm; adding the anti-nucleocapsid protein monoclonal antibody with the final concentration of 5-20 mug/mL and the anti-spinous process glycoprotein monoclonal antibody with the final concentration of 5-20 mug/mL into the uniformly mixed solution to serve as marker antibodies, and uniformly mixing for 10 min; then adding 0.1mL of BSA as a blocking agent, wherein the use mass fraction concentration range of the BSA is 1% -10%, and uniformly mixing for 30 min; adding a BSA (bovine serum albumin) uniformly mixed solution, centrifuging for 10min at the rotation speed of 12500rpm at 4 ℃, carefully discarding the supernatant, then re-suspending and precipitating by using a Tris-HCL buffer solution with the concentration of 8.0 being 40 mu L, pH, adding sucrose serving as a protective agent into the re-suspended solution, wherein the final concentration of the sucrose in mass fraction is 5-30%, and fully mixing uniformly to prepare a gold-labeled antibody;
then spraying the prepared gold-labeled antibody on a bonding pad, drying for 5h in a drying environment at the temperature of 37 ℃ to solidify the gold-labeled antibody on the bonding pad, and sealing and storing;
step three, film dispensing: preparing a film-scratching coating liquid;
the film-scratching coating solution comprises PBS buffer salt, isopropanol, Triton X-100 and trehalose; the PBS buffer salt is PBS buffer salt with the final concentration range of 0.001M-0.05M, pH-8.2; the isopropanol is isopropanol with the volume fraction final concentration range of 1-5%; the Triton X-100 uses Triton X-100 with the volume fraction final concentration range of 0.0001-0.001%; the trehalose is trehalose with a final concentration range of 0.1-1%;
diluting the anti-nucleocapsid protein monoclonal antibody and the anti-spinous-process glycoprotein monoclonal antibody to 1-2 mg/mL by using a film-scratching coating solution, scratching one end of the diluted anti-nucleocapsid protein monoclonal antibody and the diluted anti-spinous-process glycoprotein monoclonal antibody, which is close to a binding pad, on a nitrocellulose membrane in sequence to serve as a detection line, scratching the other end of the nitrocellulose membrane by using a goat anti-mouse IgG antibody to serve as a quality control line, and placing the scribed nitrocellulose membrane in a drying environment at 37 ℃ for drying for 48 hours to obtain the nitrocellulose membrane with the detection line and the quality control line.
Step four, assembling the reagent card: and (3) sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad which are processed in the first step to the third step to a bottom plate, cutting the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad into reagent strips with the width of 3.00mm, and filling the reagent strips into a box body template to prepare the kit.
9. The preparation method according to claim 8, wherein a sample application hole and an observation window are provided on a case body of the kit; the sample adding hole is positioned above the sample pad, and the observation window is positioned above the detection line and the quality control line.
10. The use of the kit prepared by the preparation method of claim 9, which comprises the following steps:
after a sample to be detected is diluted by the sample diluent, the diluted sample to be detected is dripped on a sample pad of the reagent strip through the sample adding hole, and results of a detection line and a quality control line on the reagent strip are observed through an observation window within 15-20 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011205245.XA CN112326966A (en) | 2020-11-02 | 2020-11-02 | Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011205245.XA CN112326966A (en) | 2020-11-02 | 2020-11-02 | Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112326966A true CN112326966A (en) | 2021-02-05 |
Family
ID=74324382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011205245.XA Pending CN112326966A (en) | 2020-11-02 | 2020-11-02 | Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112326966A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3889607A1 (en) * | 2020-04-02 | 2021-10-06 | Ricardo Moffa | Method for in vitro screening of patients for quick diagnosis of sars-cov-2 |
| CN113970637A (en) * | 2021-11-05 | 2022-01-25 | 山东畜牧兽医职业学院 | Canine distemper virus latex microsphere detection test strip and preparation method thereof |
| CN114002427A (en) * | 2021-12-30 | 2022-02-01 | 北京健乃喜生物技术有限公司 | Method and kit for detecting novel coronavirus antigen |
| WO2022226496A1 (en) * | 2021-04-19 | 2022-10-27 | Global Diagnostic Systems, Benefit LLC | Devices, methods, and kits for diagnosing sars cov-2 infection |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10127882A1 (en) * | 2001-06-11 | 2002-12-12 | Basf Plant Science Gmbh | Cassette for expressing transgene, useful e.g. in production of pharmaceuticals and fine chemicals, contains promoter from the LOX5 gene of Arabidopsis, provides cotyledon-specific expression |
| JP2004229648A (en) * | 2002-04-30 | 2004-08-19 | Institute Of Physical & Chemical Research | New protein and dna encoding the same |
| CN101243321A (en) * | 2005-06-21 | 2008-08-13 | 美国政府健康及人类服务部，疾病控制和预防中心 | Methods, immunoassays and devices for detection of anti-lipoidal antibodies |
| US20110206687A1 (en) * | 2009-10-20 | 2011-08-25 | Abbott Laboratories | Isolation And Purification Of Anti-IL-13 Antibodies Using Protein A Affinity Chromatography |
| US20140087398A1 (en) * | 2005-11-18 | 2014-03-27 | Arlington Scientific, Inc. | Oxidized cardiolipin and uses to detect cardiolipin antibodies |
| CN104614511A (en) * | 2014-01-14 | 2015-05-13 | 田中贵金属工业株式会社 | Immune chromatography analysis method, immune chromatography analysis device, and immune chromatography analysis reagent box |
| CN107703313A (en) * | 2017-11-23 | 2018-02-16 | 中山市创艺生化工程有限公司 | One kind detection myoglobins immunofluorescence quantitative test paper bar |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN110007095A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof |
| CN110988350A (en) * | 2019-11-08 | 2020-04-10 | 南京市产品质量监督检验院 | Quantum dot test paper capable of quantitatively detecting florfenicol residual quantity in various matrixes |
| CN111303254A (en) * | 2020-02-20 | 2020-06-19 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) antigen detection kit |
| CN111366728A (en) * | 2020-03-27 | 2020-07-03 | 重庆探生科技有限公司 | Immunochromatography kit for detecting novel coronavirus SARS-CoV-2 |
| CN111474354A (en) * | 2020-06-02 | 2020-07-31 | 益善生物技术股份有限公司 | 2019-nCoV novel coronavirus detection chromatography test strip, detection card and kit |
-
2020
- 2020-11-02 CN CN202011205245.XA patent/CN112326966A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10127882A1 (en) * | 2001-06-11 | 2002-12-12 | Basf Plant Science Gmbh | Cassette for expressing transgene, useful e.g. in production of pharmaceuticals and fine chemicals, contains promoter from the LOX5 gene of Arabidopsis, provides cotyledon-specific expression |
| JP2004229648A (en) * | 2002-04-30 | 2004-08-19 | Institute Of Physical & Chemical Research | New protein and dna encoding the same |
| CN101243321A (en) * | 2005-06-21 | 2008-08-13 | 美国政府健康及人类服务部，疾病控制和预防中心 | Methods, immunoassays and devices for detection of anti-lipoidal antibodies |
| US20140087398A1 (en) * | 2005-11-18 | 2014-03-27 | Arlington Scientific, Inc. | Oxidized cardiolipin and uses to detect cardiolipin antibodies |
| US20110206687A1 (en) * | 2009-10-20 | 2011-08-25 | Abbott Laboratories | Isolation And Purification Of Anti-IL-13 Antibodies Using Protein A Affinity Chromatography |
| CN104614511A (en) * | 2014-01-14 | 2015-05-13 | 田中贵金属工业株式会社 | Immune chromatography analysis method, immune chromatography analysis device, and immune chromatography analysis reagent box |
| CN107703313A (en) * | 2017-11-23 | 2018-02-16 | 中山市创艺生化工程有限公司 | One kind detection myoglobins immunofluorescence quantitative test paper bar |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN110007095A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof |
| CN110988350A (en) * | 2019-11-08 | 2020-04-10 | 南京市产品质量监督检验院 | Quantum dot test paper capable of quantitatively detecting florfenicol residual quantity in various matrixes |
| CN111303254A (en) * | 2020-02-20 | 2020-06-19 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) antigen detection kit |
| CN111366728A (en) * | 2020-03-27 | 2020-07-03 | 重庆探生科技有限公司 | Immunochromatography kit for detecting novel coronavirus SARS-CoV-2 |
| CN111474354A (en) * | 2020-06-02 | 2020-07-31 | 益善生物技术股份有限公司 | 2019-nCoV novel coronavirus detection chromatography test strip, detection card and kit |
Non-Patent Citations (1)
| Title |
|---|
| 国家药品监督管理局高级研修学院: "医疗器械专业技术知识", 31 July 2020, 中国医药科技出版社, pages: 211 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3889607A1 (en) * | 2020-04-02 | 2021-10-06 | Ricardo Moffa | Method for in vitro screening of patients for quick diagnosis of sars-cov-2 |
| WO2022226496A1 (en) * | 2021-04-19 | 2022-10-27 | Global Diagnostic Systems, Benefit LLC | Devices, methods, and kits for diagnosing sars cov-2 infection |
| CN113970637A (en) * | 2021-11-05 | 2022-01-25 | 山东畜牧兽医职业学院 | Canine distemper virus latex microsphere detection test strip and preparation method thereof |
| CN114002427A (en) * | 2021-12-30 | 2022-02-01 | 北京健乃喜生物技术有限公司 | Method and kit for detecting novel coronavirus antigen |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112326966A (en) | Novel rapid detection kit for coronavirus antigen, and preparation method and application thereof | |
| CN111337689A (en) | Novel coronavirus detection kit | |
| CN111413495B (en) | Novel coronavirus IgM/IgG colloidal gold detection kit | |
| CN112485436A (en) | Colloidal gold immunochromatographic test strip for detecting in-vivo neutralizing antibody of neocorona vaccine after injection and preparation method thereof | |
| CN111426829A (en) | Quantum dot microsphere immunochromatography test strip for detecting total amount of SARS-CoV-2 IgM-IgG antibody | |
| CN109900913A (en) | A kind of A type and influenza B virus antigen colloidal gold method combined detection kit and preparation method thereof | |
| CN109709339B (en) | Colloidal gold immunochromatographic test strip for detecting skeletal muscle troponin I of cattle or sheep and application thereof | |
| CN111007257A (en) | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof | |
| CN110568187A (en) | Test strip and kit for detecting group A streptococcus antigen and preparation method thereof | |
| CN111273008A (en) | Colloidal gold immunochromatography kit for rapidly detecting novel coronavirus IgM antibody and preparation method thereof | |
| CN110007095A (en) | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof | |
| CN115629212A (en) | Monkey pox virus antigen detection reagent, kit and preparation method | |
| CN116041489A (en) | Monkey pox virus binding protein and application thereof, and monkey pox virus detection kit | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN113917141A (en) | Brucella latex microsphere antibody detection card and preparation method thereof | |
| CN106188248A (en) | A kind of Epstein-Barr virus antigen preparation procedure and utilize the quick detection kit of detection Epstein-Barr virus antibody prepared by this antigen | |
| CN101706499A (en) | FLAG fusion tag colloidal gold test strip and preparation method thereof | |
| CN111562392A (en) | Novel coronavirus IgG antibody fluorescence immunoassay detection kit | |
| CN111879951A (en) | Novel coronavirus IgA/IgG antibody joint inspection immunochromatographic test strip | |
| CN107957497A (en) | A kind of bird flu H5 subtype virus antibody rapid quantitative detection reagent box and its application | |
| CN111796103A (en) | Colloidal gold test strip for detecting echinococcosis and preparation method thereof | |
| CN114324879B (en) | Novel qualitative detection kit for coronavirus neutralizing antibody based on colloidal gold double-antibody sandwich method | |
| CN103267851B (en) | A kind of kit detecting premature rupture of fetal membranes and preparation method thereof | |
| CN111735962B (en) | Novel coronavirus IgM/IgG antibody joint detection immunochromatography test strip | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application 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 |