CN111537743B - SARS-CoV-2 new coronavirus antibody detection reagent kit - Google Patents
SARS-CoV-2 new coronavirus antibody detection reagent kit Download PDFInfo
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- CN111537743B CN111537743B CN202010451090.1A CN202010451090A CN111537743B CN 111537743 B CN111537743 B CN 111537743B CN 202010451090 A CN202010451090 A CN 202010451090A CN 111537743 B CN111537743 B CN 111537743B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to a SARS-CoV-2 new coronavirus antibody detection kit. The novel coronavirus SARS-CoV-2 antibody (IgG) detection device comprises a solid carrier, a specific polypeptide combination connected to the solid carrier, and an RBD structure domain of the S protein of the novel coronavirus SARS-CoV-2 connected to the solid carrier. The kit of the invention is a novel coronavirus SARS-CoV-2 antibody detection kit which has sensitivity and specificity and can meet the requirements of epidemic prevention and clinical work, in particular the requirements of epidemiological investigation and general inspection.
Description
Technical Field
The invention mainly relates to an antibody detection kit. In particular, the invention relates to a novel coronavirus (SARS-CoV-2) IgG antibody detection kit.
Background
In 2020, the new type coronavirus (SARS-CoV-2) pneumonia (COVID-19) rolled the world, and epidemic prevention and control has become a common task for governments and people of all countries. The disease control experts provide the prevention and control measures of 'early discovery, early isolation and early treatment', and the diagnostic kit becomes an indispensable tool for epidemic prevention and control.
The diagnostic kits developed at present can be classified into two types according to the detection target, one is nucleic acid for detecting viruses, and the other is antibody for detecting viruses. However, these diagnostic kits still have the following disadvantages, so that the requirements of epidemic prevention and clinical work, particularly the requirements of epidemiological survey and general inspection, cannot be completely met: the specificity of the nucleic acid detection kit is close to 100%, but the sensitivity is low due to difficult sampling and the like, and is only 30% -50%, namely, more missed detections exist; the sensitivity of the antibody detection kit using SARS-CoV-2 component proteins such as S protein and N protein can reach more than 95%, but the specificity is low due to principle defects (especially for some patients with diseases, non-SARS-CoV-2 antibodies in the body can also be identified by the component proteins), namely, more false detections exist.
Disclosure of Invention
In view of the problems in the prior art, the inventors developed a "short peptide protein composite chip" based on "iPDMS nanomembrane" and "heterogeneous antibody kinetics", thereby solving the above technical problems.
Namely, the present invention comprises:
1. a novel coronavirus SARS-CoV-2 antibody (IgG) detection device (detection device 1 of the present invention) comprising a solid support, and the following polypeptide combinations attached to the solid support:
seq ID NO: 1 (S31, CTLKSFTVEKGIYQTSNFRV),
seq ID NO: 2 (S52, VVLSFELLHAPATVCGPKKS),
seq ID NO: 3 (N15, TPKDHIGTRNPANNAAIVLQ), and
d.SEQ ID NO: 4 (N39, ALPQRQKKQQTVTLLPAADL);
it also includes the RBD domain of the S protein of the novel coronavirus SARS-CoV-2 linked to the solid support.
2. The detection device of claim 1, wherein the solid support is polydimethylsiloxane (iPDMS membrane) with an initiator on its surface. The polydimethylsiloxane having an initiator on the surface can be found in, for example, chinese patent application publication CN 101265329A.
3. The detection device of claim 1, further comprising an N protein of the novel coronavirus SARS-CoV-2 linked to the solid support.
4. A novel coronavirus SARS-CoV-2 antibody (IgG) detection kit (detection kit 1 of the present invention) comprising the detection device according to claim 1 or 2 or 3.
5. The detection kit according to claim 4, which is used for the diagnosis of pneumonia caused by the novel coronavirus SARS-CoV-2.
In the present specification, the "diagnosis" may be confirmed or may be reference information for confirmed diagnosis, as the case may be.
6. Use of the detection device of claim 1 or 2 or 3 for the preparation of a novel coronavirus SARS-CoV-2 antibody (IgG) detection kit for the diagnosis of novel coronavirus SARS-CoV-2 induced pneumonia (COVID-19).
The detection device 1 or the detection kit 1 of the present invention can be used for diagnosing whether or not a subject organism (e.g., human) suffers from pneumonia caused by the novel coronavirus SARS-CoV-2. In particular, the method of manufacturing a semiconductor device,
when at least two of the RBD domains of the polypeptide and the S protein respond to a sample from a subject (DMI >1), determining that the subject is infected with the novel coronavirus SARS-CoV-2; otherwise, the subject is determined not to be infected with the novel coronavirus SARS-CoV-2.
In this specification, "response" means that the signal value of a positive spot read with a reading device is significantly different from the signal value of a negative spot. For example, the signal value of a positive spot read by the reading device is greater than or equal to 10, preferably greater than or equal to 20, more preferably greater than or equal to 30; and the signal value of the negative spot read by the reading device is less than 10, preferably less than 5, more preferably less than 1. The recognition device may be, for example, a microarray chip imager manufactured by abbotto biotechnology, su.
In the present specification, the solid support may be one or a plurality of solid supports, but preferably one, that is, all the polypeptides are independently attached to the same solid support. In the present invention, the solid carrier is not particularly limited as long as it is a carrier which is a solid or an insoluble material. The polypeptide can be linked to the solid support by methods known to those skilled in the art.
Examples
Example 1 preparation and validation of Polypeptides and proteins
SEQ ID NO: 1 to 4, and RBD domain of S protein of novel coronavirus SARS-CoV-2, which was synthesized by Nanjing Kingsry Biotech Co., Ltd.
Example 2 preparation of a kit (detection device)
Kit (detection device) 1
Respectively spotting the SEQ ID NO: 1-4, and RBD structure domain of S protein of novel coronavirus SARS-CoV-2, and spotting positive quality control point and negative quality control point to prepare kit (detection device) 1 (chip reaction plate in 48-well plate form).
Example 3 detection with the kit
Inspection step
1. Preparation work: the reagent and the test sample need to be balanced to room temperature before the test; serum was diluted 100-fold with serum diluent (50-fold dilution of whole blood).
2. Chip wetting: the chip reaction plate is taken out. The surface of the chip is soaked by cleaning solution for 3 minutes, and then the cleaning solution is discarded.
3. Adding a sample: 100 mul of sample to be tested is added into the reaction hole of the chip.
4. Sample incubation: the chip reaction plate was placed in a constant temperature incubator and incubated at 37 ℃ and 500rpm for 30 minutes.
5. Cleaning a sample: the sample in the reaction well of the chip is discarded, and the reaction well is rinsed with washing solution and spun off, and repeated 3 times.
6. Adding enzyme antibody: and adding 100 mu L of enzyme-labeled antibody liquid into the reaction hole of the chip, wherein the enzyme-labeled antibody is a goat anti-human IgG antibody marked by HRP.
7. And (3) enzyme-resistant incubation: the chip reaction plate was placed in a constant temperature incubator and incubated at 37 ℃ and 500rpm for 30 minutes.
8. Enzyme anti-cleaning: and discarding the enzyme-labeled antibody liquid in the reaction hole of the chip, flushing the reaction hole with a cleaning solution and throwing away, and repeating for 3 times.
9. Color development: adding 70 μ L of chromogenic substrate solution into each hole, and standing at room temperature for 5-10 min.
10. Discarding the color developing solution, prying the cover (using a straight screwdriver), rinsing with ultrapure water for 3 times, and drying in the air/blow-drying.
11. A48-well microarray chip imager (manufactured by Suzhou Albout Biotechnology Co., Ltd.) was used to photograph and the developed color signal was automatically collected.
12. And (4) judging a result:
with respect to kit 1, when at least two of the RBD domains of the polypeptide and S protein respond to a sample from a subject (DMI >1), the subject is determined to be infected (positive) with the novel coronavirus SARS-CoV-2; otherwise, the subject was judged to be not infected (negative) with the novel coronavirus SARS-CoV-2.
For 300 samples (some people have similar clinical manifestations of new coronary pneumonia such as fever and dry cough) with the exclusion of new coronary pneumonia through nucleic acid detection, imaging, epidemiology and clinical manifestations, the detection result by using the kit 1 is 299 negative cases and 1 positive cases, and the specificity is 299/300-99.7%.
The results of the detection using the kit 1 were negative in 2 cases and positive in 298 cases for 300 samples for which new coronarism was confirmed, and the sensitivity was 298/300-99.3%.
Therefore, the kit of the invention is a novel coronavirus SARS-CoV-2 antibody detection kit which has sensitivity and specificity and can meet the requirements of epidemic prevention and clinical work, in particular the requirements of epidemiological investigation and general inspection.
While embodiments of the present invention have been described above, the present invention is not limited to the specific embodiments and applications described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Claims (6)
1. A novel detection device for coronavirus SARS-CoV-2 IgG antibody, which comprises a solid carrier and the following polypeptide combination connected on the solid carrier:
a. SEQ ID NO: 1 (S31, CTLKSFTVEKGIYQTSNFRV),
b. SEQ ID NO: 2 (S52, VVLSFELLHAPATVCGPKKS),
c. SEQ ID NO: 3 (N15, TPKDHIGTRNPANNAAIVLQ), and
d. SEQ ID NO: 4 (N39, ALPQRQKKQQTVTLLPAADL);
it also includes the RBD domain of the S protein of the novel coronavirus SARS-CoV-2 linked to the solid support.
2. The detection device of claim 1, wherein the solid support is polydimethylsiloxane having an initiator on its surface.
3. The detection device of claim 1, further comprising an N protein of the novel coronavirus SARS-CoV-2 linked to the solid support.
4. A novel detection kit for a coronavirus SARS-CoV-2 IgG antibody, comprising the detection device of claim 1 or 2 or 3.
5. The detection kit according to claim 4, which is used for the diagnosis of pneumonia caused by the novel coronavirus SARS-CoV-2.
6. Use of the detection device of claim 1 or 2 or 3 for the preparation of a detection kit for novel coronavirus SARS-CoV-2 IgG antibodies for the diagnosis of novel coronavirus SARS-CoV-2 induced pneumonia (COVID-19).
Applications Claiming Priority (4)
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CN202010260996 | 2020-04-03 | ||
CN2020102609965 | 2020-04-03 | ||
CN2020102615326 | 2020-04-04 | ||
CN202010261532 | 2020-04-04 |
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CN111537743A CN111537743A (en) | 2020-08-14 |
CN111537743B true CN111537743B (en) | 2022-05-03 |
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CN115304671B (en) * | 2020-08-19 | 2023-10-17 | 重庆医科大学 | New coronavirus RBD specific monoclonal antibody and application thereof |
US20230338511A1 (en) * | 2020-08-28 | 2023-10-26 | University Of Houston System | Single-chain coronavirus viral membrane protein complexes |
CN114751980B (en) * | 2022-04-22 | 2022-10-28 | 厦门博昂生物技术有限公司 | Monoclonal antibody blocking agent for detecting neocorona antigen |
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CN2735340Y (en) * | 2003-12-25 | 2005-10-19 | 上海生物芯片有限公司 | Preparation of SARS antibody screening polypeptide chip and detecting kit |
CN101575361A (en) * | 2009-03-20 | 2009-11-11 | 北京健诺威生物科技有限公司 | Novel specific polypeptide and application thereof in preparing medicament for diagnosing, preventing and treating severe acute respiratory syndrome caused by coronaviruses |
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EP1701977A2 (en) * | 2003-12-10 | 2006-09-20 | Agency for Science, Technology and Research | Sars coronavirus s proteins and uses thereof |
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AU2004245896A1 (en) * | 2003-06-10 | 2004-12-16 | Agency For Science, Technology And Research | Method of diagnosing SARS corona virus infection |
CN2735340Y (en) * | 2003-12-25 | 2005-10-19 | 上海生物芯片有限公司 | Preparation of SARS antibody screening polypeptide chip and detecting kit |
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