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Recombinant protein and test strip for detection of antibodies of 2019 novel coronavirus through double-antigen sandwich method and preparation method and application of recombinant protein and test strip

A double-antigen sandwich, coronavirus technology, applied in the directions of viruses/phages, biochemical equipment and methods, viruses, etc., can solve the problems of large impact, poor specificity, and unsatisfactory sensitivity, and achieve high sensitivity, simple operation, and stability. Good results

Active Publication Date: 2020-06-19
GENERAL HOSPITAL OF PLA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The reported methodologies of immunological diagnosis are mainly colloidal gold method, ELISA, etc., to detect virus-specific IgM and IgG antibodies in the blood. The impact is relatively large, the specificity is poor, and the sensitivity is not ideal
The double-antigen sandwich method has high sensitivity and strong specificity, but there is no report on the ideal double-antigen sandwich raw materials for 2019-nCoV on the market and in the literature

Method used

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  • Recombinant protein and test strip for detection of antibodies of 2019 novel coronavirus through double-antigen sandwich method and preparation method and application of recombinant protein and test strip
  • Recombinant protein and test strip for detection of antibodies of 2019 novel coronavirus through double-antigen sandwich method and preparation method and application of recombinant protein and test strip

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preparation example Construction

[0034] The present invention also provides a preparation method for the test strip described in the above technical scheme, comprising the following steps:

[0035] 1) Mix and couple the activated fluorescent microspheres with the recombinant protein described in the above technical scheme to obtain the multi-dominant epitope fusion protein of 2019 novel coronavirus labeled with fluorescent microspheres;

[0036] 2) spraying the 2019 novel coronavirus multi-dominant epitope fusion protein labeled with fluorescent microspheres onto the glass fiber membrane to obtain a fluorescent microsphere pad;

[0037] 3) Spray the 2019 novel coronavirus multi-dominant epitope fusion protein and the anti-N protein antibody on the detection area and the quality control area of ​​the nitrocellulose membrane respectively to obtain a nitrocellulose membrane with the detection area and the quality control area fixed;

[0038] 4) Paste the sample absorbent pad, the fluorescent microsphere pad, the...

Embodiment 1

[0048] Preparation of reagent strips for detection of novel coronavirus 2019-nCoV antibody by double-antigen sandwich method.

[0049] 1. Preparation of novel coronavirus 2019-nCoV multi-dominant epitope fusion recombinant protein:

[0050] The dominant antigenic epitopes of the new coronavirus 2019-nCoV N protein, S protein, and M protein were screened by calculation and prediction, and linked together by a flexible polypeptide (GGGS) to form a 2019-nCoV virus multi-dominant epitope fusion protein. The amino acid sequence is as follows: The sequence is shown in SEQ ID NO.1. Entrusted Beijing Deaoping Biotechnology Co., Ltd. to produce and prepare, the purity is greater than 95%.

[0051] DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVT LLPAADLDDFSKQ GGGS DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT NGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGT GVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPG TNTSNQVAVLYQDVNCTEVPVAIHA GGGS TLAILTALRLCAYCCNIVNVSL VKPSFYVYSRVKNLNSSRVPDLLV

[0...

Embodiment 2

[0064] Application of double-antigen sandwich method for detection of novel coronavirus 2019-nCoV antibody test strips

[0065] 1. Sample pretreatment

[0066] Aspirate 20 μL whole blood sample and add to 200 μL sample diluent, mix well.

[0067] 2. Test with test strips

[0068] Use a micropipette to accurately draw 80 μL of the sample solution to be tested into the sample hole of the test strip, and act for 15 minutes at room temperature (20-25°C); insert the test paper card into the carrier of the fluorescence detector, and select the sample to be tested through the touch screen. item, press the "Start Detection" button, the fluorescence detector will automatically scan the test paper card; read or print the test results on the display screen of the instrument.

[0069] 3. Analysis of test results

[0070] Quantitative detection

[0071] After the test is completed, the instrument obtains the ratio of the time-resolved fluorescence intensity of the detection area on the...

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Abstract

The invention relates to a recombinant protein and test strip for detection of antibodies of 2019 novel coronavirus through a double-antigen sandwich method and a preparation method and application ofthe recombinant protein and test strip, and belongs to the technical field of virus detection. The amino acid sequence of the recombinant protein for detection of the antibodies of the 2019 novel coronavirus through the double-antigen sandwich method is shown in SEQ ID NO. 1. The recombinant protein is a fusion protein of multiple dominant epitopes of the 2019-nCoV, a reagent for detection of theantibodies of the 2019-nCoV through the double-antigen sandwich method can be prepared, storage at room temperature, fast and single detection with high sensitivity, high throughput and low instrument cost can be achieved at any time, the operation is simple, and convenience of clinical use can be improved greatly.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a recombinant protein, a test strip, a preparation method and an application for detecting 2019 novel coronavirus (2019-nCoV) antibodies by a double-antigen sandwich method. Background technique [0002] Coronaviruses are a large family of viruses known to cause more serious illnesses such as colds, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The new coronavirus is a new strain of coronavirus that has never been found in humans before, and 2019-nCoV is the seventh type of coronavirus isolated from humans. Related disease monitoring found many cases of viral pneumonia, all diagnosed as viral pneumonia / lung infection. [0003] After the new coronavirus infects the human body, there is generally an incubation period of 3 to 14 days (in some cases, up to 24 days). During the incubation period, there may be no clinical symptoms and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/533G01N33/543G01N33/558G01N33/569
CPCC07K14/005C07K2319/00C12N2770/20022G01N33/533G01N33/54313G01N33/558G01N33/56983G01N2333/165
Inventor 何昆仑田亚平张思兵周建平周裕军
Owner GENERAL HOSPITAL OF PLA
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