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Polypeptide-ELISA kit for detecting novel coronavirus S protein unique antibody

A coronavirus and kit technology, applied in the field of peptide-enzyme-linked immunosorbent assay kits, can solve problems such as affecting specificity, and achieve the effects of simple operation, strong specificity, and improved detection accuracy

Pending Publication Date: 2021-01-12
ZHEJIANG MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sequence comparison shows that the surface spike protein S of SARS-CoV2 has 30% to 76% identity and 46% to 86% similarity with the surface spike protein S of the other six coronaviruses mentioned above. Units such as the receptor binding domain (RBD) are used as capture antigens for serological antibody detection, and there may be cross-immune reactions that affect their specificity

Method used

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  • Polypeptide-ELISA kit for detecting novel coronavirus S protein unique antibody
  • Polypeptide-ELISA kit for detecting novel coronavirus S protein unique antibody
  • Polypeptide-ELISA kit for detecting novel coronavirus S protein unique antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Prediction and determination of the unique dominant linear B cell epitope in the spike protein S on the surface of the novel coronavirus

[0033] According to the characteristics of strong antigenicity, good surface and strong hydrophilicity of the linear B cell epitope, the amino acid sequence of the spike protein S on the surface of the new coronavirus was analyzed using bioinformatics software, and the dominant linear B cell antigen was predicted and determined. Determinant site ( figure 2 ). Use bioinformatics software to compare the amino acid sequences of the surface spike protein S of the new coronavirus SARS-CoV2 with 6 other coronaviruses known to infect humans and cause related diseases, especially the SARS-CoV with the closest homology , to clarify the unique specificity of the above dominant linear B cell epitopes. The peptides were artificially synthesized, and then diluted to 10 micrograms / ml, and coated in a 96-well microtiter plate with an ...

Embodiment 2

[0034] Example 2: Preparation of unique advantage linear B-cell antigen polypeptide on the surface of novel coronavirus spike protein S

[0035] Artificially synthesized four-segment polypeptides (SEQ ID NO.1~SEQ ID NO.4) containing unique dominant linear B cell epitope sites in the surface spike protein S of the novel coronavirus: SQCVNLTTRTQLPPAYTNSFT, HVSGTNGTKRFD, LGVYYHKNNKSWMESEFRVYSS, ALHRSYLTPGDSSSGWTAG, after purification The purity is above 95%.

Embodiment 3

[0036] Example 3: Using the kit to detect the specific antibody of the novel coronavirus surface spike protein S in the sample

[0037] Sample incubation: Take out the microtiter plate in the kit that has been coated with the linear B cell antigen polypeptide with unique specificity and advantages of SARS-CoV2 surface spike protein S. Dilute each sample 100 times with the sample diluent, add to the pre-prepared microplate wells, 100 microliters per well, set up negative control wells, positive control wells and blank wells at the same time, incubate at 37°C for 1 hour, wash with washing solution Plate 3 times.

[0038] Secondary antibody incubation: Dilute the enzyme-labeled antibody 5000 times with sample diluent, add to the wells, 100 microliters per well, incubate at 37°C for 30 minutes and wash the plate 3 times.

[0039] Color development reading: configure OPD color development solution, weigh 20 mg of enzyme substrate C, dissolve in 50 ml of enzyme substrate A, add 20 ...

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Abstract

The invention discloses a polypeptide enzyme-linked immunosorbent assay kit for detecting a novel coronavirus surface spinous process protein S unique specific antibody. The kit comprises an elisa plate coated with SARS-CoV2 surface spinous process protein S unique specificity dominant linear B cell antigen polypeptide, a sample diluent, negative control serum, positive control serum, a horse radish peroxidase labeled antibody, a concentrated washing solution, an enzyme substrate solution and a stop solution. The kit disclosed by the invention is high in specificity and good in repeatability,can be simply and conveniently used for detecting the novel coronavirus unique antibody, reduces false positive, can be used for large-scale serological detection and epidemiological investigation, and evaluates the infection condition of the novel coronavirus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polypeptide-enzyme-linked immunosorbent assay (ELISA) kit for detecting a specific antibody specific to spike protein S on the surface of a novel coronavirus (SARS-CoV2). Background technique [0002] Coronaviruses are a class of linear single-stranded positive-sense RNA viruses with enveloped genomes. The new coronavirus SARS-CoV2 is the seventh coronavirus known to infect humans and cause related diseases. It is currently in a global pandemic. The top 6 coronaviruses that can cause human diseases include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV, of which the first 4 mainly cause mild local epidemics worldwide The self-limiting disease accounts for 10% to 30% of upper respiratory tract infections in adults, while the latter two can cause severe respiratory syndrome with a fatality rate of 10% and 36%, respectively. Coronavirus surface spike protein (spik...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/58G01N33/543
CPCG01N33/6854G01N33/56983G01N33/581G01N33/54393G01N2333/165G01N2469/20
Inventor 罗永能吕建新陶薇吴泉洪艳张锋姜慧芬
Owner ZHEJIANG MEDICAL COLLEGE
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