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Novel coronavirus IgA/IgM/IgG antibody ELISA detection kit

A detection kit, coronavirus technology, applied in the direction of virus/phage, DNA/RNA fragment, virus, etc., to achieve the effect of improving sensitivity and specificity, high specificity and high accuracy

Pending Publication Date: 2020-09-08
ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a novel coronavirus IgA / IgM / IgG antibody ELISA detection kit to solve the problems in the prior art. The kit uses His+N protein gene+glycine flexible peptide+S protein gene insertion Construct a prokaryotic expression vector into the PET-28a vector to obtain the recombinant protein, and coat it on a microwell plate to form a 2019-nCoVIgA / IgM / IgG antibody kit. In addition to the advantages of ordinary ELISA detection kits, the kit It also makes up for the shortcomings of low sensitivity caused by recombinant antigens, improving the sensitivity and specificity of detection

Method used

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Embodiment 1

[0033] The preparation method of novel coronavirus S+N recombinant protein comprises the following steps:

[0034] (1) According to the N and S protein gene sequences of SARS-COV-2 reported in GenBank, design specific primers for the amplification of the N and S protein genes respectively; and transformation, the N protein gene+glycine flexible peptide+S protein gene+His gene is inserted into the PET-28a vector to obtain a plasmid containing the N and S protein gene sequences, the sequence is shown in SEQ ID No.1, the N protein gene The full length is 1257bp, encoding 419 amino acids, the full length of the S protein gene is 1479bp, encoding 493 amino acids;

[0035] (2) Use Escherichia coli to express the plasmid containing the N and S protein gene sequences. After the bacteria are sonicated, the precipitate is dissolved overnight with 50mM Tris+2M urea pH=12, centrifuged, and the supernatant is dialyzed to Protein was obtained from Tris solution with pH=9.5;

[0036] The d...

Embodiment 2

[0045] A novel coronavirus IgA / IgM / IgG antibody ELISA detection kit, comprising an enzyme label plate, a substrate solution, a sample diluent, a positive control solution, a negative control solution, a concentrated washing solution, an enzyme conjugate and a stop solution, the The microtiter plate is pre-coated with the new coronavirus S+N recombinant protein.

[0046] In this embodiment, the enzyme conjugate is horseradish peroxidase-labeled mouse anti-human IgA / IgM / IgG-HRP antibody dilution.

[0047] In this example, the novel coronavirus is coated with bovine serum albumin at a mass concentration of 5%.

[0048] In this embodiment, the coating amount of the novel coronavirus is 0.1 μg / well.

[0049] In this embodiment, the sample diluent is a phosphate buffer solution with a pH of 7.2-7.4 containing 5% bovine serum albumin by mass fraction.

[0050] In this embodiment, the concentrated washing solution is a phosphate buffer solution with a volume fraction of 0.05% Tween-...

Embodiment 3

[0055] A novel coronavirus IgA / IgM / IgG antibody ELISA detection kit, comprising an enzyme label plate, a substrate solution, a sample diluent, a positive control solution, a negative control solution, a concentrated washing solution, an enzyme conjugate and a stop solution, the The microtiter plate is pre-coated with the new coronavirus S+N recombinant protein.

[0056] In this embodiment, the enzyme conjugate is horseradish peroxidase-labeled mouse anti-human IgA / IgM / IgG-HRP antibody dilution.

[0057] In this example, the novel coronavirus was coated with bovine serum albumin at a mass concentration of 1%.

[0058] In this embodiment, the coating amount of the novel coronavirus is 0.1 μg / well.

[0059] In this embodiment, the sample diluent is a phosphate buffer solution with a pH of 7.2-7.4 containing 10% bovine serum albumin in mass fraction.

[0060] In this embodiment, the concentrated washing solution is a phosphate buffer solution with a volume fraction of 0.05% Twee...

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Abstract

The invention discloses a novel coronavirus IgA / IgM / IgG antibody ELISA detection kit, and belongs to the technical field of biological detection. The kit comprises an elisa plate, a substrate solution, a sample diluent, a positive control solution, a negative control solution, a concentrated washing solution, an enzyme conjugate and a stop solution, the elisa plate is pre-coated with a novel coronavirus S + N recombinant protein; the enzyme conjugate is horse radish peroxidase labeled mouse anti-human IgA / IgM / IgG-HRP antibody diluent, and detection is performed through immunospecific reactionof an antigen and an antibody so as to qualitatively judge whether a novel coronavirus antibody exists in a test sample or not, so that the kit has high sensitivity and high specificity; the kit performs detection through immunospecific reaction of antigen and antibody, has the characteristics of high specificity and high accuracy, and is simple and convenient in steps, reasonable in design and suitable for popularization.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a novel coronavirus IgA / IgM / IgG antibody ELISA detection kit. Background technique [0002] The common signs of the new coronavirus (2019-nCoV) are fever, fatigue, dry cough, and gradual dyspnea. Some patients have mild symptoms, or even patients without clinical symptoms, which have the characteristics of human-to-human transmission. The general incubation period is 1-14 days, and The incubation period is contagious, and asymptomatic infected persons may also become the source of infection, which is mainly transmitted through respiratory droplets and close contact infection, and the crowd is generally susceptible. [0003] At present, 2019-nCoV nucleic acid detection is the routine detection method and basis for diagnosis of COVID-19. However, due to various factors such as sample collection and storage, virus-infected sites, RNA extraction methods, and quality pro...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/58C12N15/70C12N15/62C07K19/00C07K1/22
CPCG01N33/56983G01N33/581C07K14/005C12N15/70G01N2469/20G01N2333/165C07K2319/21C12N2770/20022
Inventor 王震吕建新李凯强郝珂陈林洁李凯旋
Owner ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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