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SARS-CoV-2 antibody detection method

An antibody detection, sars-cov-2 technology, applied in the field of SARS-CoV-2 antibody detection, can solve the problem of high homology and achieve important application prospects and value

Pending Publication Date: 2020-07-31
BEIJING BIOSYNTHESIS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the detection of anti-SARS-CoV-2 N protein antibody IgM and IgG mostly uses colloidal gold chromatography, but the SARS-CoV-2 new coronavirus has a high homology with the same coronavirus, and it is prone to crossover and non-specific reactions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Antigen horseradish peroxidase (HRP) labeling

[0028] (1) Antigen horseradish peroxidase (HRP) labeling:

[0029] (1) Take 6.4 mg of HRP dry powder, add 0.6 ml of double distilled water, slowly add 60 μl of sodium periodate (0.1M) dropwise into HRP, incubate at room temperature for 20 min, and dialyze the sodium acetate solution (1 mM pH4.4) overnight;

[0030] (2) Add 1.6 mg of antigenic protein to the dialyzed HRP solution, and incubate at room temperature for 2 hours;

[0031] The amino acid sequence of the antigenic protein is shown in SEQ ID NO.1, which is prepared by the following method: inserting the human SARS-CoV Nucleocapsid gene (nucleotide sequence shown in SEQ ID NO.2) into the expression vector pET28a, Construct pET28a-SARS-CoV-N fusion expression plasmid, transform into Escherichia coli, induce with IPTG, purify and renature to obtain recombinant SARS-CoV-2 Nucleocapsid protein;

[0032] (3) Add 60 μl of sodium borohydride (4 mg / ml) to the ...

Embodiment 2

[0044] Embodiment 2: competition experiment screening

[0045] Use checkerboard titration to screen for competition experiments. First, take all Anti-SARS-CoV-2 Nucleocapsid protein-specific antibodies and configure them at a concentration of 2 μg / ml and 1 μg / ml and prepare them into coated plates. The coating and blocking process refer to the examples 1. The experiment was designed according to the checkerboard titration method. The Anti-SARS-CoV-2 Nucleocapsidprotein antibody was prepared into 20ug / ml and 0ug / ml solutions as competing antibodies, and the enzyme-labeled antigen was prepared into 2ug / ml and 1ug / ml concentrations, each of which was 50μl Perform competition detection and incubate at room temperature for 15 minutes. Please refer to Example 1 for the remaining detection, color development, and termination processes. According to the experimental results, select the antibody clone with the highest inhibition rate and the working concentration of the corresponding ...

Embodiment 3

[0049] Embodiment 3: Matrix effect detection of sample

[0050] According to the experimental results obtained in Example 2, select the dominant antibody clone and its relevant raw material working concentration, and further detect the matrix effect of the sample, the specific steps are as follows:

[0051] (1) Serum matrix effect detection

[0052] a. Sample source: Rabbits were immunized with recombinant SARS-CoV-2 Nucleocapsid protein, and the serum after immunization was taken as the positive sample in the experiment.

[0053] b. Coating: 1C7 antibody was prepared to a concentration of 2ug / ml for coating, and other methods were the same as in Example 1.

[0054] Closing: The method is the same as that of implementation case 1.

[0055] Sample dilution: Take normal human serum and dilute it with phosphate buffer saline containing 1% bovine serum albumin, the dilution gradient is shown in Table 3. At the same time, use phosphate buffer saline with 1% bovine serum albumin a...

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Abstract

The invention discloses an SARS-CoV-2 antibody detection method. The SARS-CoV-2 antibody detection method comprises the following steps: carrying out horseradish peroxidase labeling on recombinant SARS-CoV-2Nucleocapsid protein, meanwhile, selecting an anti-SARS-CoV-2 antibody or a dominant antibody to coat the enzyme-linked plate and sealing; according to a competitive ELISA principle, simultaneously adding a to-be-detected sample and an enzyme-labeled antigen into a test hole of an enzyme-linked plate, performing incubation at room temperature, thoroughly washing the plate, then adding a substrate TMB, performing color development in a dark place, finally, terminating the reaction, measuring a light absorption value at 450 nm, and evaluating the titer of the antibody in the sample by calculating the antibody inhibition rate. According to the method disclosed by the invention, relatively conservative N protein in SARS-CoV-2 and a specific antibody of the N protein are selected as rawmaterials. Compared with a traditional antibody detection method, the detection of the Anti-SARS-CoV-2 specific antibody is completed more quickly, more conveniently and more sensitively, and the method has specificity, sensitivity, accuracy and precision and has important application prospect and value.

Description

technical field [0001] The invention relates to a SARS-CoV-2 antibody detection method, belonging to the technical field of antibody detection. Background technique [0002] SARS-CoV-2 belongs to the genus β of the Coronaviridae family. A scientific research team found that the sequence homology of the entire genome of SARS-CoV-2 with SARS-CoV (Severe Acute Respiratory Syndrome) is about 76%, and the sequence homology with bat coronavirus BatCoVRaTG13 in horseshoe bats is as high as 96% At the same time, the team confirmed that the receptors for SARS-CoV-2 and SARS-CoV to enter cells are both ACE2 by comparing their seven conserved non-structural proteins. [0003] The structure of coronavirus consists of a double-layer lipid envelope, including Spike glycoprotein (S), envelope protein (Envelope protein, E), membrane protein (Membrane glycoprotein, M) and nucleocapsid protein (Nucleocapsid protein, N). Among them, S protein and N protein are the most important target pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/535G01N33/543G01N33/577
CPCG01N33/535G01N33/54313G01N33/56983G01N33/577G01N2333/165
Inventor 赵风强吴妤邬晓乐
Owner BEIJING BIOSYNTHESIS BIOTECH
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