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Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof

An enzyme-linked immunoassay and coronavirus technology, which is applied in the field of enzyme-linked immunoassay to detect neutralizing antibodies against new coronaviruses, can solve the problems of high laboratory level requirements and limited promotion and use, and achieve good repeatability, shorten reaction time, good specific effect

Active Publication Date: 2020-12-18
江苏美克医学技术有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The neutralization test using live virus cell culture has high requirements for laboratory level, which seriously limits the large-scale clinical application

Method used

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  • Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof
  • Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof
  • Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof

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preparation example Construction

[0049] The preparation method of the microtiter plate comprises the following steps: dilute the RBD antigen and NTD to 1 μg / mL-5 μg / mL with sodium carbonate-sodium bicarbonate buffer solution with a pH of 8.5-9.5, and coat the two enzymes respectively In the standard plate; add 50μL-120μL to each well, stick the cover film respectively, coat at 2-8℃ for 12h-20h, then spin dry, wash with washing solution 3-5 times respectively; add blocking solution 250 to each well -300 μL, sealed at 36-38°C for 1.5-2.5 hours, then dried, washed with washing solution for 3-5 times, and the cover film was attached to obtain the antigen-coated ELISA plate.

[0050] The preparation method of the enzyme-labeled secondary antibody comprises the following steps:

[0051] A: Add 1mL of 10-15mg / mL NaIO 4 Mix the aqueous solution with 1mL of 5mg / mL HRP aqueous solution at 2-8°C for 30min-60min to make an activated HRP solution; then, add 0.5mL of 90% ethylene glycol aqueous solution to the activated H...

Embodiment 1

[0061] This embodiment provides a kit for detecting neutralizing antibodies against novel coronavirus by enzyme-linked immunoassay. solution, stop solution and washing solution, and there are two microtiter plates, one of which is coated with the RBD antigen of the S protein; the other is coated with the NTD of the S protein; and the two microtiter plates are respectively blocked with a blocking solution; The blocking solution is a Tris-HCl solution containing bovine serum albumin, and the concentration of bovine serum albumin in the Tris-HCl solution is 3wt%; sample diluent, chromogenic solution, stop solution and washing solution are conventional solutions ; The calibrator includes two, one of which is RBD neutralizing antibody diluted with sample diluent; the other calibrator is NTD neutralizing antibody diluted with sample diluent; enzyme-labeled secondary antibody includes HRP-labeled Mouse anti-human IgG antibody.

Embodiment 2

[0063] This embodiment provides a kit for detecting neutralizing antibodies against novel coronavirus by enzyme-linked immunoassay. solution, stop solution and washing solution, and there are two microplates, one of which is coated with the RBD antigen of S protein; the other is coated with NTD of S protein; the two microplates are respectively blocked with blocking solution, The blocking solution is a PBS solution containing bovine serum albumin and casein, and the concentration of bovine serum albumin in the PBS solution is 3wt%; sample diluent, enzyme-labeled secondary antibody, chromogenic solution, stop solution and washing solution Both are conventional solutions; the calibrator includes two, one of which is RBD neutralizing antibody diluted with sample diluent; the other calibrator is NTD neutralizing antibody diluted with sample diluent.

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Abstract

The invention provides a kit for detecting a novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and a detection method thereof, the kit comprises two ELISA plates coated withantigens, one ELISA plate is coated with an RBD antigen of S protein, the other is coated with NTD of S protein; and the two ELISA plates are respectively sealed by using sealing liquid. The kit disclosed by the invention is used for detecting the novel coronavirus neutralizing antibody and evaluating the immune efficacy of the vaccine. The kit for detecting the novel coronavirus neutralizing antibody by the enzyme-linked immunosorbent assay provided by the invention applies the enzyme-linked immunosorbent assay with high sensitivity, good specificity and good repeatability, is beneficial towashing and removing non-specific binding and separating unreacted substances by immobilizing the antigen, has the characteristics of high sensitivity and good specificity, and is suitable for accurate detection of high-throughput samples.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit for detecting neutralizing antibodies against novel coronavirus by enzyme-linked immunoassay and a detection method thereof. Background technique [0002] According to the genome structure and phylogenetic analysis of coronaviruses, the Coronaviridae family is divided into four genera: α, β, γ and δ. Viruses of the genus α and β can infect mammals and humans. SARS-CoV-2 belongs to the new type of coronavirus of the genus β, round or oval, with a diameter of 60-140nm, and a crown-like shape under the electron microscope. [0003] According to reports, a research team screened 399 monoclonal antibodies from the memory B cells and plasma cells of 10 recovered patients. A variety of neutralizing antibodies. Most neutralizing antibodies, such as 2M-10B11 and ACE2-fc, target the RBD of S protein and block its binding to ACE2, showing neutralizing activi...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535
CPCG01N33/56983G01N33/535G01N2333/165G01N2469/20
Inventor 孙康俊张宗慧邓玲玲吴加一李文成
Owner 江苏美克医学技术有限公司
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