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New coronavirus neutralizing antibody detection kit based on trimer S protein RBD-ACE2 binding competition

A technology for RBD-ACE2 and antibody detection, which is applied in measuring devices, color/spectral characteristic measurement, and material analysis by observing the impact on chemical indicators, etc., can solve the tedious and time-consuming problem of inability to distinguish total binding antibodies and neutralizing antibodies and other problems, to achieve the effect of less interference factors, increase the detection rate of new crowns, and simple operation

Pending Publication Date: 2021-06-11
广州中医药大学顺德医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus neutralization test (cVNT) can detect neutralizing antibodies in the patient's blood, but it needs to be tested in a third-level biosafety laboratory, which is cumbersome and time-consuming
Enzyme-Linked Immunosorbent Assay (ELISA) and Lateral Flow Assay (LFA) Rapid Tests for Total Bound Antibody, Cannot Distinguish Total Bound Antibody from Neutralizing Antibody

Method used

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  • New coronavirus neutralizing antibody detection kit based on trimer S protein RBD-ACE2 binding competition
  • New coronavirus neutralizing antibody detection kit based on trimer S protein RBD-ACE2 binding competition
  • New coronavirus neutralizing antibody detection kit based on trimer S protein RBD-ACE2 binding competition

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Effect test

Embodiment 1

[0045] Example 1 A novel coronavirus neutralizing antibody detection kit based on trimeric S protein RBD-ACE2 binding competition

[0046] 1. Composition

[0047] 1. The solid phase carrier (96-well plate or 48-well plate) coated with the S protein RBD (GenScript Z03483) (400ng / well) of the trimeric structure, wherein the SARS-CoV-2 RBD has a His tag. The His antibody is coated on the solid phase carrier, and then the RBD is coated on the solid phase carrier by the His antibody;

[0048] 2. Biotin-labeled ACE2 protein

[0049] 3. Positive control substance: the serum of a person with high expression of neutralizing antibodies after injection of the new crown vaccine;

[0050] 4. Negative control substance: a mixture of sera from 20 healthy examinees over the age of 18 who have not been infected with the new coronavirus and other coronaviruses, and who have not been vaccinated against the new coronavirus;

[0051] 5. Enzyme-labeled secondary antibody: HRP-streptavidin;

[005...

Embodiment 2

[0065] The detection of embodiment 2 vaccine injector's blood sample

[0066] 1. Experimental method

[0067] 1. In order to test the detection effect of the kit in vaccine injectors, use the kit in Example 1 to detect clinical samples. In this example, 8 vaccinators were included, and 18 healthy subjects were also included as controls.

[0068] 2. Experimental results

[0069] The test results are shown in Table 1. The inhibition rates of the 8 vaccinators were all greater than 30%. This kit can detect neutralizing antibodies in serum.

[0070] Table 1:

[0071]

[0072]

Embodiment 3

[0073] Example 3 Detection of Blood Samples from New Coronary Rehabilitation Patients

[0074] 1. Experimental method

[0075] 1. In order to test the detection effect of this kit in patients who have recovered from COVID-19, the kit in Example 1 is used to detect clinical samples. In this example, 12 recovered patients from the new crown were included.

[0076] 2. Experimental results

[0077] The test results are shown in Table 2, 12 recovered patients from the new crown. The inhibition rates are all greater than 30%. This kit can detect neutralizing antibodies in serum.

[0078] Table 2:

[0079]

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Abstract

The invention discloses a novel coronavirus neutralizing antibody detection kit based on trimer S protein RBD-ACE2 binding competition. The kit provides reliable results in the aspects of accuracy and repeatability by using an ELISA competition method, and the detection can be completed only in a secondary biosafety laboratory. According to the present invention, the His antibody is coated in a microwell plate, and then an S protein RBD (His tag) with a trimer structure is connected so as to uniformly and tidily coat the RBD (His tag) in the microwell plate, such that the sensitivity and the specificity of the kit are improved, and the operation is simple, the result repeatability is good, and the interference factor is less. The kit can effectively monitor the vaccination reaction in clinical research and serve as an evaluation index of the later vaccine effect; the detection rate of novel coronavirus is increased, and missing detection caused by false negative of nucleic acid detection is made up; and the content of neutralizing antibodies in the body of the novel coronavirus rehabilitation patient is detected so as to judge whether rework and reproduction can be performed or not and whether a reinfection risk exists or not.

Description

technical field [0001] The present invention relates to the technical field of virus detection, in particular to a novel coronavirus neutralizing antibody detection kit based on the binding competition of trimeric S protein RBD-ACE2. Background technique [0002] The new coronavirus (SARS-CoV-2) is a new type of β-coronavirus (β-coronavirus), from the Sarbecovirus subgenus, round or oval, with a diameter of 60-140nm, and a crown-like shape under an electron microscope. The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 can interact with the human ACE2 protein, leading to endocytosis of host cells in the lung and replication of the virus. At the same time, RBD is also the main target of neutralizing antibodies. The S protein exists in the form of a homotrimer on the surface of the virus. The researchers found that the conformational form of the S protein is very unstable. Even if it does not bind to the host cell receptor ACE2, it will spontaneously change ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N21/78G01N21/31
CPCG01N33/56983G01N21/78G01N21/31
Inventor 徐建华张嘉琪刘万里邓敏王大伟李宜哲林东子何金勇颜克亮滕祥云
Owner 广州中医药大学顺德医院