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Novel coronavirus neutralizing antibody detection kit

A coronavirus and kit technology, applied in the field of cell biology and immunoassay, can solve the problems of inability to comprehensively evaluate the activity of neutralizing antibodies, high experimental safety level requirements, and high experimental operation requirements, achieving short detection time and overcoming the safety level The effect of high requirements and low laboratory requirements

Active Publication Date: 2020-12-18
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the enzyme-linked immunosorbent reaction (ELISA) method is simple and common, the proteins used are all obtained through recombinant expression. Due to the influence of the production process, the protein obtained through recombinant expression is still different from the protein in the natural state. Qualitative detection of neutralizing antibodies is often missed, and this method is only an analysis at the molecular level, and cannot fully evaluate the activity of neutralizing antibodies
[0005] The live virus infection test is the gold test standard for detecting the neutralizing antibody of the new coronavirus, but the test safety level is high, the test operation is high, and the test cycle is long
If the new coronavirus is used, it needs to be processed in a biosafety level three protection laboratory, and it is very time-consuming, usually taking 2 to 4 days to complete

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation and identification of HEK293-hACE2 cells

[0039] 1. Experimental materials

[0040] 1. Main reagents:

[0041] HEK293 cells (Thermofisher, Cat. No. A14528 ); puromycin (Sangon Bioengineering (Shanghai) Co., Ltd., Cat. No. PJ593-25mg); fetal bovine serum FBS (CellMax, Cat. No. SA212.02 ); DMEM medium (Hyclone, Cat. No. SH30022.01); 0.25% trypsin (0.25% Trypsin-EDTA, Gibco, Cat. No. 25200-056); linear polyethyleneimine (Polyethyleneimine, Linear, Polysciences, Cat. No. 23966); PB-hACE2 expression vector (provided by Acrobiosystems molecular construction department); trypan blue staining solution (Trypan Blue Solution, 0.4%, Thermofisher, Cat. No. 15250061); new coronavirus spike sugar Protein S1 (SARS-CoV-2 (COVID-19) S1 protein, Mouse IgG2a Fc Tag, Acrobiosystems, Cat. No. S1N-C5257); Negative control protein (Human Epidermal Growth Factor Receptor 2 Protein Human Her2 Protein, Mouse IgG2a Fc Tag, Acrobiosystems, Cat. No. HE2-H5255); PE-labeled ...

Embodiment 2

[0057] Example 2 Preparation and identification of PE-labeled coronavirus spike glycoprotein S1

[0058] 1. Experimental materials

[0059] 1. Main reagents:

[0060] Biotinylated SARS-CoV-2 (COVID-19) S1protein, His, Avitag™ (MALS verified), Acrobiosystems, Cat. No. S1N-C82E8); PE-labeled streptavidin Avidin (PE-Streptavidin, Jackson, Cat. No. 016-110-084); Bovine Serum Albumin (Bovine Serum Albumin, BSA, Sigma-Aldrich, Cat. No. SRE0098-100G); D-(+) - Trehalose dihydrate (Sigma-Aldrich, Cat. No. T9531-10G); Phosphate buffered saline (PBS buffer, Hyclone, Cat. No. SH30256.01).

[0061] 2. Main consumables and instruments:

[0062] Cell culture dish (Thermo Fisher Scientific, Cat. No. 150466); 15ml sterile centrifuge tube (TrueLine, Cat.No. TR2010); 50ml sterile centrifuge tube (TrueLine, Cat.No. TR2012); pipette (Rui Ning RAININ); vials (Short Xinkang Pharmaceutical Packaging Co., Ltd., Boron 260); freeze dryer (Telstar, lyobeta5PS); vortex mixer (Jiangsu Kangjian Medical ...

Embodiment 3

[0078] Example 3 Establishment of flow cytometric detection method for neutralizing antibodies against novel coronavirus

[0079] 1. Experimental materials

[0080] PE-labeled SARS-CoV-2 spike glycoprotein S1; HEK293-hACE2 cells; SARS-CoV-2 neutralizing antibody standard, Anti-SARS-CoV-2 RBD Neutralizing Antibody, Human IgG1 (Acrobiosystems, Cat.No. SAD-S35) ; negative control antibody sample, Human IgG1 (N297A) Isotype Control (Acrobiosystems, Cat. No. DNP-MB273); normal human serum (Abbkine, Cat. No. BMS0060); flow detection buffer; PBS buffer.

[0081] Ultra-clean bench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., Cat. No. DL-CJ-2ND). Carbon dioxide incubator (Thermo Fisher Scientific, Thermo 3111); LUNA II automatic counter (LUNA, LunaⅡ); Xiangyi low-speed centrifuge (Xiangyi Centrifuge Instrument Co., Ltd., L-550); Inverted microscope (OLYMPUS, CKX53) ; flow cytometer (BD FACSCelesta™ flow cytometer).

[0082] 2. Establishment of a flow cytometric detecti...

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Abstract

The invention provides a novel coronavirus neutralizing antibody detection kit. The kit comprises HEK293-hACE2 cells, PE labeled novel coronavirus spike glycoprotein S1, a novel coronavirus neutralizing antibody positive standard substance, a negative control antibody sample and a flow detection buffer solution. Also provided is a method for detecting a novel coronavirus neutralizing antibody of non-diagnostic purpose. The new coronavirus neutralizing antibody detection method established by relying on HEK293hACE2 cells and fluorescein labeled new coronavirus spike glycoprotein (namely PE-S1)can quickly complete detection of the new coronavirus neutralizing antibody in serum within 12h, and has the characteristics of strong specificity, high sensitivity, good repeatability, simple operation, low laboratory requirements, high safety and the like.

Description

technical field [0001] The invention relates to the technical fields of cell biology and immunoassay, in particular to a novel coronavirus neutralizing antibody detection kit. Background technique [0002] The novel coronavirus SARS-CoV-2 (2019-nCoV) is a virus newly discovered in 2019 that can cause viral pneumonia or lung infection in humans. The development of a new coronavirus vaccine is in full swing. At the beginning of August 2020, there were 165 vaccines in the experimental stage around the world, 26 of which have entered the clinical trial stage, and 6 vaccines have entered the third phase of clinical trials. The principle of action of the vaccine is to expose the immune system to the components of the virus and induce the body's immune system to produce a protective response against the coronavirus. After the new crown vaccine is inoculated, it will first cause the body to respond in two ways. One is that the body produces antibody molecules that can recognize vi...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/58G01N33/554
CPCG01N33/554G01N33/56983G01N33/582G01N33/6854G01N2333/165G01N2469/20
Inventor 石晓娟苗景赟
Owner ACROBIOSYSTEMS INC
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