The SeroFlash™ SARS-CoV-2 Neutralizing Antibody Assay Fast Kit is a complete set of optimized buffers and reagents designed for detecting and quantifying SARS-CoV-2 neutralizing antibody in circulation in a fast and high throughput format. The kit has the following advantages:
BackgroundCOVID-19 is an infectious disease caused by SARS-CoV-2, a new member of the same coronavirus family that caused SARS and MERS. It was found that the SARS-CoV-2 spike glycoprotein harbors a furin/PC cleavage site at the boundary between the S1/S2 subunits, which could be cleaved by furin and/or furin-like PCs secreted from host cells and bacteria in the airway epithelium [1, 2]. Unlike SARS-CoV, cell entry of SARS-CoV-2 is pre-activated by furin and/or furin-like PCs, reducing its dependence on target cell proteases for entry [3]. The cleavage activation of S-protein is well demonstrated to be essential for SARS-CoV-2 spike-mediated viral binding to ACE2, cell-cell fusion, and viral entry into human lung cells [4, 5]. It was also observed that other viruses containing a furin/PC cleavage site, such as H5N1, increased replicates and developed higher pathogenicity [6]. The SARS-CoV-2 furin/PC cleavage site has been with one core region SPRRAR│SV (eight amino acids, P6–P2′). The core region is very unique, as its P2 or P3 position is a positively charged residue (Arg), and another residue is hydrophobic (Ala). These factors allow this site to be cleaved by furin or furin-like PC and the cleavage efficiency to be facilitated by other serine proteases targeting dibasic amino acid sites such as matriptase, plasmin, human airway trypsin (HAT), and TMPRSS2. Furthermore, a serine at P6 could also highly increase the cleavage efficiency, causing increased viral replication, unrestricted organ tropism, virulence, and mortality rate as proven in H5N1 infection in mice [7]. The importance of measuring SARS-CoV-2S1/S2 site targeted cleavage PC or facilitating protease activity is emphasized by that the PC-based SARS-CoV-2 S1/S2 cleavage increases SARS-Cov-2 entry into cells and replication and eventually develops higher pathogenicity of COVID-19.
Fig 1. Inhibition of Sars-Cov-2 by neutralizing antibody.
Principles and ProcedureThis kit contains all reagents necessary for quantitatively measuring SARS-CoV-2 neutralizing antibody level. In this assay, a SARS-CoV-2 spike protein is stably pre-coated onto microplate wells. His-tagged ACE2 is bound to the coated spike protein in the presence or absence of neutralizing antibody contained in the sample. The amount of the bound ACE2, which is proportional to ACE2 inhibition intensity, is then recognized by the Neutralizing Detection Complex containing anti-His antibody and measured through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The neutralizing antibody level is inversely proportional to the optical density intensity measured. The higher the neutralizing antibody is, the lower the OD intensity is.
Starting MaterialsInput materials can be serum, plasma, and various body fluids
References:1. Bunyavanich S et al: JAMA. Published online May 20, 2020. 2. Sama IZ et al: Eur Heart l, 41: 1810–1817, 2020. 3. Wang C et al: Natural Comm. 11: 2251, 2020
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