key: cord-0775387-k9ng3nkt
authors: Zhang, Bao-zhong; Hu, Ye-fan; Chen, Lin-lei; Yau, Thomas; Tong, Yi-gang; Hu, Jing-chu; Cai, Jian-piao; Chan, Kwok-Hung; Dou, Ying; Deng, Jian; Wang, Xiao-lei; Hung, Ivan Fan-Ngai; To, Kelvin Kai-Wang; Yuen, Kwok Yung; Huang, Jian-Dong
title: Mining of epitopes on spike protein of SARS-CoV-2 from COVID-19 patients
date: 2020-07-01
journal: Cell Res
DOI: 10.1038/s41422-020-0366-x
sha: 73715ee0dc32d501fd65de3ba04fe09f2d3aecad
doc_id: 775387
cord_uid: k9ng3nkt

nan

7.0 urea Na2HPO4/NaH2PO4 buffer. Peptides conjugated to keyhole limpet haemocyanin (KLH) carrier proteins were also purchased from GL Biochem (Shanghai) Ltd. These peptide-conjugated proteins were dissolved in a pH 7.4 PBS buffer.

Epitope-specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Briefly, all peptides or recombinant proteins at a final concentration of 0.5 μg/mL in 50 mM coating buffer (pH 9.6 Na2CO3/NaHCO3) were coated on ELISA plates (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. Plates were blocked with TBS-5% (w/v) non-fat milk for 3 h at 37°C and washed four times in 0.05% Tween-20 (Sigma) in TBST. Diluted patient or mice sera were added into the wells and incubated for 1 h at 37°C. Plates were washed six times in TBS-0.05% Tween and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ThermoFisher, Catalog # 31410), goat anti-human IgM (ThermoFisher, Catalog # A18841), or goat anti-human IgA (ThermoFisher, Catalog # A18781) for 1 h at 37°C. The colour was developed using trimethyl borane (TMB) solution (Sigma) and absorbance was measured at 450 nm using an ELISA reader. Samples from nonimmunised mice or healthy volunteers were used as controls. The cut-off lines were based on the mean value plus three times the standard deviation.

The T cell responses were detected by enzyme-linked immunosorbent spot (ELISpot) kits (Dakewe Biotech Co., Ltd) following the manufacturer's protocol. Briefly, splenocytes harvested from sacrificed mice were washed and immediately transferred to anti-IFN-γ antibody pre-coated filter plates. For stimulation, splenocytes were coincubated with distinct epitopes overnight at 37℃. All samples were assayed with positive controls (Phorbol myristate acetate and Ionomycin) and cells from a reference donor. All images in different wells were captured using a CTL ImmunoSpot ELISpot Analyzer and processed using the ImmunoCapture software (Cellular Technology Ltd., USA).

ELISA data were collected on a Thermo Scientific VARIOSKAN FLASH 3001 (Ref:5250040). ELISpot assay results were captured using a CTL ImmunoSpot ELISpot Analyzer and processed using the ImmunoCapture software (Cellular Technology Ltd., USA). Data were reported as the median (indicating the range from minimum to maximum) and arithmetic mean ± standard deviation using, R software package version 3.4.1 and Microsoft office 365 Excel. All R-squared and t-test were calculated by Student's t-test using Microsoft office 365 Excel. All results were plotted in Prism 7 (GraphPad Software Inc., CA).

The genes encoding the spike RBD (amino acid residues 306 to 543 of the spike protein) and full-length NP of SARS-CoV-2 were codon-optimized using E. coli.

Detailed information on the two recombinant proteins can be found in our previous work 1 .

Serial two-fold dilutions of heat-inactivated sera (treated at 56º C for 30 min) were prepared from a starting dilution of 1:10. The serum dilutions were mixed with equal volumes of 100 TCID50 (median tissue culture infective dose) of SARS-CoV-2 as indicated. After 1 h of incubation at 37º C, 35 µL of the virus-serum mixture was added to a monolayer of Vero-E6 cells for SARS-CoV-2 infection in 96-well microtitre plates in quadruplicate. After 1 h of adsorption, an additional 150 µL of culture medium was added to each well and incubated for 3 days at 37º C in 5% CO2 in a humidified incubator. A virus back-titration was performed without immune serum to assess the input virus dose. The cytopathic effect (CPE) was read at 3 days post infection. The highest serum dilution that completely protected cells from CPE in half the wells was estimated using the Reed-Muench method and was taken as the neutralising antibody titre. Positive and negative control sera were included to validate the assay.

ELISA data were collected on a Thermo Scientific VARIOSKAN FLASH 3001 (Ref:5250040). ELISpot assay results were captured using a CTL ImmunoSpot ELISpot Analyzer and processed using the ImmunoCapture software (Cellular Technology Ltd., USA). Data were reported as the median (indicating the range from minimum to maximum) and arithmetic mean ± standard deviation using, R software package version 3.4.1 and Microsoft office 365 Excel. All R-squared and t-test were calculated by Student's t-test using Microsoft office 365 Excel. All results were plotted in Prism 7 (GraphPad Software Inc., CA). 

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Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

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Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerging microbes & infections 9