Study shows new route for dangerous coronavirus strain emergence skoltech NatureComms
) in the RPMI medium , containing 10% of FBS , 1% of penicillin-streptomycin solution , Brefeldin A and costimulatory CD28/CD49d reagent . Negative control samples were stimulated with the complete medium; for positive control, PMA/ionomycin combination was used. Surface markers were stained with fluorescent antibody panel containing CD3-APC/Fire , CD4-AF647 , CD8a-AF700 , CD45RA-PE/Dazzle , CD197-BV421 . Intracellular cytokines were stained using IL-2-FITC , IFNγ-PE , TNFα-BV785 antibodies .
A total of 16 serum samples were obtained during the first wave of the COVID-19 pandemic in spring-summer 2020 from recovered volunteers with PCR-confirmed SARS-CoV-2 infection and tested in a microneutralization assay. Microneutralization was performed with hCoV-19/St_Petersburg-3524V/2020 virus , and 30769 V and 23680 V viruses isolated from the patient S . Serum was heat inactivated , serially diluted 2-fold starting from 1/10, mixed with 25 TCID50 of virus, incubated for 1 h at 37 °C and inoculated into Vero cells in triplicates in 96-well plate. Culture media was the same as for virus isolation.
Serum samples obtained from patient S were tested for virus specific antibodies in ELISA and in microneutralization assay with either Reference or patient S viruses. ELISA was performed with “SARS-CoV-2-IgG-EIA-BEST” according to the manufacturer’s instructions.HLA genotyping was performed using a commercial kit according to the manufacturer’s instructions . HLA-A, -B, -C, -DRB1 and -DQB1 loci were genotyped with 3-field resolution.
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