Infinite 200 automated plate reader

Plasma samples were thawed on ice and centrifuged at 10,000× g in order to remove precipitates before 10-fold dilutions were made. For the detection of SARS-CoV-2-neutralizing antibodies, a surrogate virus neutralization test was conducted. This test was previously shown to robustly correlate with conventional virus neutralization tests [14 (link)]. Here, we followed the manufacturer’s specifications using RBD peptides for both the Wuhan-Hu-1 strain and the B.1.617.2 (L452R, T478K) variant (Genscript, Piscataway Township, NJ, USA). The absorbance was detected at 450 nm (A450) on the Infinite^® 200 automated plate reader (Tecan, Männedorf, Switzerland). The optical densities (OD) of the negative control values were used to calculate inhibition percentage. Results for each individual sample were calculated using the following formula: inhibition = (1 − OD_SAMPLE/OD_{NEG CTRL}) × 100. The WHO Reference Panel for anti-SARS-CoV-2 immunoglobulin (20/268) was used for the calibration and calculation of international units (IU) [13 (link)].

Plasma samples were obtained by centrifugation of anticoagulated blood from healthy donors and COVID-19 patients, respectively. For the semi-quantitative analyses of IgM and IgG specific for the nucleocapsid (N) or the spike protein subunits S1, S2 and receptor binding domain (RBD), we used the respective MILLIPLEX SARS-CoV-2 antigen panel kits (Merck) to the manufacturer’s instructions. In brief, plasma samples were diluted 100-fold in assay buffer and were incubated with antigen-conjugated beads for 2 h at room temperature with shaking at 600 rpm. After washing, PE-conjugated anti-human IgG/IgM detection antibodies were added to the samples followed by incubation for 1.5 h at room temperature and 600 rpm. Samples were then washed, immersed in sheath fluid (Merck) and analyzed on the Luminex 100/200 flow cytometer (Luminex). For the detection of trimeric spike protein specific IgA we used an enzyme-linked immunosorbent assay according to the manufacturer’s specifications (Euroimmun, Lübeck, Germany). Absorbances were detected at 450 nm (A₄₅₀) on the Infinite 200 automated plate reader (Tecan).