Cobas e801 immunoassay analyser

Serum ferritin concentration was measured by electrochemiluminescence immunoassay technology (Cobas e 801 immunoassay analyser, Roche Diagnostics Corp, Indianapolis, IN, USA). Serum sample was mixed with a biotinylated monoclonal ferritin-specific antibody and a monoclonal ferritin-specific antibody labelled with ruthenium complex to form a sandwich complex. After the addition of streptavidin-coated microparticles, the complex became bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture was aspirated into the measuring cell where the microparticles were magnetically captured onto the surface of the electrode. Unbound substances were then removed by a washing step. Application of a voltage to the electrode then induced chemiluminescent emission which was measured by a photomultiplier.

IgG and IgM titers were measured as units per ml (U/ml) which correlates 1:1 with the WHO standard unit for the SARS-CoV-2 binding antibody units per ml (BAU/ml). To this end, serum was analysed using the commercial electrochemiluminescence Elecsys Anti-SARS-CoV-2 S immunoassay (Roche, Mannheim, Germany) by a cobas^® e801 immunoassay analyser according to the manufacturer’s instructions (Roche).

IgG and IgA levels in serum were determined by anti-SARS-CoV-2 assay (Euroimmun), an ELISA which detects antibodies against the SARS-CoV-2 S1 spike domain. The assay was performed according to the manufacturer's instructions. Briefly, serum samples were diluted 1:10 in sample buffer and pipetted into rSARS-CoV-2 spike precoated strips of eight single wells of a 96-well microtitre plate. After incubation for 60 min at 37°C, wells were washed three times, peroxidase-labelled anti-IgG or anti-IgA added and incubated. After 30 min, three additional washing steps were performed before substrate was added and incubated for 15-30 min in the dark. Thereafter, stop solution was added, and optical density (OD) values measured on a POLARstar Omega plate reader (BMG LABTECH, Ortenberg, Germany) at 450 nm corrected for 620 nm. Finally, OD ratios were calculated based on the sample and calibrator OD values, where a ratio <0.8 was considered to be negative and >1.1 to be positive. To quantify antibody responses, IgG and IgM were measured as units per ml (U/ml) that correlates with the WHO standard unit for the SARS-CoV-2 binding antibody units per ml (BAU/ml). To this end, serum was analysed using the commercial electrochemiluminescence Elecsys Anti-SARS-CoV-2 S immunoassay (Roche, Mannheim, Germany) by a cobas® e801 immunoassay analyser according to the manufacturer's instructions (Roche).