Triturus elisa instrument

Diagnostic of M. pneumoniae was conducted using the Liaison M. pneumoniae IgG, IgM kit (DiaSorin) using a 1/100 dilution of the patient sera. Indirect ELISA assays were performed on 96-well plates Immulon 4 HBX 96-well plates (ThermoFisher) incubating 1 µg of each antigen at 4 °C overnight. 1/100 dilutions of each patient serum were added to the plate and detected using an anti-human IgG antibody conjugated with HRP (Thermofisher Scientific). Upon incubation for 30 min with 100 µl of substrate (Thermofisher Scientific), 100 µl of sulfuric acid 25% was added to stop the reaction and absorbance was read at 450 nm on a Triturus ELISA instrument (Grifols) device. Reference filter was set at 620 nm.

All serum samples were thawed to perform the Elecsys^® Anti-SARS-CoV-2 IgM/IgA/IgG assay on Cobas^TM e801 (09203079190, Roche Diagnostics International Ltd., Rotkreuz, Switzerland) according to manufacturer instructions. Samples were positive if the index was ≥1. The ELISA COVID-19 IgG immunoassay (G1032, Vircell, Granada, Spain) used Triturus^® ELISA Instrument (Grifols, Barcelona, Spain) according to the Vircell-adapted protocol for this analyzer; samples were positive if the index was ≥11.2. Antigens and immunoassay characteristics are shown in Supplementary Table S2.
Discordant samples between the two immunoassays were reanalyzed with other commercial immunoassays according to manufacturer instructions or by adapting its protocols to Triturus^® ELISA Instrument: ELISA Anti-SARS-CoV-2 (IgG) (EI 2606-9601 G, Euroimmun, Lubeck, Germany); LIAISON^® SARS-CoV-2 S1/S2 IgG (311450, DiaSorin, Gerenzano, Italy). Antigens and immunoassay characteristics are shown in Supplementary Table S2.