Ds2 elisa system

SARS-CoV-2 cellular response was measured at the 12-month visit using a specific quantitative IFN-γ release assay in whole blood following the manufacturer´s instructions (SARS-CoV-2 IGRA stimulation tube set, Euroimmun, Lübeck, Germany). Details are provided elsewhere.¹⁵ Briefly, lithium heparinized blood from each patient was incubated 21 h at 37 °C in the three tubes supplied: blank tube for the individual IFN-γ background and, mitogen tube for unspecific IFN-γ secretion as controls, and stimulation tube coated with antigens of the SARS-CoV-2 spike protein for specific IFN-γ secretion. The IFN-γ concentration released in the plasma fraction obtained after centrifugation of the three tubes was then measured by an enzyme-linked immunosorbent assay (Human interferon-gamma ELISA, Euroimmun, Lübeck, Germany) with an automated instrument (Dynex DS2® ELISA system) in international units per milliliter (IU/mL). IFN-γ response was defined as stimulated minus unstimulated. Results were interpreted as follows: IFN-γ[SARS-CoV-2] – IFN-γ[blank] <100 mIU/mL was considered negative, 100–200 was considered borderline, and >200 was considered positive. Upper limit of quantification achieved was 5000 mIU/mL. Concentrations of IFN-γ above the calibration curve were defined as >5000 mIU/mL.

IgG against the surface S1 domain of the spike protein (S-IgG) and the internal nucleocapsid (N) protein (N-IgG) (Euroimmun, Lubeck, Germany) were measured (in EDTA plasma samples) at hospital admission and at 1, 2, 6 and 12 months after patients’ discharge, using commercial semi-quantitative EIA kits in an automated instrument (Dynex DS2® ELISA system) following the manufacturer instructions. Antibody levels were evaluated by calculating the ratio of the optical density (OD) of the patient sample over the OD of the calibrator (sample OD/calibrator OD = S/CO [absorbance/cut-off]). Results were interpreted according to the following criteria: ratio <1.1 was defined as negative and ratio ≥1.1 as positive.

Detection of neutralizing antibodies against SARS-CoV-2 (Nab) was performed at the 12-month visit in an automated instrument (Dynex DS2® ELISA system) by means of a surrogate neutralizing antibody test (SARS-CoV-2 NeutraLISA, Euroimmun, Lübeck, Germany), that determines the inhibitory effect of antibodies that can compete with the biotinylated host-cell receptor (ACE2) for the binding to the receptor-binding domain (RBD) of the S1 subunit of SARS-CoV-2 spike protein (inhibition percentage, %IH). Results were interpreted as follows: %IH <20 was considered negative, %IH ≥20 to <35 was considered borderline, and %IH ≥35 was considered positive.