Sars cov 2 interferon gamma release assay

Using the manufacturer’s selected parts of the SARS-CoV-2 S1 domain of the spike protein (based on the Wuhan strain), 0.5 mL of whole blood was stimulated for a period of 20–24 h [ET 2606-3003, SARS-CoV-2 Interferon Gamma Release Assay (IGRA) (Euroimmun, Lübeck, Germany)]. An additional tube coated with selected parts of the SARS-CoV-2 S1 domain of the Omicron spike protein was used for stimulation (Euroimmun, Lübeck, Germany) spike. We carried out negative and positive controls in accordance with the manufacturer’s instruction and measured IFN-γ using an ELISA (EQ 6841–9601, Euroimmun, Lübeck, Germany). For analysis, we used an Aesku.Reader (Aesku.Group, Wendelsheim, Germany) and Gen5 version 2.01 software.

We also evaluated SARS-CoV-2–specific T cell responses by measuring IFN-γ production after stimulation with SARS-CoV-2 spike protein using the Euroimmun SARS-CoV-2 Interferon Gamma Release Assay (IGRA) (Euroimmun, Lübeck, Germany) according to the manufacturer’s instructions. A set of three tubes was used for each participant: (1) SARS-CoV-2 IGRA BLANK without interferon-activating substance as the individual background stimulation, (2) SARS-CoV-2 IGRA TUBE containing the S1 domain of the SARS-CoV-2 spike protein, and (3) SARS-CoV-2 IGRA STIM containing a mitogen for unspecific interferon stimulation to evaluate the viability and stimulation capacity of T cells and sufficient number of T cells in the participant’s blood sample. The response was defined as IFN-γ concentration in the TUBE sample minus that in the BLANK sample, in international units per milliliter (IU/mL). IFN-γ responses above 200 mIU/mL were defined as positive.

An automated commercial in vitro diagnostic method that measures a component of cell-mediated immune reactivity to the S1 protein of SARS-CoV-2 was performed (SARS-CoV-2 Interferon Gamma Release Assay (IGRA), Euroimmun, Lübeck, Germany). If a patient has been in contact with the S1 protein of SARS-CoV-2, their white blood cells will release IFN-gamma in response to contact with the antigen. The assay was performed using fresh whole blood collected from the study subjects using the following method. Briefly, human lithium–heparin whole blood was obtained from each patient and 500 uL was distributed and mixed on each tube of one set of stimulation tubes (#1: blank, #2: S1 domain from the spike protein, #3: mitogen causing unspecific IFN-gamma secretion from T cells). Incubation at 37 °C for 16 h was performed to promote the release of interferon gamma by T cells in response to contact with the antigen. Finally, individual supernatants of these stimulated samples were collected, and an IFN-gamma ELISA (Euroimmun) was performed following manufacturer’s instructions. Calibrated standards were included and expressed in International Units per milliliter (IU/mL).