Quan t cell sars cov 2

The IFN-γ and chemokine release assay “Quan-T-Cell SARS-CoV-2” (Euroimmun, Germany) was used to detect spike S1-specific T cells. For logistic reasons, we performed the assay for infection-naïve vaccinated individuals with PBMC and for SARS-CoV-2-infected individuals with heparin whole blood samples. The assay was conducted with three tubes, an unstimulated control (BLANK), an S1-coated tube (TUBE), and a mitogen-coated positive control (STIM). To compare PBMC and whole blood stimulations, we calculated the required cell number for PBMC stimulation based on Trucount™ analyses of 500 µl whole blood samples (mean values). Moreover, we did not compare absolute concentrations of secreted cytokines, instead we used the fold induction which was calculated for each patient individually (TUBE/BLANK). PBMC were thawed and seeded with 7×10⁵ to 1×10⁶ per tube in 500 µl medium (RPMI1640 +2mM L-Glutamine + 100 U/mL Penicillin-Streptomycin +1mM sodium pyruvate +10% FCS) or 500 µl heparin-whole blood was added to each tube. Subsequently, tubes were inverted and incubated for 20 h at 37°C with 5% CO₂. After the incubation, supernatant or plasma was collected by centrifuging the tube for 10 min at 6000g. The supernatant and plasma were frozen until further usage. Cytokines and chemokines in the supernatant or plasma were measured via Luminex-based multiplex assays.

The spike protein-specific T cell response was determined using a SARS-CoV-2 interferon (IFN)-γ release assay (IGRA) kit (Quan-T-Cell SARS-CoV-2, Euroimmun Medizinische Labordiagnostika, Luebeck, Germany). Whole blood collected in lithium heparin tubes was stimulated in blank, spike protein-, and mitogen-coated tubes for 16 h. The samples were then centrifuged to isolate and freeze the stimulated plasma, which was then subjected to an IFN-γ assay by ELISA according to the manufacturer’s protocol. The T cell response capacity was determined by subtracting the IFN-γ concentration (mIU/mL) in the test tube from that in the blank tube. According to the manufacturer, a value of >100 was regarded as a detectable response.

To evaluate cellular response against viral proteins Quan-T-Cell SARS-CoV-2 (Euroimmun, Lübeck, Germany) test was used. A cut-off value of 18.44 mIU/mL was used to define positive test values according to the manufacturer’s instructions. In the first stage, freshly drawn heparinized whole blood was incubated with the S1 antigen of the SARS-CoV-2 virus coated on the bottom of the test tube. Whole blood was also incubated in a second negative control tube (assessment of non-specific background response) and a third positive control tube (assessment of overall T cell response after stimulation). After incubation time (22-24h), serum plasma was obtained. In the second stage, an ELISA test was performed to measure the secreted IFN-γ in the first step of the test.