Ifn γ elisa max deluxe kit

IFN‐γ was measured using the IFN‐γ ELISA MAX Deluxe Kit (BioLegend) and performed according to the manufacturer's instructions. Microplates were read at 450nm immediately following the addition of stop solution (2N H₂SO₄). IFN‐γ was quantified by extrapolating from the standard curve using GraphPad Prism. Values below the lower limit of detection of the assay were recorded as 7·81 pg/ml. IL‐2 was measured using a custom Bio‐Plex Pro Human Cytokine Set (Bio‐Rad) and performed according to the manufacturer's instructions. The mean fluorescent intensity of the cytokine beads was measured on a Bio‐Plex 200 (Bio‐Rad). Cytokine concentration was calculated from control curves of standards provided in the kit. Values below the lower limit of detection of the assay were recorded as 6·28 pg/ml.

IFN-γ protein was measured by an IFN-γ ELISA MAX Deluxe kit (BioLegend, San Diego, California, USA), following manufacturer’s instructions with a few modifications: an additional point on the standard curve (1000pg/ml); a 1-hour incubation for standards, samples, and blanks; and a pre-read step (at 450nm with just the TMB substrate) to standardise development of the assay. When standard 2 reached an optical density of 0.1, stop solution was added and the plate was read at 450nm. The amount of IFN-γ in each sample was analysed using the Gen5 software 8-point standard curve. To calculate the T cell response to SARS-CoV-2, the amount of IFN-γ in the control (PBS only) sample was subtracted from the corresponding value for the SARS-CoV-2 peptide stimulated sample and reported as pg/ml of plasma. In the absence of a response to the peptides, the amount of IFN-γ was calculated as below the lower limit of detection (LLOD). Therefore, a value equal to the lowest value on the standard curve was given for that sample.