Streptavidin coupled alkaline phosphatase

Cellular immunity was assessed by a T-cell stimulation assay. PepMix™ SARS-CoV-2 peptide pools were purchased from JPT (Berlin, Germany). The pools comprised 15-mer peptides overlapping by 11 amino acids (aa) covering the entire sequences of the SARS-CoV-2 spike protein. The spike peptides were split into two sub-pools, S1 (aa 1-643) and S2 (aa 633-1273). Peptides were dissolved in dimethyl sulfoxide and diluted in an AIM-V medium for use in the ELISpot assays. For ex vivo ELISpot assays, PBMCs were thawed. A total of 1–2 × 10⁵ cells per well were incubated with SARS-CoV-2 peptides (2 μg/mL; duplicates), AIM-V medium (negative control; 3–4 wells), or PHA (L4144, Sigma, St. Louis, MO, USA; 0.5 μg/mL; positive control) in 96-well plates coated with 1.5 μg anti-IFN-γ (1-D1K, Mabtech, Stockholm, Sweden) for 24 h. After washing, spots were developed with 0.1 μg biotin-conjugated anti-IFN-γ (7-B6-1, Mabtech), streptavidin-coupled alkaline phosphatase (Mabtech, 1:1000), and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Spots were counted using a Bio-Sys Bioreader 5000 Pro-S/BR177 (Karben, Germany) and Bioreader software generation 10. T-cell responses were considered positive when mean spot counts were at least threefold higher than the mean spot counts of the unstimulated wells.

For T cell stimulation, PepMix SARS-CoV-2 peptide pools for wild type (WT) were acquired from JPT (Berlin, Germany). The spike (S) peptides are split into sub-pools S1 (aa 1–643) and S2 (aa 633–1273) for wild type (WT). Peptides were dissolved in dimethyl sulfoxide and diluted in AIM-V medium for use in enzyme-linked immunosorbent spot (ELISpot) assays as described previously (6 (link)). For ex vivo T cell IFN-γ ELISpot assay, PBMCs from test subjects after the 2^nd vaccination were thawed and processed subsequently within one day. A total of 1–2×10⁵ cells per well were incubated with SARS-CoV-2 peptides (2 µg/mL; duplicates), AIM-V medium (negative control; 3–4 wells) or phytohemagglutinin (PHA) (L4144, Sigma; 0·5 µg/mL; positive control) in 96-well plates coated with 1.5 µg anti-IFN-γ (1-D1K, Mabtech) for 24 hours. After washing, spots were developed with 0.1 µg biotin-conjugated anti-IFN-γ (7-B6-1, Mabtech), streptavidin-coupled alkaline phosphatase (Mabtech, 1:1000) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Spots were counted using a Bio-Sys Bioreader 5000 Pro-S/BR177 and Bioreader software generation 10. Data were processed as spot-forming cells (SFCs) per 10⁶ PBMCs after subtracting the spots from the negative control (mean spot numbers from three to four unstimulated wells).