SARS-CoV-2 Specific T-Cell Responses

SARS-CoV-2 specific T-cell responses were determined by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay using synthetic peptide pools of the SARS-CoV-2 spike (S1/S2) and membrane (M) protein (600 pmol/mL of each peptide; PepTivator^®, Miltenyi Biotec, Bergisch Gladbach, Germany). The peptide pools mainly consisted of 15-mer sequences with 11 overlapping amino acids. In total, 250,000 mononuclear cells isolated from patient blood were incubated with the respective peptides and added onto anti-interferon-gamma-coated strip assay plates (Merck Millipore Ltd., Tullagreen, Ireland). After 19 h of incubation, substrate solution (Oxford Immunotec, Oxford, UK) was added and spots were analyzed by an ELISpot reader (AID Fluorospot, Autoimmun Diagnostika GmbH, Strassberg, Germany). Mean values of duplicate samples were considered and a total of at least three spots after subtraction of unstimulated (background) spots (spot increment) was defined as positive [17 (link)].

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Wünsch K., Anastasiou O.E., Alt M., Brochhagen L., Cherneha M., Thümmler L., van Baal L., Madel R.J., Lindemann M., Taube C., Witzke O., Rohn H., Krawczyk A, & Jansen S. (2022). COVID-19 in Elderly, Immunocompromised or Diabetic Patients—From Immune Monitoring to Clinical Management in the Hospital. Viruses, 14(4), 746.

Publication 2022

PepTivator AID Fluorospot

Amino acids Assay Blood Cells Enzyme Enzyme linked immunospot assay Interferon gamma M protein Membrane Patient Peptides Sars cov 2 Spots

Corresponding Organization : University of Duisburg-Essen

Other organizations : Ruhrlandklinik

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Variable analysis

independent variables

Synthetic peptide pools of the SARS-CoV-2 spike (S1/S2) and membrane (M) protein (600 pmol/mL of each peptide; PepTivator®, Miltenyi Biotec, Bergisch Gladbach, Germany)

dependent variables

SARS-CoV-2 specific T-cell responses determined by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay

control variables

250,000 mononuclear cells isolated from patient blood
Incubation time of 19 h

controls

Unstimulated (background) spots

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