Quantiferon sars cov 2 kit

We evaluated the T-cell response at T2 and T3 by using the QuantiFERON SARS-CoV-2 kit (Qiagen, Hilden, Germany). This is an interferon gamma (IFN-γ) release assay, which contains heparinized antigen tubes that allow both to collect whole blood and to stimulate lymphocytes with a combination of three antigen peptides specific to SARS-CoV-2 (SARS-CoV-2 Ag1, Ag2, and Ag3). The SARS CoV-2 Ag1 tube contains CD4+ epitopes derived from the S1 subunit of the spike protein; the SARS CoV-2 Ag2 tube contains CD4+ and CD8+ epitopes from the S1 and S2 subunits of the spike protein; the SARS CoV-2 Ag3 tube contains CD4+ and CD8+ epitopes from S1 and S2 plus immunodominant CD8+ epitopes derived from the whole genome. After stimulation, plasma samples were analyzed for the detection of IFN-γ (IU/ml) using an ELISA-based platform. Samples were processed following the manufacturer’s instructions (QuantiFERON SARS-CoV-2 Starter kit, ref. 626115; QuantiFERON SARS-CoV-2 Extended kit, ref. 626215; QuantiFERON ELISA, ref. 626410; Qiagen, Hilden, Germany). Elevated response was defined as a value at least 0.20 IU/ml greater than Nil (negative control used to subtract IFN-γ not derived from SARS-CoV-2-specific T-cell stimulation) (16 (link)).

The AIM assay was also performed with whole blood antigen-specific stimulation by using heparinized antigen tubes from the QuantiFERON SARS-CoV-2 kit (Qiagen).
Whole blood samples were collected directly into the assay collection tubes, shaken, and incubated for 16–24 h, as described above. Whole-blood aliquots (120 μL) were withdrawn from Nil (negative control) and from the two Ag tubes (60 μL each, mixed together) of the QuantiFERON SARS-CoV-2 kit before centrifugation, and stained for 15 min at room temperature with the following combination of anti-human fluorescent monoclonal antibodies: CD3 BV786, CD4 BV750, CD134 BB700 (3μL, Clone L106), and CD25 BV421 (all Becton Dickinson, San Jose, CA, USA). Incubation in 2 mL of FacsLyse solution (Becton Dickinson, San Jose, CA, USA) for 10 min was used to remove red blood cells. After centrifugation at 300× g for 5 min and washing with 2 mL of stain buffer, 0.5 mL samples were acquired on the FACSymhony A3 flow cytometer and data was analyzed using FlowJo software (Tree Star) and FacsDiva software (Becton Dickinson, San Jose, CA, USA). Cells were gated on the forward scatter/side scatter cell gate, FSC-A vs. FSC-H, to exclude doublets and then on the CD3+CD4⁺ gate for the quantification of CD25⁺OX-40+ SARS-CoV-2-specific CD4 T-cells.