IFN-γ release was determined using the Human IFN-γ ELISpotPLUS HRP assay (#3420-4HST-10, Mabtech, Nacka Strand, Sweden) following the manufacturer’s guidelines. Wells were washed four times with PBS (Gibco-BRL, Waltham, MA, USA) and pre-conditioned with complete RPMI-1640 media (Gibco-BRL, Waltham, MA, USA) for 30 min. Expanded T cells were seeded in the wells at 5 × 104 with either 20 ug/ml individual peptide, 20 ug/ml peptide pool, no peptides (control) or anti-human anti-CD3 antibody as a positive control (mAb CD3-2, #3420-4HST-10, Mabtech, Nacka Strand, Sweden) and left to incubate overnight at 37 °C. The number of IFN-γ SFUs was determined using the AID iSpot ELISpot reader (Autoimmun Diagnostika GmbH, Strasburg, Germany).
Mab cd3 2
Manufactured by Mabtech
Sourced in Sweden
MAb CD3-2 is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. It is commonly used in research applications to detect and quantify T cells.
Automatically generated - may contain errors
Lab products found in correlation
Complete rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Aid ispot elispot reader, by Autoimmun Diagnostika (1 mentions)
Phytohemagglutinin l pha, by Merck Group (1 mentions)
Bzlf1, by JPT Peptide Technologies (1 mentions)
3 protocols using mab cd3 2
1
IFN-γ Release Quantification by ELISpot
2
SARS-CoV-2 ELISpot Assay Protocol
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The SARS-CoV-2 ELISpot assay was performed using ELISpotPRO 96-well plates (Mabtech, Nacka Strand, Sweden) following the manufacturer's guidelines and published protocols (Janetzki et al., 2015 (link)). Briefly, PBMCs were thawed and rested in 50-ml Corning bioreactor tubes overnight. For each subject, 100 µl of 2 × 106 PBMCs/ml cell suspension (2 × 105 PBMCs/well) were stimulated overnight (~16 h) with 100 µl of SARS-CoV-2 matrix peptide pools (2 µg/ml of each peptide; JPT, Germany), SARS-CoV-2 individual peptides (2 µg/ml; JPT), mAb CD3–2 (1:500 dilution; Mabtech), CMV peptide pool control (2 µg/ml; Mabtech), or blank control (complete RPMI 1640 medium with DMSO). The numbers of SARS-CoV-2-specific IFN-γ-secreting T cells/2 × 105 PBMCs, referred to as IFN-γ spot forming cells (SFCs), were determined using an AID iSpot Reader (AID, Strasberg, Germany) and analyzed using ELISpot 7.0 Software (AID). Wells containing SFC numbers greater than mean + 3SD of the blank controls were considered positive. A dotted line is drawn in each figure to show the positive threshold.
3
EBV-Derived Peptide Stimulation Assay
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The following stimulatory agents were used in this study: Overlapping peptide pools of EBV-derived proteins BZLF1 (59 peptides) and EBNA3A (234 peptides) (JPT Peptide Technologies, Berlin, Germany), consisting of 15mers overlapping 11 amino acids in a concentration of 1 µg/ml. The optimal assay concentration of both peptide pools was identified in previous titration experiments. Phytohemagglutinin (PHA-L) (Sigma-Aldrich Chemie, Schnelldorf, Germany) was used as a mitogen for stimulation in a concentration of 2 µg/ml. All experiments were performed in triplicates when cells were stimulated with antigen (BZLF1, EBNA3A) or in six replicates for the PHA-L-stimulated cells. RPMI-10 was added as a negative control in triplicates and anti-CD3 (in a dilution of 1:1000, mAb CD3-2, Mabtech AB, Nacka Strand, Sweden) was used as a positive control in a single well for each donor.
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