Monoclonal antibodies mabs

Lymphocyte, B-, and T-cell phenotyping were performed as previously published^{69 (link)–71 (link)} as a service at Helsinki University Hospital (analysis codes: 6258smB-Fc, 21388Tdif-Fc, and 8302LyDiff-T). Briefly, for flow cytometry, cells in EDTA samples were stained using whole blood technique, and analyzed on a FACSCanto II flow cytometer (Beckton Dickinson Biosciences, San Jose, CA, USA). Fresh heparin-blood samples or peripheral blood mononuclear cells (PBMCs) were used for B- and T-lymphocyte immunophenotyping. Four- or 10-color flow cytometry panel with monoclonal antibodies (mAbs) against the surface antigens IgM, IgD, CD3, CD4, CD8, CD16⁄56, CD19, CD21, CD27, CD33, CD34, CD38, CD45, CD56, CD57, CD133, HLA-DR, CD62L, CD45RA, and CD45RO (BD Biosciences, San Jose, CA)^{70 (link)}. The memory status of T cells was studied with the antibody panel including anti-CD45, -CD3, -CD4, -CD8 -CD45RA, and -CCR7 (R&D Systems, Minneapolis, MN, USA)^{70 (link),71 (link)}.

Splenocytes from immunized mice were cultured in flat-bottom 96-well plates (TPP, Denmark) at 1 x 10⁶ cells per well in HL-1 medium (Biowhittaker, Lonza, France), complemented with 2 mM GlutaMax (Invitrogen, Life Technologies, France), 5 x 10⁻⁵ M β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, France) in the presence of 10 μg/ml of individual peptides. After 12, 48 and 72 hours of incubation at 37°C and 5% CO₂, IL-2, TNF-α and IFN-γ were respectively quantified in the culture supernatants by ELISA as previously described [8 ]. Monoclonal antibodies (mAbs) specific to IL-2 (clone JES6-1A12 for coating and clone JES6-5H4 for detection) or IFN-γ (clone AN-18 for coating and clone R4-6A2 for detection) were from BD Pharmingen (Le pont-de-Claix, France). Anti-TNF-α mAbs (clone 1F3F3D4 for coating and clone XT3/XT22 for detection) were from eBioscience.