1). For the preparation of ELISpot plates, MAIPSWU10 Multiscreen filtration plates (Millipore, Billerica, MA, USA) were pre-wetted with 70% ethanol for ≤ 1 min and washed with sterile water. Coating antigens (TTd, DT, PT, FHA and PRN) were diluted to 0.5 μg/well in sterile phosphate buffered saline (PBS) (SVA, Uppsala, Sweden) and anti-IgG coating mAbs were diluted

to 10 μg/ml in PBS and added to the plate. Wells used as blank controls were incubated with PBS only. The plates were BEZ235 ic50 then incubated overnight (ON) or ≤ 72 h at 4 °C. The plates were washed five times with PBS and blocked with RPMI-GlutaMAX™ supplemented with 10 mM HEPES and 50 μg/ml Penicillin–Streptomycin and 10% FCS (all from Gibco Invitrogen), referred to as “R10”, for at least 30 min at room temperature (RT). After the 72 hour pre-activation period cells were harvested and washed once in R10 before

counting. The cells were resuspended and added to the plates in duplicates with 100 μl cell suspension/well (for cell concentrations see paragraph 2.7). The ELISpot plates were incubated ON in humidity at 37 °C, 5% CO2. The cells were discarded and the plates were washed with PBS (5 × 200 μl/well). Biotinylated anti-IgG detection mAbs diluted to a concentration of 1 μg/ml in PBS supplemented with 0.5% FCS were added Bortezomib to the plate wells and incubated for 2 h at RT. The plates were washed with PBS (5 × 200 μl/well) before Streptavidin conjugated with Alkaline-Phosphatase (SA–ALP)

(Mabtech) diluted 1/1000 in PBS supplemented with 0.5% FCS was added and incubated for 1 h at RT. Unbound conjugate was washed away with PBS (5 × 200 μl/well). BCIP/NBT-plus substrate (Mabtech) was filtered through a 0.45 μm-filter and 100 μl/well was added to the wells and incubated for 7 min at RT. The reaction was stopped by rinsing the plates with tap water. The plates were then left to dry ON in darkness. The protocol used was Acesulfame Potassium originally described in 2009 (Buisman et al., 2009) but was later modified for a European collaboration project (Child Innovac). Antigen coating concentrations were 1.5 μg/well for PT and 0.7 μg/well for TTd, all antigens and the coating antibody were diluted in PBS. Major changes in the Child Innovac protocol were as follows; Stimulation: The cultivation medium consisted of AIM V medium (Gibco Invitrogen) supplemented with 10% FCS without β-mercaptoethanol. Also, 0.01 μg/ml of each antigen included in the assay was added to the stimulated aliquot. Coating of plate: The plates were pre-wetted with 35% ethanol and incubated with antigen ON or longer (< 5 days).