Fig. 5

ZZTat101 is able to confer on fNY-ESO-1 the ability to induce B-lymphocytes to secrete anti-fNY-ESO-1 IgG antibodies. a PBMCs were incubated for eleven days without anti-CD40/IL-4/IL-21, with ZZTat22-57 + anti-CD40/IL-4/IL-21 or ZZfNY-ESO-1Tat22-57 + anti-CD40/IL-4/IL-21, respectively. Then, supernatants were collected and added to anti-human IgG-coated plates. After an incubation of 2 h, biotinylated f-NY-ESO-1 was added to each well. After overnight incubation, an acetylcholinesterase-labeled streptavidin conjugate was added and enzymatic activity was determined using Ellman's reagent. b PBMCs were incubated for eleven days with or without anti-CD40/IL-4/IL-21, ZZfNY-ESO-1Tat22-57 + anti-CD40/IL-4/IL-21, respectively. PBMCs were transferred from culture plates to ELISPOT plates previously coated with fNY-ESO-1. After an additional 24-h incubation at 37 °C, a biotinylated anti-human IgG was added. Spot-forming cells were then detected using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP as substrate. Spot-forming cells were quantified with an automated ELISPOT reader