Also consistent with the murine system, no significant responses were detected for any antigens when the ELISpot wells were precoated with anti-His mAb to capture the His-tagged IVTT proteins. The background responses with the unpurified or bound IVTT products were greater than for purified IVTT products, Figure 6 . Antigen-specific IFN-c ELIspot responses by human PBMC stimulated with IVTT-proteins. PBMCs were cultured with IVTTproduced FluM, FluHA, pp65 and EBNA3A purified using (A) NI-NTA nickel resin or (B) MagneHis Ni-particles; (C) unpurified; associated to (D) ProteinG beads or (E) Polybeads; or (F) added to wells precoated with Anti-His. Negative controls were medium only, unpurified whole extract, and whole extract associated to Polybeads or Protein G beads; positive control was CEF peptide pool. IFN-c ELIspot responses (spot forming cells, SFC) of cultured PBMCs were analysed after 36 hrs stimulation. Data are presented as mean +/-standard deviation of 10 volunteers. *P,0.05, **P,0.01 and ***P,0.001 compared to negative controls. doi:10.1371/journal.pone.0027666.g006