In both human/virus and murine/parasite models, IVTT products purified by either NI-NTA resin or MagneHis Niparticles were highly effective inducers of antigen-specific IFN-c and TNF-a responses in vitro. In the human/virus but not murine/ parasite model, antigen-specific IFN-c and TNF-a responses could also be induced by unpurified IVTT reactions or IVTT products associated to Polybeads or Protein beads, indicating that the E. coli and LPS background of the whole extract was not sufficient to mask the immunogenicity of the IVTT recombinant products. Addition of carboxylate beads to unpurified IVTT products increased the immunogenicity of the IVTT viral products by up 1.8-fold for IFN-c, 2.6-fold for IL-10 and 7-fold for TNF-a. The most robust and specific antigen-specific IFN-c and TNF-a responses (by CBA) were detected at the highest dilution of IVTT product, where background E. coli responses were lowest. This is a favorable scenario for proteomic-wide scale cellular screening, as the use of highly diluted IVTT products is more cost-effective. Unexpectedly, poor results were obtained with ELIspot wells precoated with anti-IFN-c mAb as well as anti-HIS mAb to bind the HIS tag on the IVTT product. In vivo studies with PyCSP IVTT products confirmed that the target protein was produced and that the IVTT produced proteins were immunogenic.