T-cell antigen discovery (T-CAD) assay: a novel technique for identifying T cell epitopes

The identification of T cell epitopes is a critical step in evaluating and monitoring T cell mediated immune responses. Here, we describe a novel technique for simultaneously identifying class I and class II MHC restricted epitopes using a one-step protein purification system. This method uses Ni/chelate coated magnetic beads and magnetic separation to isolate poly-histidine tagged recombinant antigen from bacterial lysates. These beads, once coated with antigen, are also used to deliver antigen to APC where it is processed and presented to T cells. A colorimetric assay and ovalbumin specific, lacZ inducible, T cell hybridomas were used to validate the system. Further, using PSA specific hybrids, generated from T cells isolated from PSA secreting tumors, both class I and class II MHC restricted epitopes of PSA were identified. Additional characterization has shown that these peptides contribute significantly to the overall PSA specific response in vivo, and may represent the dominant epitopes of PSA.