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

• Published in: Journal of immunological methods Vol. 256; no. 1; pp. 107 - 119
• Main Authors: Turner, Michael J., Abdul-Alim, C.Siddiq, Willis, Richard A., Fisher, Terrence L., Lord, Edith M., Frelinger, John G.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.2001; Elsevier
• Subjects: Animals; Antigen Presentation; Antigen-Presenting Cells - immunology; Biological and medical sciences; Cell Line; Cells, Cultured; Diverse techniques; Epitopes, T-Lymphocyte - immunology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Histidine; Histocompatibility Antigens Class I - immunology; Histocompatibility Antigens Class II - immunology; Hybridomas; Immunobiology; Lymphocyte Activation; Lymphoid cells: ontogeny, maturation, markers, receptors, circulation and recirculation; Major histocompatability complex; Mice; Microbiology; Microspheres; Molecular and cellular biology; Peptides - genetics; Prostate-specific antigen; Prostate-Specific Antigen - genetics; Prostate-Specific Antigen - immunology; protein purification; Recombinant Fusion Proteins - immunology; Sequence Deletion; T cell epitopes; T-Lymphocytes - immunology; Transfection; DHFR; NF-AT; T cell epitopes; APC; Prostate-specific antigen; MHC; Ova; Major histocompatability complex; T-CAD assay; PSA; Purification; Immune response; Antigenic determinant; Major histocompatibility system; Use; Class II histocompatibility antigen; Cellular immunity; Method; Protein; Histidine; Aminoacid; T-Lymphocyte; Bacteria; Class I histocompatibility antigen; Tumor cell
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00436-7

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, lac Z 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.