High throughput epitope identification and t cell receptor specificity determination using loadable detection molecules

A method for detection of one or more antigenic peptide responsive T cells in a sample, the method comprising:

• i. providing loadable detection molecules comprising at least one peptide-free MHC class I molecule and at least one detectable label,
• ii. providing at least one antigenic peptide,
• iii. contacting the loadable detection molecules with the at least one antigenic peptide to provide loaded detection molecules comprising at least one peptide-MHC (pMHC) class I molecule,
• iv. contacting the loaded detection molecules with the sample, and
• v. detecting binding of the loaded detection molecules to the one or more antigenic peptide responsive T cells,
• wherein each of the at least one antigenic peptide is represented by:
• at least two different detectable labels, or
• at least one detectable label which is a nucleic acid label,
• wherein the peptide-free MHC class I molecule comprises a heavy chain comprising an alpha-1 domain and an alpha-2 domain connected by a disulfide bridge, said heavy chain comprising an amino acid sequence selected from:
• (a) SEQ ID NO:
• 1, or
• (b) an amino acid sequence having at least 80% sequence identity to the sequence in (a),
• wherein a mutant cysteine residue is positioned in the alpha-1 domain at amino acid residue 84 or 85 and a mutant cysteine residue is positioned in the alpha-2 domain at amino acid residue 139.