The MHC presents peptide fragments of antigens in an allele-specific manner to T cells. Out of an enormous pool of potential peptides generated during processing, only a select subset are presented as epitopes on the cell surface, and therefore have the potential to elicit T-cell responses. The dominant peptide selected from HEL by I-Ak comprises amino acids HEL 48-62. Previous data have suggested that the primary anchor of the HEL48-62 epitope is D52. This is supported by experiments in which alanine substitution of this residue drastically decreased the binding affinity of the peptide for I-Ak. A number of preliminary experiments have been performed to identify the major epitopes of A52 HEL and have indicated that the 48-62 A52 peptide is undetectable. Instead, 44-61 A52 has been identified as the predominant I-Ak epitope derived from A52 HEL by 2-D HPLC and mass spectrometry. Initial I-Ak competitive binding experiments show that the dominant 44-61 A52 epitope d eriv ed from A52HEL has a greater relative affinity for I-Ak than the mutant 48-62 A52 peptide. The relative affinity of the 44-61 A52 peptide is equal to that of the dominant wildtype processed HEL 48-62 peptide. In this project we are trying to develop methods to reduce the complexity of the antigenic peptide mixtures by using 2-D HPLC, the first dimension being ion-exchange chromatography and the second, capillary RP-HPLC.
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