To choose ligands with the highest and lowest affinities, the relative MHC binding affinities of the variants were determined using the purified I-Aᵁ molecule and a prototype labeled peptide (9.4Ala, 9.4Phe and 9.4Tyr). These variant peptides with higher T-cell stimulatory capacities bind more effectively than 9.4Lys (ASQKRPSQR) in concordance with earlier cellular assays.