Schematic diagram of the synthesis and characterization of a chemical conjugate between an antibody Fab' fragment and a recombinant MHC class I, beta2M, peptide complex. (A) Purified HLA-A2/Flu-MA peptide_58-66 complexes containing a heavy chain substituted with a cysteine residue near the C-terminus were first derivatized with a bismaleimide polyethylene oxyde linker and then coupled to the reduced cysteine of a purified Fab' fragment. (B1) Elution profile from a Superdex 200 FPLC column of the purified HLA-A2/Flu peptide complex. (B2) Purification of the Fab-HLA-A2/Flu conjugate with a yield of approximately 30%. (B3) Elution profile of the purified Fab-HLA-A2/Flu conjugate with an apparent M_r of approximately 95 kDa. (C) SDS-PAGE showing that the purified Fab-HLA-A2/Flu conjugate gives under nonreducing conditions a major band with an apparent M_r of 82 kDa (lane 3), while as control proteins, intact mAb gives a band of 150 kDa (lane 1) and the F(ab')₂ fragment a band of 100 kDa (lane 2). Under reducing conditions, the conjugate gives a single band with an apparent M_r of 57 kDa (lane 6) while intact mAb (lane 4) gives two bands of 50 and 25 kDa and F(ab')₂ fragment a band of 25 kDa (lane 5).