Recombinant antigen expression on yeast surface. The extracellular domain of A33 (A, B) or full length NY-ESO-1 (C-F) was cloned into the pYD1 vector and protein expression induced for at least 24 h (B, D, F). Yeast induced to express an irrelevant antigen served as negative controls (A, C, E). Samples were either stained with a monoclonal anti-A33 antibody (A, B), a monoclonal anti-NY-ESO-1 antibody (C, D) or serum from patients known to react with NY-ESO-1 protein at a dilution of 1:100 (E, F). All assays were carried out at least in triplicate. Isotype-matched control antibodies did not show any specific binding (data not shown).