Visualizing variability in human tetramer reagents using IP-FCM.

<p>(A) Flow cytometric assessment of commercial batches of human tetramers using antibodies detecting conformational determinants expressed by folded MHC class I molecules. (B) Western blot quantification of monomeric heavy chains present in the tetramer reagents analyzed in panel A. (C) Absence of significant correlation between heavy chain concentration and detection of correctly folded class I molecules in the tetramers measured by IP-FCM. (D) Flow cytometric assessment of commercial batches of human pentamers by IP-FCM. (E) Comparison of two batches of HLA-A2:surviving peptide with a correctly folded commercial standard. Top row, examples of flow cytometry characterization of tetramer confonformation; bottom row, quantification of triplicate estimates with tight error estimates. (F) Negative control mouse heavy-chain tetramer showing absence of reactivity with human-specific heavy chain antibodies. (G) Detection of survivin-specific T cells in HLA-A2 transgenic mice using HLA-A2:surviving tetramer reagent and an irrelevant HLA-A2:TRP2 tetramer control.