Contamination of synthetic HuD protein spanning peptide pools with a CMV‐encoded peptide

To detect HuD‐specific T cells in patients with Hu‐antibody associated paraneoplastic neurological syndromes (Hu‐PNS), we used short‐term stimulation assays with HuD protein spanning peptide pools (PSPP) with purities of at least 70% and found reproducible false‐positive CD8+ T‐cell responses in three of 127 individuals (two healthy controls and one Hu‐PNS patient), which all shared HLA‐A*2402 and HLA‐B*1801. After testing the 15‐mer peptides of the HuD antigen separately, we discovered that the same three 15‐mers yielded the CD8+ T cell response in those three individuals. This highly unusual result could not be reproduced when using new batches of peptides with a higher level of purity (>82% and >95%). Therefore, we assumed this response was not directed against the HuD peptides and analyzed the HuD 15‐mers by Fourier transform ion cyclotron resonance (FT‐ICR) tandem mass spectrometry (MS/MS), which showed the presence of a cytomegalovirus (CMV)‐encoded peptide (AIAEESDEEEAIVAY) as a contaminant. The three responding individuals all were CMV‐seropositive and the contaminating peptide appeared to fit in the binding groove of HLA‐B*18. Our data reveal that synthetic PSPP may contain immunogenic contaminations which may cause false positive results in T‐cell stimulation assays. © 2008 International Society for Advancement of Cytometry