HCV Antigen Tetramer Synthesis

Biotinylated E2 monomers were synthesized by the laboratory of Dr. Arash Grakoui as previously described [25 (link)]. Tetramers were prepared by adding phycoerythrin (PE)- (Sigma-Aldrich) or allophycocyanin (APC)-labeled ExtrAvidin (Thermofisher), 5 times using 7.5 μl with 10 min incubation at room temperature each time. Tetramer staining was performed using 0.5 μL of Tetramer-PE and 0.5 μL of Tetramer-APC per sample. The MHC class I monomers were synthesized by either the National Immune Monitoring Laboratory (Montréal, QC, Canada) or the NIH Tetramer Core Facility (Emory University, Atlanta, GA) and were as follows: HLA-B7 restricted HCV Core peptide aa 41–49 (GPRLGVRAT), HLA-A1 restricted HCV NS3 peptide amino acids (aa) 1436–1444 (ATDALMTGY) [A1/NS3-1436], HLA-A2 restricted HCV NS3 peptide aa 1073–1081 (CINGVCWTV) [A2/NS3-1073], HLA-B27 restricted HCV peptide NS5B peptide aa 2841–2849 (ARMILMTHF) [B27/NS5B-2841]. Tetramers were prepared by adding 10 μl of phycoerythrin (PE)-labeled ExtrAvidin (Sigma, St. Louis, MO) five times, with 10 min incubation at room temperature after each addition.