Metal-conjugated neutravidin for MHC multimer assays using mass cytometry (TECH3P.939)

Abstract Antigen-specific T cells in blood can be detected through the use of soluble MHC-peptide ligands that engage αβTCR. The fluorescent tetramer assay, developed in part by John Altman et al. (1) of the Vaccine Research Center, has become a standard tool for immunologists. Evan Newell et al. ( 2) adapted this peptide-MHC tetramer technology to mass cytometry for the purpose of screening of up to 109 different peptide-MHC tetramers in a single human blood sample, as well as analyzing another 23 markers of T-cell phenotype and function using a recombinant form of streptavidin conjugated to metal tags (Maxpar® kits, Fluidigm CA). We will describe the workflow for enumeration and identification of CMV-specific CD8+ T-cells with Neutravidin-metal reagent complexed with HLA-A*0201 CMV pp65 biotinylated monomer (MBL International, MA) simultaneously with metal-labeled surface markers, cisplatin dead-cell identifier, and the use of metal barcoding of several samples into one. There are 198 different biotinylated monomers, commercially available from MBL International, which can be combined with up to 35 isotope-tagged Neutravidin reagents to design a highly multiparametric assay. 1. Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94-96 (1996) 2. Newell, E. W. et al. Combinatorial tetramer staining and mass cytometry analysis facilitate t-cell epitope mapping and characterization. Nat. Biotechnol. 31, 623-629 (2013).