Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies.

TitleQuantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies.
Publication TypeJournal Article
Year of Publication2013
AuthorsDolan BP
JournalMethods in molecular biology (Clifton, N.J.)
Volume960
Pagination169-77
Date Published2013
ISSN1940-6029
Abstract

Accurately determining the number of peptide-MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining the number of peptide-MHC complexes on the surface of cells grown in tissue culture that express an endogenous protein from which the peptide is derived. The procedure requires a monoclonal antibody with the ability to distinguish MHC class I molecules presenting the peptide of interest from other peptide-MHC complexes. Fluorescence signal measured on antibody-labeled cells can be compared to fluorescent-calibrated beads to determine the relative number of antibodies bound to the cell surface and hence the number of specific peptide-MHC complexes expressed by the cell. As new monoclonal antibodies with TCR-like specificity for peptide-MHC complexes are created, this method will be helpful in quantifying the exact numbers of complexes generated by cell types and relating these numbers to physiological outcomes of T cell activation.

DOI10.1007/978-1-62703-218-6_14
Alternate JournalMethods Mol. Biol.