title: Quantitating MHC class I ligand production and presentation using TCR-like antibodies.
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.
authors: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. dolanbr@mail.nih.gov.
authors: Brian P
authors: BP
authors: Dolan
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D017951:Antigen Presentation; D002460:Cell Line; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D008024:Ligands; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D010047:Ovalbumin