title: Epitope mapping using gram-positive surface display.
abstract: Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibody's binding characteristics will still be the main features determining the assay's reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.
authors.affiliation: School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Rockberg
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Barbara
authors.initials: B
authors.lastname: Hjelm
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry / Q000379:methods; D057089:Pathology, Molecular; D019151:Peptide Library; D019076:Transgenes / Q000235:genetics
chemical_list: D000939:Epitopes; D019151:Peptide Library