Epitope mapping using gram-positive surface display
2010 (English)In: Current Protocols in Immunology, ISSN 19343671, no SUPPL. 90, 9.9.1-9.9.17 p.Article, review/survey (Refereed) Published
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 antibodys binding characteristics will still be the main features determining the assays 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.
Place, publisher, year, edition, pages
Wiley-Blackwell, 2010. no SUPPL. 90, 9.9.1-9.9.17 p.
antibody, cell surface, epitope, epitope mapping, Gram-positive, Staphylococcus carnosus
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
IdentifiersURN: urn:nbn:se:kth:diva-60006DOI: 10.1002/0471142735.im0909s90ScopusID: 2-s2.0-77956490637OAI: oai:DiVA.org:kth-60006DiVA: diva2:477071
QC 201201132012-01-122012-01-122012-01-13Bibliographically approved