title: Molecular Methods and Protein Synthesis for Definition of Autoantibody Epitopes.
abstract: Epitope mapping is the process of experimentally identifying the binding sites, or "epitopes," of antibodies on their target antigens. Understanding the antibody-epitope interaction provides a basis for the rational design of potential preventative vaccines. Islet autoantibodies are currently the best available biomarkers for predicting future type 1 diabetes. These include autoantibodies to the islet beta cell proteins, insulin and the tyrosine phosphatase islet antigen-2 (IA-2) which selectively bind to a small number of dominant epitopes associated with increased risk of disease progression. The major epitope regions of insulin and IA-2 autoantibodies have been identified, but need to be mapped more precisely. In order to characterize these epitopes more accurately, this article describes the methods of cloning and mutagenesis of insulin and IA-2 and subsequent purification of the proteins that can be tested in displacement analysis and used to monitor immune responses, in vivo, to native and mutated proteins in a humanized mouse model carrying the high-risk HLA class II susceptibility haplotype DRB1*04-DQ8.
authors.affiliation: Diabetes and Metabolism Unit, School of Clinical Sciences, Southmead Hospital, University of Bristol, Bristol, UK. karen.elvers@bristol.ac.uk.
authors.forename: Karen T
authors.initials: KT
authors.lastname: Elvers
authors.affiliation: Diabetes and Metabolism Unit, School of Clinical Sciences, Southmead Hospital, University of Bristol, Bristol, UK.
authors.forename: Alistair J K
authors.initials: AJ
authors.lastname: Williams
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D059811:HLA-DRB1 Chains / Q000276:immunology*; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D059811:HLA-DRB1 Chains; C527195:HLA-DRB1*04 antigen; D007328:Insulin; D050505:Mutant Proteins; C516862:PTPRN protein, human; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
keywords: Circular dichroism; Epitope analysis; FPLC; Mass spectrometry; Mutagenesis; Protein expression; Protein purification; Reverse phase HPLC