Molecular Methods and Protein Synthesis for Definition of Autoantibody Epitopes.
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.
['Elvers|Karen T|KT|', 'Williams|Alistair J K|AJ|']
D000818:Animals D001323:Autoantibodies D056148:Chromatography, Reverse-Phase D002942:Circular Dichroism D018604:Epitope Mapping D000939:Epitopes D059811:HLA-DRB1 Chains D006801:Humans D007328:Insulin D013058:Mass Spectrometry D051379:Mice D050505:Mutant Proteins D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
Mutagenesis Circular dichroism Epitope analysis FPLC Mass spectrometry Protein expression Protein purification Reverse phase HPLC