BACKGROUND: Prediction, risk assessment, and diagnosis of autoimmune diseases often rely on detection of autoantibodies directed to multiple target antigens, such as the 65-kDa isoform of glutamic acid decarboxylase (GAD65-abs) and the tyrosine phosphatase-like protein islet antigen-2 (IA2-abs), the two major subspecificities of islet cell antibodies (ICAs) associated with insulin-dependent diabetes mellitus. We hypothesized that a combination of autoantigens in a fusion protein unifying the important immunodominant epitopes could provide an efficient target for cost-effective, one-step screening of sera. METHODS: Chimeric proteins composed of GAD65 and IA2 residues were constructed, analyzed for their immune reactivity with monoclonal antibodies and sera, and used in a diagnostic assay with (35)S-labeled protein as antigen. RESULTS: Length and order of GAD65 and IA2 sequences were critical for conservation of the conformational epitopes in the fusion protein. Among four chimera tested, only IA2((606-979))/GAD65((1-585)) retained wild-type-like folding of GAD65 and IA2 domains and yielded a stable protein after baculovirus expression. Reactivity of GAD65 antibody- and IA2 antibody-positive sera from patients newly diagnosed with insulin-dependent diabetes mellitus, from ICA-positive prediabetics, and from ICA-positive first-degree relatives demonstrated conservation of the relevant autoreactive epitopes. The assay based on the in vitro translated fusion antigen had a sensitivity and specificity identical to those for detection of GAD65- and IA2-abs based on the two separate GAD65 and IA2 proteins. CONCLUSIONS: Autoantigens such as GAD65 and IA2 can be combined successfully in a fusion protein of similar immune reactivity. This allows simultaneous detection of GAD65- and IA2-abs in a one-step screening assay and cost-effective identification of positive individuals at risk of diabetes or at onset of disease.
BACKGROUND: Prediction, risk assessment, and diagnosis of autoimmune diseases often rely on detection of autoantibodies directed to multiple target antigens, such as the 65-kDa isoform of glutamic acid decarboxylase (GAD65-abs) and the tyrosine phosphatase-like protein islet antigen-2 (IA2-abs), the two major subspecificities of islet cell antibodies (ICAs) associated with insulin-dependent diabetes mellitus. We hypothesized that a combination of autoantigens in a fusion protein unifying the important immunodominant epitopes could provide an efficient target for cost-effective, one-step screening of sera. METHODS: Chimeric proteins composed of GAD65 and IA2 residues were constructed, analyzed for their immune reactivity with monoclonal antibodies and sera, and used in a diagnostic assay with (35)S-labeled protein as antigen. RESULTS: Length and order of GAD65 and IA2 sequences were critical for conservation of the conformational epitopes in the fusion protein. Among four chimera tested, only IA2((606-979))/GAD65((1-585)) retained wild-type-like folding of GAD65 and IA2 domains and yielded a stable protein after baculovirus expression. Reactivity of GAD65 antibody- and IA2 antibody-positive sera from patients newly diagnosed with insulin-dependent diabetes mellitus, from ICA-positive prediabetics, and from ICA-positive first-degree relatives demonstrated conservation of the relevant autoreactive epitopes. The assay based on the in vitro translated fusion antigen had a sensitivity and specificity identical to those for detection of GAD65- and IA2-abs based on the two separate GAD65 and IA2 proteins. CONCLUSIONS: Autoantigens such as GAD65 and IA2 can be combined successfully in a fusion protein of similar immune reactivity. This allows simultaneous detection of GAD65- and IA2-abs in a one-step screening assay and cost-effective identification of positive individuals at risk of diabetes or at onset of disease.
Authors: C Tiberti; L Yu; F Lucantoni; F Panimolle; I Spagnuolo; A Lenzi; G S Eisenbarth; F Dotta Journal: Clin Exp Immunol Date: 2011-12 Impact factor: 4.330