ELISA based on peptide antigens reproducing cross-reactive viral epitopes to detect antibodies in latent autoimmune diabetes in adults vs. type 1 diabetes

Abstract

Diagnosis of Latent Autoimmune Diabetes in Adults (LADA) is based on the adult-age, anti-islet autoantibodies, and temporary insulin-independence. As in Type-1-Diabetes (T1DM), autoimmunity may trigger LADA and enteroviruses-infections can play a role. Anti-human Glutamic-Acid-Decarboxylase (hGAD) autoantibodies are accepted clinical biomarkers, but do not discriminate LADA vs. T1DM. The hypothesis is that protein antigens detecting anti-hGAD antibodies do not expose epitopes specific for different disease forms.We investigated the diagnostic value of autoantibodies in LADA vs. T1DM to peptides of hGAD65/67 isoforms, and Enterovirus-Coxsackie-B4 (CVB4), as antigens sharing the epitope PEVKXK (X: E/T) included in CD8 T-cell CVB4 epitope restricted by diabetes-associated HLA-A2.1. Statistically significant differences of IgM and/or IgG in LADA and T1DM vs. controls were identified. In LADA IgMs to GAD65/67 peptides are diagnostics, IgGs to GAD65/67 peptides correlate with anti-CVB4 peptide antibodies. IgM and/or IgG to all tested peptides can predict LADA, monitoring CVB4 infected patients, improving LADA vs. T1DM stratification. • A customized SP-ELISA based on synthetic peptides Ac-hGAD65(250-273)-NH2 (1), Ac-hGAD67(258-281)-NH2 (2), and Ac-CVB4P2C(28-50)-NH2 (3) is described. • The method was designed to detect specific IgM and/or IgG in LADA, T1DM, vs. controls • Final aim is improvement of LADA vs. T1DM patient stratification.

Keywords

• Customized peptide-based solid-phase Enzyme Linked ImmunoSorbent Assay