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

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.

Cross-reactive peptide epitopes of Enterovirus Coxsackie B4 and human glutamic acid decarboxylase detecting antibodies in latent autoimmune diabetes in adults versus type 1 diabetes.

BACKGROUND: Diagnosis of latent autoimmune diabetes in adults (LADA) is usually based on the adult age, anti-pancreatic islet cell antibodies detection, and insulin independence. This study investigates the diagnostic value of antibodies against human glutamic acid decarboxylase (hGAD) peptides in LADA and type 1 diabetes mellitus (T1DM) patients, and their cross-reactivity with an Enterovirus Coxsackie B4 (CVB4) shared epitope. METHODS: Sera from 27 LADA patients, 23 T1DM patients, and 24 controls were tested in ELISA for antibodies against hGAD peptides and a selected sequence of P2C protein of CVB4 (CVB4P2C). Diagnostic power of peptides was analyzed by ROC-curve analysis and cross-reactivity among peptides evaluated. RESULTS: IgM and IgG antibodies showed significant differences between LADA and T1DM versus controls for all peptides. Antibody responses present high agreement among peptides for IgM and IgG-isotypes in T1DM, which is not reproduced in LADA. IgM antibodies showed high predicting diagnostic power particularly in LADA (sensitivity > 85%, specificity 95.8%). CONCLUSIONS: Our study highlights the usefulness of peptides as diagnostic antigens in T1DM and LADA, and extends previous findings by comparing IgM and IgG-isotype antibodies in the same population. Additionally, results highlight the role of the entourage in the shared sequon PEVKXK in GAD and CVB4P2C particularly in IgMs identification.

Antibody Epitope of Human α-Galactosidase†A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease.

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (KD =39×10-9 m), which is nearly identical to that of the full-length enzyme (KD =16×10-9 m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.