Poster Presentation The 13th International Congress of the Immunology of Diabetes Society 2013

Rapid assays for detection of type 1 diabetes associated autoantibodies in organ donors (#85)

Taina Härkönen 1 2 , Mevlida Kararic 1 2 , Heikki Hyoty , Mikael Knip 1 2 3 4
  1. Children's Hospital , University of Helsinki and Helsinki Unversity Central Hospital, Helsinki
  2. Diabetes and Obesity Research Program , University of Helsinki , Helsinki, Finland
  3. Folkhälsan Research Center, Helsinki, Finland
  4. Department of Pediatrics Tampere, Tampere University Hospital, Finland

Diabetes-associated autoantibodies are essential in the prediction of type 1 diabetes. Autoantibodies are also used to identify autoantibody-positive organ donors in large international studies (e.g. nPOD and PEVNET) which aim at uncovering pathogenic changes in pancreas and related organs in order to understand the mechanisms of beta-cell destruction. The current study set out to develop rapid assays for autoantibodies to glutamic acid decarboxylase 65 (GADA), islet antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) by modifications of both our in-house radioimmunoassay (RIA) and a commercial ELISA (RSR Limited, United Kingdom). We have analyzed 100 diabetic children and non-diabetic siblings for IA-2A and GADA both with conventional and rapid assays. The RIA was modified into a rapid assay by incubating the labeled antigens and serum for one hour at RT instead of overnight at 4 °C. Immune complexes were bound to Protein-A Sepharose at 4 ºC for one hour. The analysis showed a strong correlation between the conventional and rapid assays (r=0.92 for IA-2A and r=0.89 for GADA). The rapid assays were further optimized by incubating serum and labeled antigen for 45 min at RT and immune complexes precipitated for 30 min at 4 ºC to obtain results within two hours. Antigen labels were made ready, standards and controls were pipetted on plates beforehand and all were kept at -80 ºC. The results showed a strong correlation with the conventional assays. The RSR ELISAs were first performed according to the manufacturer’s instructions. The results were in good concordance with the RIA results. Then assays were modified by shortening the incubation times and rising the temperature. Our preliminary results indicate that all three ELISA assays can be accomplished in less than 2 hours and the results correlate well both with the RIA and the conventional ELISA results.