Recombinant human insulin

Pancreatic autoantibodies (IAA, GADA, IA2A and ZnT8A) were determined in serum at diagnosis, using previously described standardized radio-assays [13 (link)]. IAA were determined using a competitive fluid-phase radio-assay which uses [¹²⁵I]-labelled, recombinant human insulin (PerkinElmer Inc., Waltham, MA, USA) as antigen. GADA, IA2A and ZnT8A were determined by means of standardized radio-binding assays using in vitro transcribed and translated [³⁵S]-labelled recombinant human full-length GAD65, IA2ic (amino acids 605–979) and ZnT8 antigen. The ZnT8A assay simultaneously measures autoantibodies against both variants of the COOH-terminal domain (Arg325/Trp325). Antibody results for GADA, IA2A and ZnT8A are expressed as a semi-quantitative index calculated using a dilution curve of a positive sample. All cut-off values were set at the 99th percentile of the control population. Our laboratory has participated in different islet autoantibody standardization program (IASP) workshops, the last one in 2018. Specificity was 100% for all four antibody assays and sensitivity was 65% for IAA, 68% for GADA and 62% for IA2A and ZnT8A.

Streptozotocin, nicotinamide and anti-insulin antibodies (AB 260137) used in this study were obtained from Sigma-Aldrich, USA. The Optium Xceed glucometer and dosing strips were purchased from Abbott, Brazil. Thionembutal was supplied by the Abbott laboratory, USA. The blood laboratory test kits were supplied by Gold Analisa Diagnostics Ltda., Brazil. Recombinant human insulin was obtained from PerkinElmer, Shelton, CT, USA. All reagents used had the best possible quality.