Development of an immunoassay for the simultaneous detection of GADA and ZnT8A in autoimmune diabetes using a ZnT8/GAD65 chimeric molecule

TRABUCCHI, ALDANA; BOMBICINO, SILVINA S.; SABLJIC, ADRIANA V.; MARFÍA, JUAN IGNACIO; TARGOVNIK, ALEXANDRA; IACONO, RUBÉN F.; MIRANDA, MA. VICTORIA; VALDEZ, SILVINA N.

Frontiers in Immunology

Frontiers

Año: 2023

Introduction: The combined presence of autoantibodies to the 65 kDa isoformof glutamic acid decarboxylase (GADA) and to the islet-specific cation effluxtransporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss ofimmune tolerance and the clinical manifestation of autoimmune diabetesmellitus (DM). The screening of GADA and ZnT8A could help to reach to acorrect diagnosis and to start an early and adequate treatment. The aim of thestudy was to develop an immunoassay for the simultaneous detection of theseautoantibodies using a chimera molecule that includes the immunodominantregions of ZnT8 and GAD65, expressed by baculovirus-insect cells system.Materials and Methods: ZnT8/GAD65 was expressed using the Bac to Bac™baculovirus expression system. The recombinant chimera was purified by anHis6-tag and identified by SDS-PAGE and western blot analysis, and by an indirectELISA using specific antibodies against ZnT8 and GAD65. A fraction of ZnT8/GAD65 was biotinylated. A bridge ELISA (b-ELISA) was developed using ZnT8/GAD65 immobilized in polystyrene microplates, human sera samples fromhealthy individuals (n = 51) and diabetic patients (n = 49) were then incubated,and afterwards ZnT8/GAD65-biotin was added. Immune complexes wererevealed with Streptavidin-Horseradish Peroxidase. Results were calculated asspecific absorbance and expressed as standard deviation scores: SDs.Results: ZnT8/GAD65 was efficiently produced, yielding 30 mg/L culturemedium, 80% pure. This recombinant chimera retains the immunoreactiveconformation of the epitopes that are recognized by their specific antibodies, soit was used for the development of a high sensitivity (75.51%) and specificity(98.04%) b-ELISA for the detection of ZnT8A and/or GADA, in a one-stepscreening assay. The ROC curves demonstrated that this method had highaccuracy to distinguish between samples from healthy individuals and diabeticpatients (AUC = 0.9488); the cut-off value was stablished at 2 SDs.Conclusions: This immunoassay is useful either to confirm autoimmune diabetesor for detection in routine screening of individuals at risk of autoimmune DM. AsDM is a slow progress disease, remaining asymptomatic for a long preclinicalperiod, serological testing is of importance to establish a preventive treatment.