Detection of Diabetes Autoantibodies (ZnT8A and GADA) by Bridge Fluorophore-Linked Immunosorbent Assay Using Baculovirus Expressed ZnT8/GAD65 Chimera

Background: Type 1 Diabetes Mellitus (T1DM) is preceded by a prolonged asymptomatic autoimmune phase during which islet autoantibodies represent the earliest detectable biomarkers of disease. Although Radioligand Binding Assays (RBAs) remain the reference method for autoantibody detection, their use is limited by the requirement of radioactive reagents, specialized facilities and high operational costs, highlighting the need for sensitive, non-radiometric assays suitable for large-scale screening. Objective and Methods: In this study, we developed a bridge Fluorophore-Linked Immunosorbent Assay (b-FLIA) for the simultaneous detection of glutamic acid decarboxylase autoantibodies (GADA) and zinc transporter 8 autoantibodies (ZnT8A) using a recombinant ZnT8/GAD65 chimeric antigen expressed in a baculovirus–insect cell system. Assay performance was evaluated using sera from healthy controls and diabetic patients previously characterized by RBA. Results: The b-FLIA achieved an area under the receiver operating characteristic curve of 0.9867, with 90.5% sensitivity and 100% specificity for the detection of ZnT8A and/or GADA. Furthermore, the assay showed an almost perfect agreement with RBA (κ = 0.912) and displayed excellent analytical precision (intra-assay CV 1.34%, inter-assay CV 2.88%). Conclusions: These findings demonstrate that the combination of a baculovirus-expressed ZnT8/GAD65 chimeric antigen with a bridge fluorescence immunoassay constitutes a practical and sensitive platform for multiplex detection of diabetes-associated autoantibodies, with potential application in routine laboratories and future population-based screening programs.