2.5.3 Bridge ELISA (b-ELISA)
The protocol employed was based on that previously described (Villalba et al., 2007; Trabucchi et al., 2019; Trabucchi et al., 2022), with minor modifications. Briefly, polystyrene microplates were coated ON at 4 °C with 0.1 ug of purified ZnT8/GAD65 chimera per well. After washing with PBS, adding 200 μL of blocking solution per well for 1.5 h, and washing five times with PBS-T, 50 ul samples were added in duplicate. After 1 h of incubation at room temperature, plates were washed again and 56.0 ng of ZnT8/GAD65-biotin per well were added. One hour later, another round of washing steps were done, and the bound ZnT8/GAD65-biotin was detected by the addition of Streptavidin-Horseradish Peroxidase (Jackson ImmunoResearch Laboratories, Inc.). Finally, microplates were washed five times with PBS-T plus one final washing step with PBS. The color reaction was elicited by the addition of the chromogenic substrate incubated for 15 minutes in the dark and was then stopped with 4N H2SO4. The oxidized substrate was measured at 450 nm with an ELISA plate. The blank control was made by replacing serum samples with PBS‐MT. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs = (A‐Ac)/SDc, where Ac is the mean specific absorbance of 25 healthy control sera and SDc its standard deviation. The cut‐off value of the assay was set at SDs = 2.