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.