2.4 Identification of the recombinant ZnT8/GAD65 protein expressed in Sf9 cells by indirect ELISA.
The identification of purified chimera was evaluated by means of indirect enzyme linked immunosorbent assay (ELISA) using mouse monoclonal antibodies against GAD65 and a rabbit serum against ZnT8. For this purpose, polystyrene microplates (Maxisorp, NUNC) were coated ON at 4 °C with 0,1 ug of purified ZnT8/GAD65 chimera per well, washed three times with phosphate buffered saline (PBS: 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4), blocked for 1.5 h with 200 μL of blocking solution (3% skim milk in PBS) per well, and washed five times with PBS containing 0.05% Tween 20 (PBS-T). Samples diluted 1/100 in 3% skim milk in PBS-T (PBS-MT) were evaluated five times, and microplates were incubated for 1 h. After another round of 5 washes with PBS-T the bound specific antibodies were detected by the addition of peroxidase-conjugated goat antibodies to mouse IgG or with peroxidase-conjugated goat antibodies to rabbit IgG diluted 1/3000 in PBS-MT. Following washing (five times with PBS-T plus one final wash with PBS), the chromogenic substrate was added (3,3′,5,5′-tetramethyl-benzidine/H2O2 mixture; Single Component TMB Peroxidase EIA Substrate Kit, BioRad), and plates were incubated for 15 min in the dark. The color reaction was stopped with 4N H2SO4. The oxidized substrate was measured at 450 nm with an ELISA plate reader MultiskanFC (Thermo Scientific Labsystems). The blank control was made by replacing serum samples with PBS‐MT. Results were expressed as specific absorbance (A = the mean absorbance of each sample minus the mean absorbance of the blank control).