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).