ELISA Evaluation of Refolded Antibody

To reassure the ability of the refolded product to neutralize antigens, ELISA analyses were performed. 96 well ELISA plates were either coated with 100 ng/well PT-gliadin or coated with 1% w/v PEG 6000 as negative control. We described the coating protocol as well as the ELISA in detail in our previous study [11 (link)]. To reduce unspecific interactions, samples containing refolded tscFv or tscFv IBs were diluted with Tris-buffered saline (24.8 mM Tris, 136.9 mM NaCl and 2.7 mM KCl, pH 8.0) containing 0.05% Tween 20 (TBST). 100 μL sample/well were incubated for an hour at 25 °C and 450 rpm. Every well was washed three times with 300 μL TBST. Subsequently, 100 μL of a 1:1000 dilution of Anti-Chicken IgG (H + L), F(ab′)2 fragment-Peroxidase antibody produced in rabbit (Sigma, Vienna, Austria) with TBST were added per well and incubated at 37 °C and 450 rpm for an hour (THERMOstar microplate incubator, BMG Labtech, Ortenberg, Germany). Each well was washed four times with 300 μL TBST. A color reaction was mediated by the addition of 100 μL premixed 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Scientific, Vienna, Austria), which reacted with the peroxidase. After 15 min, 50 μL of 0.9 M HCl were added as stop reagent. Absorbance was measured at 450 nm in a Multiskan FC Microplate Photometer (Thermo Scientific, Vienna, Austria).