Streptavidin conjugated to hrp

Polystyrene high-binding microtiter plates (Corning, Corning, NY, USA) were coated with 100 µL of rEGF at 5 µg/mL and incubated overnight at 4 °C. After washing three times with PBS, 0.1% Tween 20 (PBST), wells were blocked with 3% skimmed milk in PBS (300 µL/well) for 1 h at RT. Biotinylated nanobodies were serially diluted and incubated for one hour at RT. After washing, streptavidin conjugated to HRP (Biotechne R&D Systems, Minneapolis, MN, USA) diluted 1:200 was added for one hour at RT. The reaction was visualized, stopped, and measured, as described above. The KD estimation was carried out by following the method and fitting function described in [37 (link)]. A linear regression analysis using this function was performed using the MyCurveFit web server (https://mycurvefit.com/, last accessed on 4 May 2023).

Immulon 4 plates was coated with OvTrop (10 μg/well) or (KLH) (10 μg/well). Another set of plate for measuring the Der p 10-specific IgE response was coated with Der p 10 (5 μg/well). Both sets were incubated overnight at 4C, then blocked with 200 μl of blocking buffer (PBS 1X, 0.05% Tween-20 containing 5% BSA). The IgE detection ELISA plate was placed at 4C until later use. Three pools of 50 μl of undiluted IgG-depleted (using Pierce Protein A/G Ultralink Resin (ThermoFisher, Waltham, USA) sera was added to both OvTrop and KLH antigen-containing wells and incubated overnight at 4°C. 50uL of each pool were transferred to the Der p 10-coated plate, following incubation for 1 hour at RT. After washing, 50 μl of 1:250 (diluted in PBS 1X, 0.05% Tween-20 containing 1% BSA) biotinylated anti-mouse IgE (Invitrogen, Carlsbad, USA) were added to the appropriate wells and incubated 1 hour at RT. Following an additional wash step, 50μl of streptavidin-conjugated to HRP (R&D Systems, Minneapolis, USA) was added diluted 1:100 and incubated for 30 minutes at RT. A final wash step was performed and 50μl of 1Step Ultra TMB-ELISA Substrate (ThermoFisher) was added to each well and the colorimetric reaction was stopped after 10 minutes 30 minutes by adding 25 μl of H₂SO₄. The plate was then read at 450 nm.