Nunlock microplates

With modifications, the ELISA was performed according to the protocol of Zhang et al. (2019) [17 (link)]. Briefly, 400 Nunlock microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with antigen (cBP, barley protein fractions) (50 μg/mL). After the incubation, blocking, and washing steps, the human sera (FA-PS or nA-PS) were added to the plate for overnight incubation with the anti-human IgE biotin-labeled antibodies (ab99807; Abcam, Cambridge, UK) for 1.5 h at 37 °C. After washing, the plate was incubated with streptavidin-HRP (ab 7403; Abcam, Cambridge, UK) for 0.5 h at 37 °C. After the final wash step, the TMB substrate (Abcam, Cambridge, UK) was added to develop a color reaction which was stopped by adding 50 μL H₂SO₄ (2M). The optical density (OD) was measured at 405 nm using the Jupiter UVM−340 spectrophotometer (ASYS Hitech GmbH, Eugendorf, Austria). Each barley fraction was subject to reaction with the FA-PS and nA-PS sera as a negative control in triplicates. A mean OD value ± SD was considered positive if it exceeded the mean of the negative controls by more than three standard deviations.

Total IgE was determined by sandwich ELISA, using a commercial test (Cat. No. 157718; Abcam, Cambridge, UK) following the manufacturer’s protocol. Specific to OVA, serum IgA and IgG and feces IgA were measured by indirect ELISA (16). The antigen (1 µg/well in 10 mM PBS, pH 7.4) was coated onto 400 Nunlock microplates (Greiner Bio-One Gmbh, Frickenhausen, Germany) and incubated at 37 °C for 1 h. Next, the plates were blocked with 1.5% gelatin diluted in PBS and incubated under the same conditions. Following blocking, the plates were washed with PBST (PBS + 0.05% Tween 20). Serial dilutions of serum and feces extracts (50 µL) were added to the plates and incubated for 1 h at 37 °C. After a washing step, the plates were incubated with HRP-labeled antibodies specific to mouse IgG (1:1000) or IgA (1:1000) for 1 h at 37 °C. The plates were washed and incubated for 1 h at RT with ABTS (Roche Diagnostics GmbH, Mannheim, Germany). The absorbance was measured at 405 nm on a Jupiter UVM spectrophotometer (ASYS-Hitech GmbH, Eugendorf, Austria). The endpoint titers (EPTs) were expressed as the reciprocal dilution of the last sample dilution of 0.1 OD above the negative control [42 (link),43 (link),44 (link),45 (link)].