4.4. Total Immunoglobulin E (IgE) Levels in Serum

Total serum IgE levels were assessed using an ELISA kit (eBioscience, San Diego, CA, USA). In brief, 96-well plates (NUNC, Penfield, NY, USA) were coated with eBioscience’s IgE capture antibody (88-50460-88, San Diego, CA, USA) and incubated overnight at 4 °C. After the washing, the plates were incubated at room temperature for 2 h with blocking buffer. Standard IgE was serially diluted and added to the appropriate wells to generate a calibration curve. Serum samples were added to each well. The plates were incubated at room temperature for 2 h on a shaker, followed by two washes. Each well was incubated with a biotinylated antibody designed to detect IgE (cat. 88–50460–88, eBioscience, San Diego, CA, USA). The plates were shaken for 1 h at room temperature. Each well was treated with avidin-horseradish peroxidase (HRP) after four washes. The incubation was performed in a shaker at room temperature for 30 min. After washing the plates four times, the substrate solution was applied, and the plates were incubated for 15 min at room temperature. The absorbance was measured at 450 nm after the addition of the stop solution.