Ap conjugated mouse anti human ige antibody

To ensure the binding of the protein in the native state to the ELISA (Enzyme-linked immunosorbent assay) plate, Thermo Scientific™ Nunc™ Immobilizer™ Amino surface plates were used. Plates were incubated with 2 μg of purified recombinant Jug r 3 (2 μM) in 100 mM sodium carbonate, pH 9.6 at room temperature (RT) for 1 hour. Remaining electrophilic groups were quenched by reaction with 10 mM ethanolamine. Sixty μM of OLE were added and incubated overnight at 4 °C. According to the manufacturer’s protocol subsequent steps were performed. After washing, patients’ serum samples (1:10 diluted in TBST/0.5 BSA) were added to the wells and incubated ON at 4 °C. As negative controls, normal human serum (NHS), and OLE were tested in parallel. Afterwards AP-conjugated mouse anti-human IgE antibody (1:1000; 2 h RT; BD Biosciences) was added. Bound IgE was detected by colorimetry using SIGMA FAST^TM p-nitrophenyl phosphate substrate tablets (Sigma-Aldrich) and measured at 405 nm. The mean value of the negative controls plus 2x standard deviations were subtracted. The two tailed paired t-test was used for comparison of IgE binding to Jug r 3 with Jug r 3-OLE, respectively. P-values below 0.05 were considered statistically significant. Analyses were performed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). All sera were tested in duplicates.

For detection of Bos d 5-specific IgE in sera of milk allergic patients (10 patients positive and 10 patients negative to oral cows milk allergen challenge; sera were retrospectively collected in accordance with the Helsinki Declaration of 1975 and under approval of the ethical committee of the Bambino Gesù Pediatric Hospital, Rome; individual informed consent from all donors was collected by Dr. Alessandro Fiocchi, Children’s hospital Bambino Gesú, Rome, Italy), duplicate wells of MaxiSorp 96 well flat-bottom plates (Nunc, Rochester, NY, USA) were coated with 100 µl/well of apo-Bos d 5 or holo-Bos d 5 diluted at 5 µg/ml in coating buffer and incubated overnight at 4 °C. After 2 h blocking at room temperature with 200 µl Tris buffered saline (TBS)+ 0.05% Tween 20 (TBST), wells were incubated with 100 µl of human serum diluted 1:5 in TBST overnight at 4 °C. Alkaline-phosphatase (AP) conjugated mouse anti-human IgE antibody (BD Pharmingen) diluted at 1:1000 in TBST was used as secondary antibody. AP substrate (100 µl/well; SIGMAFAST p-Nitrophenyl phosphate Tablets, Sigma) was added to detect bound AP conjugated antibodies. The optical density was measured at 405 nm using an Infinite M200Pro microplate reader (Tecan, Austria).