Monoclonal anti human ige alkaline phosphatase antibody

Either 0.5 µg of purified Hel a 6 or 5 µg of sunflower pollen extract was spotted onto nitrocellulose membrane and blocked with 3% BSA. Strips were then exposed to sera of 39 sunflower-sensitized patients at 1:10 v/v at 4 °C for overnight. Bound IgE-antibodies were detected with Monoclonal Anti-Human IgE–Alkaline Phosphatase antibody produced in mouse (Sigma-Aldrich, Catalogue no. A3076) at 1:1000 v/v and NBT-BCIP substrate (Abcam).

MicroWell 96-Well Microplates (Nunc, Thermo Fisher Scientific) were coated with either nHel a 6 (5 ng/μl) or sunflower pollen extract in a bicarbonate buffer (pH 9.2) followed by blocking with 3% BSA (Sigma). Wells were then exposed to patient sera in 1:5 v/v at 4 °C for 16 h. Bound IgEs were detected by Monoclonal Anti-Human IgE−Alkaline Phosphatase antibody produced in mouse (Sigma-Aldrich, Catalogue no. A3076) at 1:1000 v/v and p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma-Aldrich). The reaction was stopped with 3 N NaOH, and absorbance was measured at 405 nm. An individual patient serum having P/N value [ratio between A₄₀₅ of a patient serum (P) and the mean of healthy controls (N)] more than 2.0 was considered as positive for that allergen.