• 1-fluoro-2-nitro-4-azidobenzene (FNAB, IUPAC nomenclature: 4-azido-1-fluoro-2-nitrobenzene) can be purchased from Apollo scientific ltd, UK (cat no. 248-878-6). Alternatively, it can be made from 4-Fluoro-3-nitroaniline by a simple diazotization reaction as described earlier (6). CAUTION. FNAB is explosive and should be handled with care, especially when using large quantities. FNAB can be stored safely in dark in a loosely capped bottle at 4°C in a refrigerator.
• Polystyrene microtiter plates, (Greiner Labortechnik, Germany).
• Anti-rabbit IgG, (Sigma Aldrich, cat. no. I3382) CAUTION Store all reagents at 2-8ºC. If slight turbidity occurs upon prolonged storage clarify the solution by centrifugation before use.
• Rabbit IgG, (Sigma Aldrich, cat. no. I4508).
• Anti-rabbit IgG-alkaline phosphatase conjugate, (Sigma Aldrich, cat. no. R3115).
• Human IgE ELISA Quantitation Set (Cat. no. E80-108, Bethyl laboratories, USA). CAUTION Store all reagents at 2-8ºC. Do not freeze reagents. All reagents must be kept at room temperature (20-25⁰C) before use.
Components supplied in the kit are:
Affinity purified Goat anti-Human IgE Coating Antibody A80-108A, 1 ml at 1 mg/ml
Human IgE Calibrator, RC80-108-6, 1.0 ml.
HRP Conjugated Goat anti-Human IgE Detection Antibody A80-108P, 0.1 ml at 1 mg/ml.
• Epicoccum nigrum was prepared according to published procedure.
• Bovine serum albumin (BSA), Sigma (USA). BSA solution should be prepared in PBS. CRITICAL: BSA solution should be filtered prior to use to avoid microbial contamination.
• p-nitrophenyl phosphate (Sigma Aldrich, USA cat. no. P7998). Store in cool place. Recommended storage temperature: -20 °C keep container tightly closed in a dry place. CAUTION: Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
• Phosphate buffered saline (PBS) was prepared by mixing 0.85% NaCl to 0.01 M phosphate buffer (pH 7.2). To make 0.01 M phosphate buffer add 1.217 g Na2HPO4, 0.379 g NaH2PO4. Add distilled water to make 1 l solution.
• Washing buffer was made by adding 0.1% Tween 20 to PBS.
• Substrate dye buffer - It is prepared by adding 1 mg of p-nitrophenyl phosphate to 1ml of 50 mM bicarbonate buffer, pH 9.6 containing 0.02% MgCl2. It was used for colour development.
• Stop Solution -2 M NaOH.