• 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 (4,5) CAUTION. FNAB is explosive and should be handled with care, especially when using large quantities. We did not encounter any untoward incident while working with FNAB in last 12 years.
• Polystyren microtiter plates (Greiner Labortechnik, Germany).
• Anti-human 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.
• Human IgG, (Sigma Aldrich, cat. no. I4506).
• Anti-human IgG-HRP conjugate, (Sigma Aldrich, cat. no. A8419 ).
• Human IgE ELISA Quantitation Set (Cat. no. E80-108, Bethyl laboratories, USA). CAUTION Store all reagents at 2-8 degree Celsius. Do not freeze reagents. All reagents must be kept at room temperature (20-25 degree Celsius) 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
• Bovine serum albumin (BSA), Sigma (USA). CRITICAL BSA solution should be filtered prior to use to avoid microbial contamination.
• o-phenylenediamine dihydrochloride (OPD), (Sigma Aldrich, USA cat. no. P1526). Store in cool place. Recommended storage temperature: -20 °C; Keep container tightly closed.
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 Sodium phosphate dibasic , 0.379 g Sodium phosphate monobasic. Add distilled water to make 1 liter solution.
• Washing buffer was made by adding 0.1% Tween 20 to PBS.
• Substrate dye Substrate dye buffer was prepared by mixing 12 ml of citrate buffer (0.025M citric acid and 0.05M Disodium phosphate, pH 5), 5 µl of Hydrogen peroxide (30% w/v), and 4 mg of o-phenylenediamine dihydrochloride.
• Stop Solution- 5% Sulfuric acid