• 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 (1,7) CAUTION. FNAB is explosive and should be handled with care, especially when using large quantities.
• Polystyren microtiter plates (Greiner Labortechnik, Germany).
• Anti-human IgG, (Sigma Aldrich, cat. no. I3382) CAUTION Store all reagents at 2-8 degree celsius. 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 ).
• Rabbit IgG, (Sigma Aldrich, cat. no. I4508)
• 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 degree celsius; 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% sodium chloride to 0.01 M phosphate buffer (pH 7.2). To make 0.01 M phosphate buffer add 1.217 g disodium phosphate and 0.379 g monosodium phosphate to 500 ml of distilled water. Add distilled water to make the volume upto 1 liter.
• Washing buffer was made by adding 0.1% Tween 20 to PBS.
• Substrate -dye buffer was prepared by mixing 12 ml of citrate buffer (0.025M citric acid and 0.05 M sodium phosphate dibasic , pH 5), 5 µl of hydrogen peroxide (30% w/v), and 4 mg of o-phenylenediamine dihydrochloride.
• Stop Solution- 5% sulphuric acid