Streptavidin horseradish peroxidase

An in-house ELISA assay was developed [21 ], briefly, Nunc Maxisorp 96-well plates were coated with 100 µL/well of anti-IL-6 coating antibody at (1 µg/mL) in standard assay diluent (PBS) and stored overnight at 4°C. All antibodies and conjugated streptavidin-horseradish peroxidase were purchased from Biosource, SARL, Belgium. The plates were blocked with 300 µL/well of standard assay diluent containing 5 g/L BSA (Fraction V; Sigma-Aldrich, Dorset, UK) and left for 2 h at room temperature. The plates were then washed with PBS containing 0.1% Tween 20 v/v, followed by the addition of diluted standards or samples. Immediately following this biotinylated IL-6 detection antibody (0.4 µg/mL) in standard assay diluent containing 5 g/L BSA was added and the plates left to stand for 2 h at room temperature. The plates were then washed before the addition of streptavidin-horseradish peroxidase in standard assay diluent containing 5 g/L BSA and the plate left to incubate for 20 min at room temperature. After washing the plates, o-phenylenediamine in substrate buffer (0.05 M phosphate-citrate buffer (pH 5.0) containing 0.03% sodium perborate) was added. The reaction was stopped by the addition of 1 M sulphuric acid and the plates were read at 450 nm, referenced at 630 nm.