Goat anti mouse horseradish peroxidase hrp antibody

Enzyme-linked immunosorbent assay (ELISA) experiments were essentially performed as described previously (4 (link)). Briefly, 3.3 pmol biotinylated oligonucleotides were bound per well to streptavidin-coated microtiter plates (Pierce Cat# 15125 Thermo Scientific) and incubated for 1 h in 100 μl PBS at room temperature (prepared from 20× solution Pierce, no potassium). Upon washing of the plates three times with PBS + 0.05%Tween-20 (PBS-T) plates were incubated for 2 h with 1H6 antibody at half maximal binding concentration (50 ng/ml) in 400 mM sodium PBS + 0.5% BSA at room temperature. Plates were washed three times with PBS-T, followed by incubation with goat-anti-mouse horseradish peroxidase (HRP) antibody (Sigma) at 50 ng/ml in PBS + 0.5% BSA. After washing five times with PBS-T, 100 μl of the HRP substrate TMB (3.3΄,5.5΄-tetramethylbenzidine, Merck Millipore) in was added to each well. Reactions were stopped with 100 μl 0.3 M Sulphuric Acid and signal intensity was detected at λ = 450 nm using a Multiskan absorption meter (Thermo Scientific). Absorbance was calculated after correcting for background from negative controls. All ELISA experiments were performed at least three times and each reaction was performed in triplicate.