1 step slow tmb elisa

Serine 506-phosphorylated and non-phosphorylated forms of the 21-amino acid topo I peptide described above (see Antibodies section) were synthesized by Biopeptide Co., Inc. (San Diego, CA). Immulon H2B ELISA plates (Thermo Scientific) were coated overnight at 4°C with 100 μl of peptides resuspended at 2 μg/ml in coating buffer (50 mM NaHCO₃, pH 9). Plates were washed with washing buffer (Tris-buffered saline [TBS] pH 7.5, 0.5% Tween) and blocked with TBS containing 1% bovine serum albumin (BSA). Plates were washed once with washing buffer and then incubated for 1 h with serial dilutions (in duplicate) of pAb506P in TBS/1%BSA, washed again, and incubated for 1 h with goat anti-rabbit-HRP. Following another wash step, plates were incubated with HRP substrate 3,3’,5,5’-tetramethyl benzidine (1-step SLOW TMB ELISA, Thermo Scientific) according to the manufacturer’s instructions. Absorbance at 450 nM was read.

Corning 96 well plates were left uncoated or were coated with 10µg/mL of native or citrullinated histone, or the same concentration of PAD4 present in the citrullinated histone solution in phosphate buffered saline (PBS) overnight at 4 °C. After washing 3 times (0.1% Tween 20 in PBS), wells were incubated with block solution (5% nonfat dried milk in 0.2% Tween 20 in PBS) at room temperature for 2–4 h, then with serum (prepared as in [44 (link)]) diluted 1:200 in block solution for 2 h at room temperature. Wells were then washed 5 times, incubated with anti-human IgG-HRP diluted 1:5000 in 5% nonfat dried milk in 0.2% Tween 20 in PBS for 2 h at room temperature, washed again 5 times, and exposed to 1-Step Slow TMB-ELISA (Thermo Fisher Scientific) for 5–10 min, then stopped with 0.18 M sulfuric acid. Plates were read at 450 nm with 540 nm plate correction using a Synergy 2 plate reader (BioTek). Samples were run in duplicate. Absorbance values from uncoated wells for each sample were subtracted from coated wells for each sample to reduce the effects of non-specific IgG binding to the plastic. Absorbance values from PAD4 coated wells were subtracted from citrullinated histone coated wells to normalize for anti-PAD4 IgG binding.