Horse radish peroxidase conjugated goat anti mouse

Polystyrene plates where coated with poly-l-lysine overnight at 4 °C. After washing with PBS, plates were coated with 20 μg/ml calf thymus DNA diluted in ddH₂O at 37 °C for 2 h. Plates were washed with PBS and blocked with 2% bovine serum albumin (BSA) in PBS at room temperature for 1 h. Serum samples were diluted 1:50 in 2% BSA in PBS and incubated overnight at 4 °C or at room temperature for 2 h. Plates were washed five times in PBS, and horse radish peroxidase-conjugated goat anti-mouse (GE Healthcare, Chicago, IL, USA) was added and incubated at room temperature for 1 h. After washing five times with PBS, 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Waltham, MA, USA) substrate solution was added and incubated for 15 min. Reactions were stopped with 2 m HCl and measured to determine the optical density at 450 nm.