Avidin biotinylated horseradish peroxidase complex

Recombinant mouse Cst6 and human CST6 (both from R&D Systems, Minneapolis, MN, USA) were used as a standard and as a negative control, respectively, in concentrations varying from 5 to 0.156 ng/ml. The wells of a 96-well plate were coated overnight at 4°C with polyclonal rabbit anti-human CST6 antibody (2 (link)), followed by incubation with 1% bovine serum albumin (ICN Biomedicals, Aurora, OH, USA) and 1% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Subsequently, standards, controls, and samples (undiluted up to 32× diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30 min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min. The above incubation steps were performed at 37°C and separated by repeated washing steps with PBS/0.05% Tween-20. Chromogenic substrate 1-step Ultra TMB (Thermo Fisher Scientific) was used as substrate for detection and the reaction was stopped by adding 4 M H₂SO₄. Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).