Avidin horseradish peroxidase hrp

The coating buffer was PBS, the blocking buffer was 2% w/v skim milk in PBS, the washing buffer was PBS containing 0.05% v/v Tween 20 (PBS-T). Reagent dilutions were prepared in 2% w/v skim milk in PBS-T (PBS-MT). Avidin–Horseradish Peroxidase (HRP) was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). The 3,3′,5,5′-tetramethyl-benzidine/H₂O₂ mixture (Single Component TMB Peroxidase EIA Substrate Kit, BioRad, Hercules, CA, USA) was employed as the chromogenic substrate. Except otherwise indicated, incubations were performed at RT, washing steps were performed with PBS-T and 50 μL per well were added in each incubation step.

PBS was used as the microplate coating buffer. Two percent w/v skim milk in PBS (PBS-M) and PBS containing 0.05 % v/v Tween 20 (PBS-T) were used as blocking solution and washing buffer, respectively. Sample or reagent dilutions were prepared in 2 % w/v skim milk, 0.05 % v/v Tween 20 in PBS (PBS-MT). Immunopure Avidin was purchased from Pierce, and Avidin–Horseradish Peroxidase (HRP) and rabbit anti-human IgG-HRP were purchased from Jackson ImmunoResearch Laboratories, Inc. The 3,3’,5,5’-tetramethyl-benzidine/H₂O₂ (Single Component TMB Peroxidase EIA Substrate Kit, BioRad, Hercules, CA, USA) was employed as the chromogenic substrate and the SuperSignal ELISA Pico (ThermoScientific, Rockford, IL, USA) as the chemiluminescent substrate. Except when otherwise indicated, incubations were performed at RT, washing steps were performed with PBS-T and 50 μL per well were added in each incubation step.