Reagents
PBS was used as the microplate coating buffer. PBS-M and PBS-T were used as blocking solution and washing buffer, respectively. Sample or reagent dilutions were prepared in 3 % w/v skim milk, 0.05 % v/v Tween 20 in PBS (PBS-MT). Streptavidin–Horseradish Peroxidase (HRP) and rabbit anti-human IgG-biotin were purchased from Jackson ImmunoResearch Laboratories, Inc. (USA). 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. 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.