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.