Peroxidase conjugated affinity purified goat anti rabbit secondary antibody

Monoclonal antibodies (100 μL/well) against the Ad5 hexon protein were coated onto 96-well plates at 4 °C overnight. After blocking with 5% bovine serum albumin/PBST (0.05% Tween-20 in 20 mM PBS) at 37 °C for 1 h, the plates were incubated with the adenoviruses at 37 °C for 2 h. The samples were then incubated with a rabbit polyclonal antibody (1: 2000; ab2435; Abcam) against TRAIL at 37 °C for 1 h. The plates were incubated with a peroxidase-conjugated affinity-purified goat anti-rabbit secondary antibody (1: 500; Proteintech, Rosemont, IL). After each step, the plates were washed three times with PBST. The color visualization was performed using tetramethylbenzidine. The reaction was stopped with 2 M H₂SO₄ and analyzed at two wavelengths (450–630 nm) with an ELX800 Universal Microplate Reader (Bio-Rad Laboratories).

Ninety-six-well plates coated overnight at 4°C with 100 μl/well of a mouse monoclonal antibody (Clone: sc-51588, Santa Cruz Biotechnology, CA, USA) raised against sTRAIL (100 ng/ml) (freeze-dried powder, Sino Biological Inc., Beijing, China) in PBS were blocked with 2% BSA/PBS-T (20 mM PBS containing 0.05% Tween-20). Serial dilutions of TRAIL protein and rAd vectors were then added in excess and incubated at 37°C. The plates were then incubated with a 1/2000 dilution of rabbit polyclonal antibody (Santa Cruz Biotechnology, CA, US) to TRAIL at 37°C. The plates were then incubated with a 1/5000 dilution of a peroxidase-conjugated affinity-purified goat anti-rabbit secondary antibody (Proteintech Group, Rosemont, IL, USA). After every step the plates were washed three times with PBS-T and eventually developed with tetramethylbenzidine (TMB), stopped with 2 M H₂SO₄, and analyzed at double wavelengths 450-630 nm with an EL × 800 Universal Microplate reader (Bio-Rad Laboratories).