El 800 universal microplate reader

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).

For ELISA, flat-bottom Immune FEP-101 96-well plates (JET BIOFIL, Guangzhou, China) were coated overnight with 0.1 μg per well of the anti-TRAIL mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Plates were washed three times in PBS containing 0.2% Tween20 (PBST), blocked with PBST containing 2% BSA and then incubated in triplicate with diluted plasma or diluted tissue homogenate supernatant for 2 h at 37 °C. Plates were washed again and incubated with a 1/2000 dilution of a rabbit polyclonal antibody (ab2435; Abcam, Cambridge, MA, USA) to TRAIL for 2 h at 37 °C. The plates were then incubated with a 1/5000 dilution of a horse radish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech Group, Inc., Chicago, IL, USA). After the final wash, plates were developed with 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB, QIAGEN, Hilden, Germany) for 20 min at room temperature and stopped after 10 min by adding 50 μl of 2M H₂SO_4. Analysis was performed using double wavelengths 450–630 nm with an EL × 800 Universal Microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).