TNF-α, IL-6 and IL-8 ELISA

ELISAs were performed as per suppliers instructions with some minor modifications. Briefly, immuno Maxisorp plates (Nunc art NO. 439454) were coated with coating antibody in PBS, sealed and left to incubate over night at room temperature (RT). Between each step, wells were washed using 0.05% Tween 20 in PBS. After coating, cells were blocked for at least 1.5 h using block buffer (0.5% BSA in PBS for TNF-α and IL-6 and 1% BSA with 0.05% NaN₃ in PBS for IL-8).

First incubation: wells were either incubated with standards (0–1000 pg/mL for TNF-α and IL-6 and 0–2000 pg/mL for IL-8) in 1:2 serial dilutions in assay diluent (0.5% BSA and 5% FBS in PBS for TNF-α and IL-6 and 0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline for IL-8) in duplicates or with samples (supernatant of incubation assay) in triplicates for 1 h (TNF-α and IL-6) or 2 h (IL-8) at RT in the dark. Second incubation: cells were incubated with detection antibody in block buffer for 1 h (TNF-α and IL-6) or 2 h (IL-8) at RT in the dark. Third incubation: wells were incubated with streptavidin-HRP solution in block buffer for 1 h at RT in the dark.

Plates were developed by adding 1-step ULTRA TMB ELISA substrate and the reaction was stopped after all concentrations of the standard showed coloration, with the same volume of Stop Solution (H₂SO₄, 1.8 M for TNF-α and IL-6 and 1 M for IL-8). Optical density was measured at 450 nm using a spectrophotometer.