Human tnf α uncoated elisa kit

TNF-α concentrations were measured in sera collected from healthy controls and patients with COVID-19. The human TNF- α uncoated ELISA kit (Invitrogen, Bender Med Systems GmBH, Vienna, Austria) was used to measure TNF-α levels within a detection range of 0.0128–5000 pg/mL. Standard curves were generated by making serial dilutions of a known concentration of TNF-α. The absorbance of sera from patients with COVID-19 and healthy controls were measured using an ELISA microplate reader (Infinite F50 Plus, Tecan, Männedorf Switzerland). The absolute concentration of TNF- α was calibrated using standard curves as suggested by the manufacturer.

The THP-1 cell line was used as a cell model. THP-1 cells were differentiated by 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (SIGMA; P1585) for 24 h. After washing non-adherent cells with RPMI-free serum, 100 ng/ml of lipopolysaccharide (LPS) (SIGMA; L2654) was used as a stimulator to mimic the inflammatory condition, and treatment of LPS 100 ng/ml alone in differentiated THP-1 cells was considered as the positive control. The cells were treated with the drug with or without the presence of LPS and incubated at 37°C for 6 or 24 h. The cell medium was then collected and stored at −20°C. The level of TNF-α released by treated cells was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The supernatants were analyzed in Nunc MaxiSorp® flat-bottom 96-well plates (Invitrogen, ThermoFisher; #442402) using a human TNF-α uncoated ELISA kit following the manufacturer’s protocol (Invitrogen, Thermofisher; #88-7346). The OD value was measured with the Infinite 200Pro OD reader, using the Tecan i-control program at 450 and 570 nm wavelengths.