Polyvinyl chloride elisa microplate

Levels of proinflammatory cytokines in each group were quantitatively detected by the indirect ELISA technique (Gan and Patel, 2013 (link)). Briefly, samples were diluted to 50 μL containing the same amount of proteins using bicarbonate/carbonate coating buffer (Sigma–Aldrich). The dilutions were then loaded in polyvinyl chloride ELISA microplate (Corning), sealed and incubated overnight at 4 °C. The plate wells were washed 3 times, and the remaining protein-binding sites in the coated wells were blocked by adding 200-μL blocking buffer (1% bovine serum albumin in PBS, 0.3% solution of H₂O₂) for 1 hour at room temperature. Afterward, 50 μL of monospecific antibodies were added and incubated for 4 hours at 37 °C. The plate wells were then washed 3 times and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, followed by another 3 times of washes. Finally, the plate was developed by incubating with 3,3′,5,5′-tetramethylbenzidine solution (Thermo fisher) for 30 minutes at room temperature, and reaction was stopped with 50 μL of sulfuric acid. The optical density was read at 450 nm on a spectrophotometer (Bio-Rad), and values were calculated and expressed as percentage changes versus control group.