Quantification of Cellular mCRP by ELISA

mCRP was detected in cellular supernatants (previously centrifuged at 1,000 g for 10 min) by an ELISA assay following the protocol recently described by Zhang et al. [52 (link)]. For this purpose, mouse anti-human CRP mAb CRP-8 (Sigma-Aldrich, C1688) was immobilized as capture antibody at 1:1,000 in coating buffer (10 mM sodium carbonate/bicarbonate, pH 9.6) overnight at 4°C. After washing three times for 2 minutes each with TBS, non-specific binding sites were blocked with filtered 1% BSA-TBS for 1 hour at RT. Samples diluted 1:100 in blocking buffer were added into wells for 1 hour at RT. Then, washing step was repeated and samples were incubated with sheep anti-human CRP polyclonal antibody (1:2,000 in blocking buffer) (BindingSite), prior incubation with a HRP-labeled donkey anti-sheep IgG (1:10,000 in blocking buffer) (Abcam). Signaling was detected with VersaMax Microplate Reader and The OD value of each sample was calculated as OD₄₅₀–OD₅₇₀ nm.