Figure 2

Specificity of EpCAM ElISA. (A) Dot Blot analysis of used detection antibodies to react with spiked native recombinant EpEX standard (10 ng) in assay buffer (PBS/1%BSA), serum (pool of 6 healthy donors) and ascites (pool of 12 negative patients). Capture and detection antibodies specifically react with EpEX in all biological matrices used and gave low background in serum and ascites (B) Detection antibodies bind specifically to EpCAM and EpEX-TM in established HEK-293FT cell lines expressing different variants of EpCAM or the control protein YFP (Western Blot). Results were confirmed by sandwich ELISA in cell extracts and respective supernatants. Concentrations are calculated for 1 μg total cellular protein extract. sEpCAM levels in supernatants of the different HEK cell lines were calculated with regard to cell number, culture volume and time. (C) Western Blot analysis of EpCAM expression in different human cancer cell lines under standard culture conditions (D) Concentrations of sEpCAM in supernatants were calculated with regard to cell number, culture volume and time. HRT-18 ^Apo and HRT-18 ^Nec indicate supernatants of HRT-18 cells that underwent apoptosis (100 μM A23187) or necrosis (100 mM H₂O₂).