Figure4: (A) RP215-specific epitope activities of the shed media from OC- 3-VGH and C-33A cells cultured in serum-free and serum supplemented media for 48 hrs by using RP215/RP215-HRP-based enzyme immunoassays. Absorbance at 450nm was plotted against serial dilutions of the shed media; (■) and (●) represent activities in serum-supplemented media for OC-3-VGH and C-33A cells, respectively;(□) and (○) represent corresponding activities in serum-free media; (▲) represents the initial activities of 10AU/mL CA215. Fresh RPMI-1640 culture medium containing 10% fetal calf serum was used as the negative control (OD=0.1); Data presented have been substrated from the negative control; (B) Relative human IgG concentrations in shed culture media of OC-3-VGH and C-33A cancer cells under serum-supplemented and serum-free conditions for 48 hrs using the anti-human IgG-Fc Mab as the probe on ELISA. (■) and (●) represent human IgG activities in serumsupplemented media for OC-3-VGH and C-33A cells, respectively; (□) and (○) represent corresponding activities in serum-free media. Fresh cell culture medium (RPMI plus 10% bovine calf serum) was used as the negative control (▲). (C) Western blot assay to detect protein bands from cultured cancer cells under serum-free and serum-supplemented conditions. Upper panels are protein bands of 60 KDa detected under reducing SDS-PAGE using RP215 as the probe. From left to right: Lane 1: OC-3-VGH cells (serum plus), Lane 2: OC-3-VGH cells (serum-free), Lane 3: C-33A cells (serum plus), Lane 4: C-33A cells (serum-free), Lane 5: FS293 cells (serum plus), Lane 6: FS293 cells (serum-free), Lane 7: RPMI medium control, Lane 8: serum-free medium control. Lower panels are protein bands of 60 KDa detected under reducing SDS-PAGE using anti-human IgG-Fc Mab as the probe. From left to right: samples from Lane 1 to 8 are identical to those described in the upper panel.