Figure 2.

Antibodies from OC ascitic fluids react with tumor cells.

(a) Western blot of lysates from OVCAR-3 (upper panel) and SKOV-3 (lower panel) cells. Each sample was incubated with equal amount of Igs purified from ascites from OC patients (a-d) or from noncancerous controls (e-h). Immunoreactivity was detected with HRP-conjugated anti-human secondary antibody. After detection of ascitic IgG immunoreactivity, the membranes were stripped and probed with anti-α-tubulin antibody to assess the loading of lysate among the samples. (b) Immunofluorescence of OVCAR-3 and SKOV-3 cells with Igs purified from ascites. The cellular localization of TAAs was detected with Cy5-conjugated anti-human IgG (green). Nuclei were stained with propidium iodide (red). (a-d) Representative images of OVCAR-3 cells incubated with (a-c) Igs from OC ascites (a) without cell permeabilization, showing surface staining; (b) after cell fixation, showing nuclear staining; and (c) after permeabilization, showing cytoplasmic staining; (d) image of fixed and permeabilized OVCAR-3 cells incubated with Igs from noncancerous controls, showing a lack of any detectable signal. (e, f) Immunofluorescence analysis of fixed and permeabilized SKOV-3 cells incubated with Igs from ascites from patients with (e) OC and (f) noncancerous conditions, confirming the difference in reactivity.