Fig. 2

The reactivity of patient-derived monoclonal antibodies with HRGEC. a Immunofluorescence staining for the binding of LN1–4 to HRGEC. HRGEC were fixed with 4% paraformaldehyde, blocked by buffer containing 3% BSA/PBS, and then incubated with LN1–4 or IgG1/IgG3 isotype controls (10 μg/ml), and FITC-conjugated goat anti-human IgG. Finally, the results were detected by a fluorescence microscope (× 100). For an accurate comparison of fluorescence signals, each image was taken with the same exposure time. A representative result from 3 experiments is shown. b Utilizing cell-based ELISA, LN1–4 and IgG1/IgG3 isotype controls were analyzed at the indicated concentrations for their bindings to HRGEC. The mean and SD are given. c Flow cytometry for the binding of LN1–4 to HRGEC. HRGEC were suspended with RPMI 1640 and incubated with LN1–4 or IgG1/IgG3 isotype controls (10 μg/ml) and then incubated with AF 488-conjugated mouse anti-human IgG. Stained cells were re-suspended in cold staining buffer and analyzed with a FACSCalibur cell analyzer. One of two experiments with similar results is shown