Analysis by fluorescence microscopy and flow cytometry of monoclonal antibodies produced against cell surface antigens.

The reactivity of seven monoclonal antibodies against the surface antigens of the murine myeloma cell line Sp2/O-Ag 14 was simultaneously analyzed by fluorescence microscopy and flow cytometry. To 1 X 10(6) Sp2/O-Ag 14 cells were added 200 microliters of the monoclonal antibody and the mixture was incubated at 4 degrees C for 30 min. After washing twice with PBS, the Sp2/O-Ag 14 cells were incubated at 4 degrees C for 30 min with a 1:400 dilution of fluoresceinated goat anti-mouse antibodies. Sp2/O-Ag 14 cells were ready for analysis after washing the cells 3 X with PBS. By fluorescence microscopic analysis different patterns of reactivity with monoclonal antibodies were detected. These patterns were identified as: smooth annular, dot-like annular, dot-like patches, diffuse and homogeneous. The observed patterns may represent different cell surface epitopes being recognized by the monoclonal antibodies. Flow cytometry analysis with the EPICS V system showed reactivity of seven monoclonal antibodies with Sp2/O-Ag 14 cell surface epitopes, which ranged from 79 to 90%. Compared to fluorescence microscopy, flow cytometry provides a faster, more sensitive and more accurate quantitative measurement of the reactivity of different monoclonal antibodies against Sp2/O-Ag 14 cell surface antigens.