Determination of B3-antigen expression by ELISA
The expression of the B3 antigen on the different MCF-7 transfectants was assayed by ELISA. Equal numbers of MCF-7 transfectant cells and control cells grown in 96-well plates were formaldehyde-fixed (fixation does not affect ELISA with B3 antigen; U. Brinkmann, unpublished) and incubated with various concentrations of (murine) MAb B3. Goat anti-mouse IgG⟨peroxidase⟩ was used as secondary antibody and antibody binding was colorimetrically detected with ABTS as described in Materials and Methods. The control cell lines KB and OVCAR3 do not express B3 antigen. In agreement with B3-immunofluorescence analyses of the MCF-7 transfectants (not shown), no differences in ELISA signals were observed, indicating the B3-antigen expression of the MCF-7 transfectants remains unchanged.