H.Burtscher1, O.Mundigl2, M.Grote2, U.Brinkmann2

Flow cytometry is an established method for fast and ac-curate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration stan-dards. A critical step for quantitation remains the produc-tion of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody proper-ties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono-and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/ HER2 or ErbB3/HER3 and with an additional digoxi-genin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated anti-gens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 1 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular anti-bodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments