Quantification of cell surface proteins with bispecific antibodies

Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined rati...

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Bibliographic Details
Published in:	Protein engineering, design and selection Vol. 26; no. 10; pp. 645 - 654
Main Authors:	Panke, C., Weininger, D., Haas, A., Schelter, F., Schlothauer, T., Bader, S., Sircar, R., Josel, H.P., Baer, U., Burtscher, H., Mundigl, O., Grote, M., Brinkmann, U., Sustmann, C.
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 01-10-2013
Subjects:	Antibodies, Bispecific - immunology Cell Line Flow Cytometry - methods Fluorescent Dyes - metabolism Humans Intracellular Space - metabolism Membrane Proteins - immunology Membrane Proteins - metabolism Original digoxigenin bispecific antibodies ErbB cMET receptor quantification
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Summary:	Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production 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 properties. 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 digoxigenin-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 antigens 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 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 antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 Edited by Anna Wu
ISSN:	1741-0126 1741-0134
DOI:	10.1093/protein/gzt035