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Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Rajan, Malini and Mortusewicz, Oliver and Rothbauer, Ulrich and Hastert, Florian D. and Schmidthals, Katrin and Rapp, Alexander and Leonhardt, Heinrich and Cardoso, M. Cristina (2015):
Generation of an alpaca-derived nanobody recognizing γ-H2AX.
5, In: FEBS open bio, pp. 779-88, ISSN 2211-5463, [Article]

Abstract

Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

Item Type: Article
Erschienen: 2015
Creators: Rajan, Malini and Mortusewicz, Oliver and Rothbauer, Ulrich and Hastert, Florian D. and Schmidthals, Katrin and Rapp, Alexander and Leonhardt, Heinrich and Cardoso, M. Cristina
Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX.
Language: English
Abstract:

Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

Journal or Publication Title: FEBS open bio
Volume: 5
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 09 Nov 2015 08:06
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