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

Rajan, Malini ; Mortusewicz, Oliver ; Rothbauer, Ulrich ; Hastert, Florian D. ; Schmidthals, Katrin ; Rapp, Alexander ; Leonhardt, Heinrich ; Cardoso, M. Cristina (2015)
Generation of an alpaca-derived nanobody recognizing γ-H2AX.
In: FEBS open bio, 5
Article, Bibliographie

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 ; Mortusewicz, Oliver ; Rothbauer, Ulrich ; Hastert, Florian D. ; Schmidthals, Katrin ; Rapp, Alexander ; Leonhardt, Heinrich ; Cardoso, M. Cristina
Type of entry: Bibliographie
Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX.
Language: English
Date: 2015
Journal or Publication Title: FEBS open bio
Volume of the journal: 5
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.

Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 09 Nov 2015 08:06
Last Modified: 09 Nov 2015 08:06
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