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

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

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.

No MeSH data available.


Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.
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f0025: Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.

Mentions: The main objective of this work was the generation and characterization of γ-H2AX specific VHHs to be used in vitro and in living cells. The generated VHHs were found to be functional in vitro as well as in vivo. In addition, the functionality in living cells was assessed by the localization of the fluorescently tagged γ-H2AX-VHH-3 (chromobody) at sites of DNA damage. The ability to identify native proteins within the intact cellular context is the ultimate test for the application of VHHs in living cells. However, during in vivo characterization we found that alternative epitope recognition and epitope masking limit their in vivo applications. These two pitfalls must be considered in the future development and screening of live-cell biomarkers and are summarized in Fig. 5.


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

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588710&req=5

f0025: Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.
Mentions: The main objective of this work was the generation and characterization of γ-H2AX specific VHHs to be used in vitro and in living cells. The generated VHHs were found to be functional in vitro as well as in vivo. In addition, the functionality in living cells was assessed by the localization of the fluorescently tagged γ-H2AX-VHH-3 (chromobody) at sites of DNA damage. The ability to identify native proteins within the intact cellular context is the ultimate test for the application of VHHs in living cells. However, during in vivo characterization we found that alternative epitope recognition and epitope masking limit their in vivo applications. These two pitfalls must be considered in the future development and screening of live-cell biomarkers and are summarized in Fig. 5.

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

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.

No MeSH data available.