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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.


Related in: MedlinePlus

Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18]. The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.
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f0005: Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18]. The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.

Mentions: An alpaca (Llama pacos) was immunized with γ-H2AX peptide coupled to KLH (keyhole limpet hemocyanin) (Fig. 1A). About two months later, serum was isolated by centrifugation of blood (2000 rpm, 10 min and 4 °C).


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)

Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18]. The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18]. The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.
Mentions: An alpaca (Llama pacos) was immunized with γ-H2AX peptide coupled to KLH (keyhole limpet hemocyanin) (Fig. 1A). About two months later, serum was isolated by centrifugation of blood (2000 rpm, 10 min and 4 °C).

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.


Related in: MedlinePlus