Generation of an alpaca-derived nanobody recognizing γ-H2AX.
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.
['Rajan|Malini|M|', 'Mortusewicz|Oliver|O|', 'Rothbauer|Ulrich|U|', 'Hastert|Florian D|FD|', 'Schmidthals|Katrin|K|', 'Rapp|Alexander|A|', 'Leonhardt|Heinrich|H|', 'Cardoso|M Cristina|MC|']
Post-translational modifications Chromobodies Laser microirradiation Alpaca heavy chain antibodies CKM, casein kinase 2 mutant DNA repair ELISA, enzyme linked immunosorbent assay FRAP, fluorescence recovery after photobleaching GFP, green fluorescent protein H2AX, histone H2AX HEK293, human embryonic kidney 293 cells KLH, keyhole limpet hemocyanin Live cell microscopy MDC1, mediator of DNA damage checkpoint-1 MEF, mouse embryonic fibroblast RFP, red fluorescent protein VHH, variable domain of heavy-chain antibody XRCC1, X-ray repair cross-complementing protein 1 siRNA, short interfering RNA