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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
Published on: September 19, 2018
A novel antibody-based approach to detect the functional ERCC1-202 isoform
Mei-Shiue Kuo1, Julien Adam1, Nicolas Dorvault1
1INSERM U981, Gustave Roussy, 94805, Villejuif, France; Faculté de médecine, Université Paris-Sud, 94270, Kremlin-Bicêtre, France.
Abstract:
ERCC1/XPF endonuclease plays an important role in multiple DNA repair pathways and stands as a potential prognostic and predictive biomarker for cisplatin-based chemotherapy. Four distinct ERCC1 isoforms arising from alternative splicing have been described (201, 202, 203 and 204) but only the 202 isoform is functional in DNA excision repair, when interacting with its obligate partner XPF. Currently, there is no tool to assess specifically the expression of ERCC1-202 due to high sequence homology between the four isoforms. Here, we generated monoclonal antibodies directed against the heterodimer of ERCC1 and its obligate interacting partner XPF by genetic immunization. We obtained three monoclonal antibodies (2C11, 7C3 and 10D10) recognizing specifically the heterodimer ERCC1-202/XPF as well as the ERCC1-204/XPF with no affinity to ERCC1 or XPF monomers. By combining one of these three heterodimer-specific antibodies with a commercial anti-ERCC1 antibody (clone 4F9) unable to recognize the 204 isoform in a proximity ligation assay (PLA), we managed to specifically detect the functional ERCC1-202 isoform. This methodological breakthrough can constitute a basis for the development of clinical tests to evaluate ERCC1 functional proficiency.
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