A 96-well plate-based enzyme-linked immunosorbent assay (ELISA) for quantifying gamma-H2AX as a biomarker to monitor DNA damage was developed and compared with gamma-H2AX detection by both Western blot (WB) and quantitative immunofluorescence assay (IFA). Using a pair of high-affinity gamma-H2AX antibodies for protein capture and detection and a synthetic peptide calibrator, this chemiluminescence ELISA can quantify gamma-H2AX concentrations as low as 16 pg/mL in crude extracts from cancer cells and solid tumors. The assay has a lower limit of detection of less than 4 pg/mL, upper limit of quantitation of 2000 pg/mL, and coefficient of variation of 20%. Treatments under evaluation using the gamma-H2AX ELISA include the topoisomerase 1 inhibitors topotecan and irinotecan, the apoptosis-inducing biomolecule TRAIL (TNF-related apoptosis-inducing ligand), and ionizing irradiation. The gamma-H2AX ELISA showed utility for drug discovery screening, molecular pharmacology studies, and pharmacodynamic monitoring. In a mouse xenograft model (A375 melanoma), the assay detected dose- and time-dependent changes in gamma-H2AX in response to treatment with irinotecan administrated alone or in combination with the poly(ADP-ribose) polymerase inhibitors ABT-888 (veliparib), AZD-2281 (olaparib), or MK-4827. Overall quantitative correlation was 0.96 between the ELISA and IFA, and 0.66 between ELISA and WB. This assay format should prove useful in situations when higher throughput and accurate quantitation are needed and gamma-H2AX analysis does not need to be restricted to nuclear foci.

Funded, in part, by NCI Contract No. HHSN261201100001E and by the Center for Cancer Research, Intramural Program of the National Cancer Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A46.