Comparison of different immunoassays for γH2AX quantification

Annika Reddig, Dirk Roggenbuck, Dirk Reinhold


Background: One of the most sensitive methods for DNA double-strand break (DSB) detection is the assessment of phosphorylated histone protein 2AX (γH2AX), which can be visualized as γH2AX focus surrounding the damage site. Quantification of γH2AX offers a variety of potential diagnostic applications being on the brink of introduction into clinical routine. Therefore, further assay optimization and automation is required to enable standardized, high-throughput γH2AX analysis. To quantify γH2AX levels, different immunoassay techniques can be performed, each showing specific characteristics regarding assay sensitivity and sample processing. Although microscopic foci quantification is considered the most sensitive approach, data of direct comparison of multiple γH2AX immunoassay techniques are rare. In the current study we compared γH2AX quantification by different methods, including automated fluorescence microscopy, flow cytometry as well as immunoblotting. Further, we discussed assay-specific advantages and disadvantages.
Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were exposed to various concentrations of the DNA DSB-inducing, cytostatic drug etoposide for one hour. Subsequently, γH2AX levels were assessed by flow cytometry, immunoblotting and automated microscopy.
Results: Automated fluorescence microscopic foci quantification revealed the lowest limit of detection (LoD) (0.53 µM etoposide). More than 10-fold higher etoposide concentrations were required to distinguish γH2AX values form background signal by fluorescence intensity-based methods like flow cytometry. Immunoblotting showed the poorest LoD of all three techniques.
Conclusions: In contrast to flow cytometry and immunoblotting, automated fluorescence γH2AX foci quantification showed the lowest LoD. This low LoD allows the assessment and follow-up of patients with respective antitumor therapy. Thus, to apply the most suitable γH2AX analysis, specific assay characteristics must be considered.