New ELISA Kit Measures Drug-Induced DNA Breaks

A gamma H2AX pharmacodynamic assay kit for the study of double strand DNA breaks through the detection of the phosphorylated histone, gamma-H2AX, is now available for drug developers as well as biotech and other life science researchers.

The ubiquitous histone H2AX is a 14-kDa member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes.

Serine 139 within this motif becomes rapidly phosphorylated by kinases to yield a form known as gamma-H2AX in response to double-strand DNA damage and apoptosis.

There are over 21 anticancer drugs that are known to result in gamma-H2AX formation. As a result, gamma-H2AX is an ideal marker to measure molecular responses to a large number of drugs. While many of these drugs have already received regulatory approval, and are currently being used to manage various types of cancers, they are the subject of ongoing clinical studies to evaluate their efficacy when used alone or in combination with molecularly targeted drugs.

While methods such as Western blots and immunohistochemistry are widely used to detect gamma-H2AX, results are difficult to validate to regulatory standards, and the ELISA method is the most quantifiable and easiest assay to validate.

The new AMS Biotechnology (Abingdon, United Kingdom) pharmacodynamic HT gamma-H2AX assay measures gamma-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences in gamma-H2AX levels in human peripheral blood mononuclear cells, cultured cells, tissue biopsies, and is expected to be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.

The ELISA test utilizes immobilized gamma-H2AX antibodies in the wells of a 96-well plate that capture gamma-H2AX from sample lysates. Incubation with a H2AX detecting antibody, followed by addition of a goat anti-mouse HRP (horseradish peroxidase) conjugate and a chemiluminescent HRP substrate yields relative light units (RLU) that directly correlate with the amount of gamma-H2AX in the sample.

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