A52

Development of MULTI-ARRAY and MULTI-SPOT immunoassays for cell cycle targets

Ilis Davydov, Chun-Jiang Zhang, Laura Schaefer, Jenny Ly, Paula Eason, John Kenten, Robert Umek, Jacob Wholstadter

Meso Scale Discovery, Gaithersburg, USA

The cell cycle is controlled by an ordered set of interactions among signaling molecules and structural proteins. Deregulation of the cell cycle can result in an uncontrolled increase in cell divisions, a hallmark of carcinogenesis. Many molecules involved in cell cycle regulation can serve as biomarkers for cancer progression and for effects of anti-cancer therapies. We developed highly sensitive electrochemiluminescence immunoassays for the detection of post-translational modifications of several key proteins involved in cell proliferation phospho-Histone H3 (Ser10), phospho-Rb (Ser608), phospho-Rb (Ser780), phospho-Aurora A (Thr288), phospho-p53 (Ser15), ubiquitinated p53 and ubiquitinated MDM2. We also present an assay to detect DNA-binding activity of p53 in nuclear extracts. The assays employ Meso Scale Discoverys patterned array technology in 96-well MULTI-SPOT plates. In many cases we demonstrate parallel detection of the post-translationally modified and total protein pools in the same well. The assays are sensitive, quantitative, involve simple protocols, and results obtained from treated and untreated cells agree with those obtained by traditional Western blot analysis.