Antibody arrays for quantitative measurement of phosphorylation Free

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Intracellular signaling modulates cellular functions and aberrations in signaling are heavily implicated in numerous cancers. Considerable attention has been focused on the development of therapeutic inhibitors that modulate the phosphorylation of important cellular targets. In order to accurately and quantitatively measure the phosphorylation status of target or effector, it is essential to have robust tools that can be broadly applied in the validation of biomarkers and/or the discovery and development of therapeutic compounds. The “sandwich” immunoassay is the preferred format but available assays that measure phosphorylation are limited due to a paucity of sandwich antibody pairs and difficulties in generating appropriate phosphorylated protein standards. We have developed a broadly applicable, immunoassay system for the rapid development of quantitative “sandwich” immunoassays. The approach involves generating capture antibodies to linear peptide epitopes near sites of phosphorylation. The selected epitopes are available to the antibody due to a unique sample preparation method that fragments proteins in the sample prior to analysis. Targeted peptide fragments are captured and an anti-phospho detection antibody is used to complete the “sandwich” and determine level of modification. Internal assay calibration is based on synthetic phospho peptide standards. Our assay system enables highly quantitative, specific measurements of phosphorylation in a multiplexed format. We have demonstrated the feasibility of this approach using the mitogenactivated protein kinase (MAPK) pathway that includes extracellular-regulated proteinkinases (ERK1/2). Both mouse 3T3 cells and human A431 cells grown in culture were stimulated with epidermal growth factor (EGF) to active receptor tyrosine kinases and downstream signaling pathways. A time course of site-specific, quantitative measurement of phosphorylation was shown for several proteins in multiple signaling pathways including the MEK/ERK, PI3K, JNK/SAPK and p38MAPK pathways and compared to unstimulated controls. Assay trends were validated by comparison with traditional Western blots . This approach has been used to specifically measure >30 phosphotyrosine sites on various targets and is being evaluated for the measurement of other phosphoresidues.