Abstract 5159: Quantitative immunoassays to measure total Akt-1 and phospho-Akt(Ser473) in cell and tissue lysate models.
Authors: Ellington Allison , Webb Seamus , Miller Thomas , Eason Paula , Dzantiev Leonid , Ranganathan Sripriya , Schaefer Laura , Oberoi Pankaj , Wohlstadter Jacob N. 
Published: August 2014

Abstract
Akt1, Akt2, and Akt3 kinases are critical effectors of the activated tyrosine receptor kinase/phosphoinositol 3-kinases, influencing cell growth, proliferation, and survival. Akt1-specific substrates have been implicated in cell cycle progression and cell motility, processes that underlie increased proliferation and metastatic spread of cancer cells. In this study, we report development and analytical validation of singleplex immunoassays for quantification of total Akt1 and phospho-Akt(Ser473) using fit-for-purpose and CLSI principles. We also demonstrate the ability of these assays to quantify total Akt1 and phospho-Akt(Ser473) in tumor-derived cell lines and mouse xenograft tumor lysates. These assays allow cancer researchers to correlate characteristics of cancer cells with absolute protein levels.
The Akt1 singleplex assays were developed using MSD's MULTI-ARRAY® technology. A monoclonal mouse antibody specific for total Akt1 was used to capture Akt1 in the solid phase, and MSD SULFO-TAGTM-labeled antibodies specific for an alternate epitope of total Akt1 or phospho-Akt(Ser473) were used as assay-specific reporters. Both assays were calibrated using full length recombinant human Akt1 protein expressed in baculovirus and phosphorylated in vitro by sequential incubation with phosphoinositide-dependent protein kinase-1 (PDK1) and MAP kinase-activated protein kinase 2 (MAPKAPK2). Akt calibrator concentrations were assigned following multi-day testing. Akt1 concentrations were confirmed by amino acid analyses, and units of phosphorylation (UP473) were assigned. The functional performance of the Akt1 calibrator was verified to be equivalent across three lots.
Both assays demonstrated excellent sensitivity (lower limits of detection were 0.028 pg total Akt/well for the total Akt1 assay and 0.005 UP473/well for the phospho-Akt(Ser473) assay) and inter-plate reproducibility (coefficients of variation were below 12.1% for both assays). Calibration with recombinant protein enabled the absolute quantification of both total and phospho-Akt(Ser473) levels in cultured cell lysates, including MCF-7, Jurkat, NIH3T3, rat L6, and COS7, as well as epithelial, renal cell, and kidney carcinomas. Dilutional linearity and spike recovery testing demonstrated minimal matrix effects and accurate quantitation in these samples. (Recovery values in the range of 80-120% of the expected values were observed.) The assays showed no significant cross-reactivity with Akt2 or Akt3 or with the active forms phospho-Akt(Ser472) and phospho-Akt(Ser474).
These assays can accurately quantify total Akt1 and phospho-Akt(Ser473) in multiple cell models.
Citation Format: Allison Ellington, Seamus Webb, Thomas Miller, Paula Eason, Leonid Dzantiev, Sripriya Ranganathan, Laura Schaefer, Pankaj Oberoi, Jacob N. Wohlstadter. Quantitative immunoassays to measure total Akt-1 and phospho-Akt(Ser473) in cell and tissue lysate models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2013-5159