Abstract 3346: Development of a multiplex screening panel for Akt signaling pathway biomarkers in cell and tissue lysate models

• Published in: Cancer research (Chicago, Ill.) Ill.), 2014-10, Vol.74 (19_Supplement), p.3346-3346
• Main Authors: Miller, Thomas W., Tressler, Karen, Dunty, Jill, Eason, Paula, Dzantiev, Leonid, Ranganathan, Sripriya, Schaefer, Laura, Stewart, David, Oberoi, Pankaj, Wohlstadter, Jacob
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2014-3346

Major challenges face the large-scale implementation of intracellular tumor biomarkers for clinical diagnosis and therapeutic development. These challenges include non-quantitative results, insufficient assay sensitivity, and lack of multiplexing capability. For many studies, sample volumes are limited (e.g., tumor lysates); however, it is essential to extract as much biomarker information as possible from a given sample while maintaining the quality and consistency necessary to sustain ongoing studies. To address these challenges, MSD® has developed multiplex panels for assaying cell signaling biomarkers using fit-for-purpose methods that emphasize optimal multiplex combinations and rigorous development of critical reagents. Here we report the development and verification of a multiplex screening panel of immunoassays for simultaneous measurement of total and phosphorylated analytes of the Akt signalling pathway using MSD technology. Markers in the panel include phosphorylated and total forms of GSK3B, p70S6K, FOXO3a, PTEN, Akt, S6RP, PRAS40, and ERK1. We demonstrate the ability of these assays to measure analyte levels in multiple tumor-derived cell lines and human tissue samples (normal and tumor) with excellent sensitivity and performance. Most analytes can be quantified using no more than 10 µg of sample. By using recombinant proteins to calibrate some of the assays, we were able to quantify analytes in cultured cell lysates, including MCF-7 and Jurkat T-cells. Lack of analyte specificity is a well-known issue when multiplexing intracellular signaling analytes, especially those in a common signaling pathway such as the Akt pathway. Through rigorous optimization, we achieved non-specific binding less than or equal to 1% for all analytes in this panel. Spike recovery and dilution linearity were characterized to demonstrate matrix tolerance in tumor cell line and tissue lysates as well as dynamic ranges between 3 and 4 logs, allowing quantification of analytes at different abundance levels without using multiple dilutions. In conclusion, MSD MULTI-SPOT® Akt signaling assay panels have been developed and verified for measurement of intracellular tumor biomarkers of relevance to clinical diagnosis, therapeutic development, and treatment of various cancers. The accuracy, reliability, ease of use, and high-throughput features of these multiplex assays make them ideally suited for use in large-scale clinical studies. Citation Format: Thomas W. Miller, Karen T