Abstract 6293: Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins

Abstract

Abstract The PI3K-Akt signaling pathway modulates cell growth, survival and apoptosis, and this pathway is frequently altered in human cancers, contributing resistance to radiation and chemotherapy treatment. Akt is a target for specific inhibition and recently, a number of small molecules have been developed to improve the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2, and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of the Multiple Detection Approach to characterize the PI3K-Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immunoassays or immunoassay with total protein on the Simple Western platform using chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight via capillary electrophoresis, followed by immobilization via UV-crosslinking. Next, PI3K-Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies, to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe were removed after the detection step with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading, and/or other systematic errors. These results present the utility of the Multiple Detection Approach to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies. Citation Format: Irina G. Kazakova, Crystal Tran, Anita Silver, Jessica Dermody. Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6293.