A novel capillary nano-immunoassay towards rapid, quantitative measurement of cell cycle biomarkers in clinical trials


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Eleanor Tiplady1,Krishna Yalla1,Eilidh McCulloch1,Alan Bilsland1,Kathrin Heinzmann2,Fiona Thomson1
1University of Glasgow,2Cancer Research UK

Abstract

Background

Pharmacodynamic protein biomarkers for cancer treatments are commonly measured by ELISA or immunostaining techniques but suitable antibodies are not always readily available.  Low-abundance targets, scarce samples and the need to use minimally invasive sample procedures can be further complications for assay development with clinical samples. We aim to develop quantitative methods for detecting CyclinB1 and MCM2 (phospho S53), key signalling hubs of cell cycle pathway regulation, using a novel capillary nano-immunoassay method: Simple Western (ProteinSimple).  We will explore the feasibility of applying Simple Western assays for measurement of biomarkers in human samples for the study of novel cell cycle inhibitors in clinical trials.

Method

Cdc7, CyclinB1 and MCM2 (phospho S53) protein levels were measured in lysates from HCT116 colorectal cancer cell line (both untreated and treated with a novel cell-cycle inhibitor) by Simple Western. In brief, protein lysates were denatured as for a normal western blot and plated on a 12-250kDa separation plate (ProteinSimple). This was placed into a ProteinSimple Wes machine, which carried out electrophoresis reactions within specially designed capillaries, allowing accurate estimation of signal intensity for each sample and antibody combination. Results were analysed using Compass (ProteinSimple) and Prism 8 (GraphPad).

Results

Quantitative Wes assays were successfully developed for CyclinB1 and MCM2 (phospho S53), with linear detection in HCT116 lysates at a range of 50-450 µg/ml and 20-100 µg/ml respectively. A ~3-fold increase in CyclinB1 abundance was measured in HCT116 cells in response to a cell-cycle inhibitor.  These biomarkers were detected at low abundance in human PBMCs, and analysis of target expression in other biological matrices from patients is in progress.

Conclusion

Simple Western assays allow highly sensitive and higher-throughput detection of protein biomarkers for clinical trial samples. This new technology has allowed the development of assays for targets that were previously unquantifiable using traditional immunostaining techniques.