Abstract
Abstract The detection and study of proteins and their interactions utilizing small samples of cells or tissues is an ongoing problem in cancer research. Recent advances in the early detection of many cancers have resulted in smaller sized tumors for biopsy and evaluation. This has created a need for sensitive and robust assays for pathway related proteins. One mission for the Antibody and Protein Purification Unit (APPU) of the National Cancer Institute has been to evaluate emerging technologies and create new applications for these technologies. Using Acoustic Membrane MicroParticle (AMMP) assays on the ViBE Workstation (Bioscale, Lexington, MA) for solution phase immunoassays, the APPU has developed several applications which support the great utility and breadth of use for this technology in translational research. AMMP assays employ a homogenous, sandwich assay format with paired antibodies, one coupled to a magnetic bead and the second tagged for capture on the sensor, and the analyte forming a complex which interacts with a vibrating membrane sensor. The assay measures the concentration of the analyte by a resonant frequency shift of the membrane caused by the protein interaction – without matrix interference or signal scatter seen in optical techniques. A highly sensitive assay for Prolactin showed no serum interference, which is a common problem in optical immunoassays for circulating peptide hormones. An assay for the important marker C-Met required less than 125 nanograms of cell lysate protein for detection and quantitation. An AMMP assay was further able to quantitate C-Met on the surface of Hep2 cells using an antibody capture method. In an application for detection and quantitation of a member of the MAPK kinase family, an ultrasensitive assay for the detection of MEK1 was developed as well as a highly specific solution phase assay for pMEK1 (S218/S222) as shown by cognate phosphopeptide inhibition. And finally in an application which we believe to be truly unique, a solution heterodimer assay for the ERK1/MEK1 interaction was shown to be susceptible to inhibitor treatment. In conclusion, the AMMP technology was able to perform a wide range of applications required by translational research studies. The assays showed superior lower limit of detection and were reproducible, only requiring nanogram quantities of lysate proteins. The ability to measure protein/protein interactions in solution phase is especially useful for drug development and evaluation of drug effects in targeted therapies. Citation Format: Christopher McAndrew, Christopher Heger, Ashley Saab, W Matthew Dickerson, Paul K. Goldsmith. Development of applications for acoustic membrane microparticle assay technology for translational medicine. [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 3208. doi:10.1158/1538-7445.AM2013-3208
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