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Bioluminescent Protease Assays

(Developmental Center-Driven Research Project)

Stanford: R Shinde, CH Contag; 

UCD: SW Kang, K Lam; LLNL: AL Gryshuk, J Perkins

This project focuses on detecting picogram levels of active proteases in biofluids as indicators of disease states. Characterizing and quantifying ultra-low levels of protease activity will create new opportunities for the diagnosis and staging of cancer. It will enable early disease detection and assessment to dramatically effect therapeutic outcome. Biofluids (serum/plasma, urine, saliva etc.) do not contain genome or transcriptome data. Since they interface with tissues, these fluids may hold information pertaining to disease states. Proteases are part of the serum proteome and are associated with pathologic conditions (cancer, inflammation, infection and cardiovascular disease). Serum protease profiling using sensitive assays could potentially provide diagnostic and prognostic data. Since bioluminescence does not require excitation, and has a high signal-to-noise compared to fluorescence and other techniques, it is possible to detect extremely low levels of active proteases. We are exploiting this sensitivity to probe low levels of active proteases in serum and correlate them with disease state. The goal therefore is to develop a sensitive assay to detect serum-based active protease with a PSA prostate cancer assay serving as the initial model protease target.

We have shown this past year that bioluminescence can be used to detect enzyme activity at pg/ml levels. In addition, we have demonstrated the capability of detecting PSA concentrations at BioProt.jpgthe 100 picogram level in human serum (see figure). We also demonstrated that the active PSA sub-population can be determined in human plasma (female) by spiking the biofluid with varying active PSA concentrations. The peptide sequence used in the bioluminescence assay has lower enzyme turnover and higher specificity. Optimizing the sequence will allow for detection of lower amounts of PSA in the serum.