2014 AAPS, SAN DIEGO, CA

Michael Tanen, Russell Weiner, Omar Laterza
Molecular Biomarkers & Diagnostics, Merck Research Laboratories, Rahway NJ 07065

Purpose:  

Quanterix’ single molecule array Simoa technology is a fully automated immunoassay platform that utilizes arrays of femtoliter sized reaction chambers that can isolate and detect single molecules bound to paramagnetic beads. This method can measure proteins in the femtomolar range, up to 1000-fold improvement in sensitivity over traditional ELISA. Here we describe the development and optimization of a cytokine assay using the Quanterix platform.

Method:  

The Quanterix platform is based on the use of capture antibodies coupled to paramagnetic beads and biotinylated detection antibodies, which in turn are detected by the action of a reporter enzyme: streptavidin β-galactosidase (SβG) and resorufin β-D-galactopyranoside as the fluorescence substrate. We executed a 2-variable design of experiments (DOE) strategy experiment in which multiple concentrations of detection antibody and SβG were tested. These are the 2 most critical variables to optimizing Simoa assays.

Results:  

Under the optimal conditions identified (0.6 μg/mL detection antibody and 150 pM SβG), we were able to achieve an LOD of 1.4 fg/mL. This represents a 150-fold increase in sensitivity as compared to a current ultrasensitive assay developed on the Mesoscale Discovery platform.

Conclusions:  

assays, with homebrew capabilities that allow for the use of proprietary reagents and control of the assay development and optimization process. The fully automated platform allows for rapid development time and efficiencies in sample analysis.