Acquisition of a Single Molecule Counting Platform for Use in Quantifying Low Abundance Signaling Molecules

Washington University, Saint Louis MO

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT: A variety of plasma hormones, cytokines, and other analytes circulate in the low picogram or femtogram per milliliter range and accurate quantification of these important interorgan signaling factors is technically challenging. Many of these analytes also change dramatically requiring analysis by methods with a wide dynamic range. Moreover, the ease of genetic engineering of laboratory mice has led to this species becoming a critical research tool for studying human disease. However, even terminal blood collection in mice yields insufficient plasma or serum to quantify multiple circulating factors in each mouse using many currently available methods. More sophisticated technology such as bead-based antibody single molecule counting (SMC) is emerging as a superior method for quantifying low abundance and dynamically- regulated analytes in minimal sample volumes. SMC technology couples an immunoassay with a fluorescence- based signal generation and detection system and instrument design minimizes background signal while amplifying the signal from the analyte. This Shared Instrumentation Grant requests funds to purchase the MilliporeSigma SMCxPRO platform that has sensitivity at the fg/mL level, one of the widest dynamic ranges of signal detection currently available, and is adjustable to a variety of very small sample volumes. It is much easier to operate and maintain than the previous model based on the same technology. Moreover, kits are available for a wide variety of analytes and the system is adaptable for custom assays with any pair of validated antibodies, making it highly flexible. The instrument will be sited, operated, maintained, and administered within the intellectual and administrative infrastructure of the Core Laboratory for Clinical Studies (CLCS) at Washington University School of Medicine. This core facility supports clinical and animal research by providing cost-effective laboratory testing for Washington University faculty as well as external investigators and is supported in part by the Washington University Diabetes Research Center. This system will provide a platform for a host of studies aimed at assessing the concentration of low abundance cytokines and hormones involved in endocrine processes, metabolism, inflammation, immune system signaling, and novel mechanisms of interorgan communication conducted across multiple departments within the School of Medicine. Precise measurements of these analytes will be possible using lower volumes of samples than allowed with previously used methods. Thus, acquisition of the MilliporeSigma SMCxPRO Immunoassay system will greatly benefit the extensive community of NIH-funded researchers at Washington University leading to improved understanding of diseases such as diabetes and metabolic syndrome, potentially leading to new therapies and better health outcomes in the future.