Zepto-mole ultra-high sensitivity detection of physiologic protein-protein interactions in the tcr/cd3 multiprotein complex using multiplex immunoprecipitation detected by electrochemiluminescence 4775

Abstract Description Complex protein-protein interaction (PPI) networks orchestrate cellular functions and control the specificity of signal transduction. Distinct PPI network signatures may distinguish disease from healthy conditions. This study focuses on the technical/analytical development and application of an ultrasensitive ELISA plate-based procedure that can be used for PPI analyses from tiny biosamples using a Mesoscale QuickPlex platform, focusing on the T cell antigen receptor (TCR)/CD3 due to its essential role in immunological tolerance and immunity. Our assay demonstrated exceptional sensitivity, enabling the detection of zepto-moles of native multiprotein complexes from physiological sources. Capture reagents across a range of affinities could be employed with highest affinity correlating with highest sensitivity. When TCR/CD3 complexes marked leukemia in a mouse transplant model, these were successfully detected in blood samples from pre-symptomatic mice as early as three days post-tumor injection. In the CD3δ KO mouse model of immune deficiency, our assay determined how the subunit loss was accommodated in the sparse and poorly functional peripheral T cell population. We propose that our assay will allow detailed network analysis, distinguishing qualitative, quantitative, and kinetic variations in multiprotein signaling under different conditions. Such knowledge could substantially impact strategies for pharmacological targeting. Funding Sources R01GM103841 Topic Categories Technological Innovations in Immunology (TECH)