Development of a single-molecule, real-time, label-free, mass-resolved immunoassay

Protein-protein interactions govern the biomolecular logic of immunity, signalling, and disease. Elementary rates of association, dissociation, and inhibition underpin our understanding of life processes and remain a limiting factor at the frontier of therapeutic discovery. Yet existing assays infer these processes indirectly through ensemble-averaged signals or molecular labels that obscure native dynamics. Here, we introduce a label-free, real-time, mass-resolved single-molecule immunoassay based on interferometric scattering microscopy (iSCAT) that directly observes individual protein-protein binding events in complex biological samples. By detecting light scattered from single proteins as they bind to an antibody-functionalized surface, we resolve discrete antibody-antigen interactions with single-molecule sensitivity and molecular-weight discrimination. Using IgM as a model system, we demonstrate real-time detection of individual binding events, with measured association rates that scale linearly with concentration over three orders of magnitude. Direct counts of IgM binding events in human serum yield quantitative concentrations that agree with bulk measurements obtained by enzyme-linked immunosorbent assay (ELISA). This bioaffinity iSCAT platform unifies molecular specificity, label-free detection, real-time kinetics, and mass-resolved single-molecule sensitivity, enabling direct access to protein-protein recognition processes and establishing a general framework for quantitative, single-molecule immunoassays.