Attomolar fecal cytokine profiling reveals gut immune dynamics and disease states.

The gut modulates systemic health, influencing immune, neurological, and cardiovascular processes. While fecal sequencing of microbial nucleic acids provides a non-invasive view of microbial composition, sensitive measurement of host-derived signals in stool remains limited. Here we introduce DIGEST (Digital Immunoassay for Gut-Environment Single-molecule Targets), an ultrasensitive digital immunoassay that quantifies proteins in fecal extracts to attomolar levels. In mice, longitudinal profiling during a high-fat diet perturbation revealed coordinated host cytokine responses that occurred within 24 hours, with sustained elevation after diet withdrawal, enabling non-invasive tracking of within-subject immune dynamics. Application of DIGEST to quantify a panel of host inflammatory cytokines in patients with inflammatory bowel disease distinguished active ulcerative colitis from quiescent disease and non-IBD controls (AUC=0.98). In advanced melanoma patients receiving PD-1 blockade, pretreatment fecal IL-23 concentrations discriminated responders from non-responders with an AUC of 0.87. Together, these results establish DIGEST as a generalizable platform for sensitive, non-invasive quantification of host protein activity at the gut interface, with broad applications in basic science discovery, disease surveillance, and therapy response prediction.