P017 An activity-based chemiluminescence assay targeting granzyme A for the detection of Inflammatory Bowel Diseases

Abstract

Abstract Background Inflammatory Bowel Diseases (IBD) represent a pressing global health concern lacking precise diagnostic tools for accurate patient stratification. Conventional methods, such as colonoscopy are extremely invasive whilst generic inflammatory markers (such as CRP or Calprotectin), are not specific enough and pose limitations in assessing disease activity, urging the need for novel, non-invasive diagnostic approaches which can rapidly inform clinicians on the inflammatory status of a patients bowels. Methods Our study focused on developing a non-invasive chemiluminescence assay targeting active granzyme A (GzmA) in stool supernatants. Employing a multifaceted approach encompassing functional, chemical, and imaging assays on human cells and gut tissue biopsies, we discerned elevated GzmA levels predominantly secreted by CD8+ T cells in inflamed gut tissues. Results The design of GzmA-INH, a specific inhibitor for GzmA, underscored the prevalence of active extracellular GzmA and it’s role in driving inflammation in the guts - revealing promising potential as a novel IBD biomarker. Further, we synthesized GzmA-CL, a highly reactive and selective chemiluminescent reporter for GzmA. Integrating GzmA-CL into a robust antibody-coated 96-well plate assay (Figure 1a), we analyzed 30 IBD biosamples along with healthy controls. Notably, substantially higher GzmA levels were observed in IBD patients, with discernible distinctions between ulcerative colitis (UC) and Crohn’s disease (CD) (Figure 1b). These levels correlated positively with established biomarkers, including faecal calprotectin and immune cell counts. Conclusion Our investigation highlighted active extracellular GzmA as a noteworthy biomarker in gut inflammation. The rapid, selective, and sensitive nature of our chemiluminescence assay presents promising prospects for precise IBD diagnosis and advancements in personalized medicine.