Endoscopy and the use of fecal calprotectin (fecal CP) are among the least favored methods for assessing disease activity by inflammatory bowel disease (IBD) patients; the handling/processing of fecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin ELISA, CPa9-HNE, with the aim of quantifying neutrophil activity and NETosis and proposing a non-invasive method for monitoring disease activity in IBD patients.
In vitro cleavage was performed by mixing calprotectin (S100A9/S100A8) with human neutrophil elastase (HNE), and a novel HNE-derived calprotectin neo-epitope (CPa9-HNE) was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis (UC,
CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were four fold higher in CD (p<0.0001) and UC (p<0.0001) patients than in HS. CPa9-HNE correlated well with the SES-CD score (r=0.61, p<0.0001), MES (r=0.46, p=0.0141), and the full Mayo score (r=0.52, p=0.0013). CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: AUC=0.82 (p=0.0003), UC: AUC=0.87 (p=0.0004). The performance of CPa9-HNE was equipotent or slightly better than that of fecal CP.
Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.
Calprotectin; IBD; biomarkers; inflammation; neutrophil elastase; neutrophil extracellular traps (NETs); neutrophil granulocyte.
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