Fig 1: Ex vivo cultures of neutrophil granulocytes demonstrating CPa9-HNE biomarker level fold-change compared with inactive neutrophils, reflecting true neutrophil activity. CPa9-HNE biomarker levels after A] 4 h of cultivation with neutrophil agonists B] and 24 h of cultivation; MRP8/14 biomarker measurements after C] 4 h of cultivation with neutrophil agonists D] and 24 h of cultivation. Definition: negative control [only tissue], S. aureus [tissue + Staphylococcus aureus], inactive neutrophils [tissue + neutrophils], neutrophils + S. aureus [tissue + neutrophils + Staphylococcus aureus], neutrophils], neutrophils + PMA [tissue + neutrophils + phorbol myristate acetate], neutrophils + Cal iono [tissue + neutrophils + calcium ionophore]. Error bars represent the standard error of the mean [SEM].
Fig 2: Biomarker levels of CPa9-HNE, faecal CP, and MRP8/14 serum calprotectin stratified according to endoscopic disease severity based on the A-C] SES-CD for Crohn’s disease [n = 54], D-F], full Mayo score for ulcerative colitis [n = 43], and G-I] MES score for ulcerative colitis [n = 30]. Data are depicted as interquartile range [IQR] with 10–90 percentile. Statistical differences were calculated using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. CP, calptotectin; ANOVA, analysis of variance; SES-CD, Simple Endoscopic Score for Crohn’s Disease; MES, Mayo Endoscopic Score.
Fig 3: Biomarker levels of CPa9-HNE in Crohn’s disease [CD: n = 54] and ulcerative colitis [UC: n = 43] vs. healthy subjects [HS: n = 23]. A-C] CPa9-HNE biomarker and D-F] MRP8/14 biomarker were quantified in serum from ulcerative colitis and Crohn’s disease and compared with those of the healthy subjects. Data are depicted as interquartile range [IQR] with 10–90 percentile. Asterisks [*] depict significant differences between HS, CD, and UC patients, calculated using one-way ANOVA, **p <0.01. ***p <0.001, ****p <0.0001. ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance.
Fig 4: Overview of the calprotectin sequence of both A] S100A9 and B] S100A8 dimers. The calprotectin fragments generated by human neutrophil elastase [HNE]-mediated cleavage and selected for ELISA development are depicted by an arrow down [?], where the neo-epitope selected for ELISA development is depicted by the color grey. Relative peptide abundance of selected HNE-derived calprotectin neo-epitope cleavage fragments; antibody specificity test and biological evaluation of the CPa9-HNE ELISA. C] The relative peptide abundance for the CPa9-HNE sequence was tested using mass spectrometry in samples containing HNE-cleaved calprotectin, full-length calprotectin, or HNE. Furthermore, D] antibody specificity was tested against the selection peptide, elongated peptide, truncated peptide, non-sense peptide, and finally E] biological evaluation of the CPa9-HNE assay was tested in samples containing HNE + calprotectin, calprotectin [Control 1], and HNE [Control 2]. Error bars represent the standard error of the mean [SEM]. ELISA, enzyme-linked immunosorbent assay.
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