Abstract
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.
Highlights
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies
Our study presents the multiplex quantification of inflammatory proteins in stool using ultra-high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode
We used the correlation between feces fresh weight (FW) and the total protein content measured by BCA to determine the amount of stool collected on a swab (Figure S1 and Table S3)
Summary
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies. Inflammatory bowel disease (IBD) and food allergies are a highly prevalent and diverse group of intestinal disorders, potentially resulting in chronic inflammation of the gastrointestinal tract (e.g., Crohn’s disease, ulcerative colitis, and celiac diseases). Name Alpha-1-antitrypsin isoform 1 Immunoglobulin heavy constant alpha 1 Immunoglobulin heavy constant alpha 1 and 2 Immunoglobulin heavy constant alpha 2 Eosinophil cationic protein Eosinophil-derived neurotoxin Myeloperoxidase Calprotectin 1
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