Simultaneous quantitative profiling of clinically relevant immune markers in neonatal stool swabs to reveal inflammation

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.生命早期出现的异常免疫应答，可能诱发炎症性肠病（Inflammatory Bowel Disease, IBD）与食物过敏（如乳糜泻，Celiac Disease）。粪便免疫标志物水平可对炎症应答（如食物过敏、自身免疫反应）进行分类，且此类应答与初始微生物定植过程同步发生。免疫亲和检测（immunoaffinity assays）通常被用于定量血液/血清中的循环免疫蛋白标志物。然而，目前尚无可靠的多重检测方法可用于定量粪便中的免疫蛋白水平。本研究开发了质谱检测方法，可同时对134份新生儿粪便拭子样本中的粪便钙卫蛋白（calprotectin）、髓过氧化物酶（myeloperoxidase）、嗜酸性粒细胞衍生神经毒素（eosinophil-derived neurotoxin）、嗜酸性粒细胞阳离子蛋白（eosinophil cationic protein）、α1抗胰蛋白酶1（alpha 1 antitrypsin 1）以及适应性免疫效应因子（adaptive immunity effectors）进行定量检测。本研究优化了样本提取与蛋白水解流程，并从响应线性（>100，通常为0.04~14.77 µg/mg总蛋白）、决定系数（coefficient of determination, R²>0.99）、检测限（limit of detection, LOD：0.003~0.04 µg/mg总蛋白）、定量限（limit of quantification, LOQ：0.009~0.122 µg/mg总蛋白）以及稳健性等方面对该多重检测方法进行了验证。日内精密度与日间精密度的中位数变异系数（CV）分别为9.8%与14.1%。本研究通过定量母乳来源的IGHA2，以区分胎便（meconium）与粪便样本，并检测首次食物摄入情况。对生命早期免疫标志物进行谱型分析，可反映肠道稳态失衡，或可用于炎症性肠病与食物过敏的症状前干预。