Department of Cell Biological Science, Faculty of Advanced Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 001-0021, Japan.
Anal Biochem. 2013 Dec 15;443(2):124-31. doi: 10.1016/j.ab.2013.08.021. Epub 2013 Aug 28.
Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.
小肠隐窝底部的潘氏细胞分泌 α-防御素，有助于先天免疫并塑造肠微生物群的组成。由于缺乏灵敏的测定法来定量腔内分泌的 α-防御素，因此人们努力建立分泌的 α-防御素与疾病之间的关系受到了阻碍。在这里，我们报告了一种用于检测不同来源的鼠潘氏细胞 α-防御素肠肽 4（Crp4）的高灵敏度夹心酶联免疫吸附测定法（ELISA），包括从胃到远端结肠冲洗的腔内容物和粪便颗粒。从分泌 Crp4 特异性 mAb 的 10 个大鼠杂交瘤中选择了一对单克隆抗体（mAb），对其进行了优化，以用于夹心 ELISA 中的 Crp4 检测和特异性。在 CD1 小鼠中，腔内分泌的 Crp4 水平从近端小肠的 6.8±5.2ng/ml 逐渐增加到远端小肠的 54.3±10.3ng/ml，并且该肽在结肠腔和粪便中均被检测到。与野生型对照相比，IL10 基因缺失（IL10null）小鼠的粪便中分泌的 Crp4 显著减少，IL10null 小鼠是炎症性肠病（IBD）的模型。这种 Crp4 夹心 ELISA 可准确测定腔内分泌的 α-防御素作为潘氏细胞功能和肠完整性的生物标志物，可用于多种疾病状态，例如 IBD、传染病、移植物抗宿主病和与肠道微生物失调相关的肥胖症。
