A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin
A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin
复制标题
DOI：
10.1016/j.ab.2013.08.021
复制
发表时间：
2013-12-15
影响因子：
2.9
通讯作者：
Ayabe, Tokiyoshi
中科院分区：
文献类型：
--
作者：
Nakamura, Kiminori;Sakuragi, Naoya;Ayabe, Tokiyoshi
小肠隐窝底部的Paneth细胞分泌α -防御素，这有助于先天免疫和塑造肠道微生物群的组成。建立分泌的α -防御素与疾病之间关系的努力一直受到缺乏敏感的测定方法来量化腔内α -防御素的阻碍。在这里，我们报告了一种高灵敏度的三明治酶联免疫吸附试验（ELISA），用于不同来源的小鼠Paneth细胞α -防御素-4 (Crp4)，包括从胃冲洗到远端结肠和粪便颗粒的管腔内容物。从10个分泌Crp4特异性单克隆抗体的大鼠杂交瘤中选择一对单克隆抗体（mab），优化其在夹心ELISA中检测和特异性Crp4。CD1小鼠肠内Crp4水平从近端小肠的6.8 +/- 5.2 ng/ml逐渐升高到远端小肠的54.3 +/- 10.3 ng/ml，并在结肠管腔和粪便中检测到该肽。与野生型对照相比，IL10缺失小鼠（炎症性肠病（IBD）模型）粪便中分泌的Crp4显著减少。这种Crp4夹心ELISA能够准确测定肠道α -防御素作为多种疾病状态下Paneth细胞功能和肠道完整性的生物标志物，如IBD、传染病、移植物抗宿主病和与肠道微生物群失调相关的肥胖。(C) 2013爱思唯尔公司版权所有。
Paneth cells at the base of small intestinal crypts secrete alpha-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted alpha-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal alpha-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell alpha-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 alpha-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. (C) 2013 Elsevier Inc. All rights reserved.