A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Abstract

Paneth cells at the base of small intestinal crypts secrete -defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish relationship between secreted -defensins and disease have been hampered by a lack of sensitive assays to quantify luminal -defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell -defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon, as well as 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 gradually increased from 6.8 ± 5.2 ng/ml in proximal 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 enable accurate determinations of luminal -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.

Decreased secretion of Paneth cell α‐defensins in graft‐versus‐host disease

“…Fecal samples were prepared as previously described . In brief, samples were air‐dried, powdered using a bead beater‐type homogenizer (Beads Crusher μT‐12; TAITEC, Saitama, Japan), and vortex mixed with phosphate buffered saline for 1 h at 4°C.…”

“…In the sandwich ELISA, we used 2 anti‐Crp1 monoclonal antibodies, designated 77‐R5 and 77‐R20, generated following immunization of rats with synthetic oxidized Crp1. The sandwich ELISA was carried out as previously described . In brief, 100 μL of samples were added to the wells coated with Crp1 monoclonal antibody (77R‐5, 1 μg/mL) and incubated at 25°C for 2 h. After washing, 100 μL of 0.5 μg/mL biotinylated detection antibody (77R‐20, 0.5 μg/mL) was added at 25°C for 1 h. Subsequently, the wells were incubated with 100 μL of Streptavidin‐HRP conjugate (GE Healthcare Biosciences, Piscataway, New Jersey, USA) at 25°C for 1 h. After final washes, 100 μL of TMB chromogen substrate buffer was added and incubated at 25°C for 30 min.…”

“…However, it is not easy to evaluate Paneth cell numbers or composition of intestinal flora in fecal samples. We have recently developed a highly sensitive sandwich enzyme‐linked immunosorbent assay (ELISA) for the mouse Paneth cell α‐defensin cryptdin (Crp) in fecal pellets . It is thus important to determine whether fecal levels of defensins could be surrogate markers of Paneth cell loss and dysbiosis.…”

Analysis of Serotonin in Human Feces Using Solid Phase Extraction and Column-Switching LC-MS/MS

“…Fecal samples, collected in a dedicated container with a lid, were rst stirred, dispensed, and frozen at −80°C, then lyophilized and powdered. 10) Most were lyophilized by a freeze dryer FDU-2110 (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and powdered using a crusher Multi Beads Shocker ® MB 2000 (Yasui Kikai Corporation, Osaka, Japan), then stored at −80°C. A portion of each sample was lyophilized and powdered by TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan), and used for the quanti cation of serotonin.…”

“…ese studies have mainly focused on the composition and function of gut microbiota and analyses of substances derived from the hosts in feces have also been reported. For example, a quantitative analytical method using a sandwich enzyme-linked immunosorbent assay for α-defensins, which are produced by Paneth cells and are thought to regulate the growth of gut microbiota, has been developed, 10) and studies concerning the involvement of α-defensins in diseases and health have been reported. 11,12) e "brain-gut interaction" in which the brain and gut a ect each other is important because of its implications in various diseases.…”