Initial Validation of Cytokine Measurement by ELISA in Canine Feces

Abstract

Measurement of fecal cytokines has been used as a marker of intestinal inflammation in people and correlates with endoscopic findings. The aim of this study was to evaluate the use of canine-specific enzyme-linked immunosorbant assays (ELISAs) for quantification of cytokines in canine fecal samples as a non-invasive biomarker. Interleukin (IL)-6,-8,-10,-23/12p40 and TNF- were assessed by using spiked fecal samples from 3 healthy dogs. Standard curve validation was performed, and the impact of time to freeze, duration of storage and number of freeze-thaw cycles on cytokine concentration were also examined. All the cytokines assayed could be detected, with varying accuracy. The mean coefficient of variation (CV) for all standard curves ranged from 2.95%-9.8%. The mean intra-assay CV ranged from 3.1%-11.14%, and inter-assay CV from 4.36%-18.83%. Recovery of IL-23 was poor (7.23%-17.12%), precluding further interpretation of stability studies. Mean recovery did not appear to be affected by time to freeze and repeat freeze-thaw cycles in all cytokines investigated. Recovery for all cytokines after short-term storage of 30 days at −80˚C showed a recovery of <70% or >130%. In conclusion, although fecal IL-6,-8,-10, and TNF- could be used as biomarkers of intestinal inflammation in the dog, the quality of laboratory performance and poor recovery at lower concentrations limit their application. Bench-top and freeze-thaw stability was acceptable, and samples should ideally be analyzed within a week. Investigation involving dogs with acute and chronic inflammatory intestinal disease is required to determine the role of this methodology in a clinical setting.

Galectin-3 Regulates Indoleamine-2,3-dioxygenase-Dependent Cross-Talk between Colon-Infiltrating Dendritic Cells and T Regulatory Cells and May Represent a Valuable Biomarker for Monitoring the Progression of Ulcerative Colitis

“…Blood and fecal samples were prepared as previously described [14]. IDO activity was determined by spectrophotometric assay for KYN in the serum and fecal samples of patients with UC and healthy controls [15].…”

Indoleamine 2,3-dioxygenase-dependent expansion of T-regulatory cells maintains mucosal healing in ulcerative colitis

“…Blood and fecal samples were prepared as previously described. 21 The serums and supernatant fluids were collected and stored at −80°C until they were measured by enzyme-linked immunosorbent assay (ELISA). IDO activity was determined by spectrophotometric assay for kynurenine in the serum and fecal samples of patients with UC and healthy controls.…”

Fecal Galectin-3: A New Promising Biomarker for Severity and Progression of Colorectal Carcinoma

“…Stools (1–10 g) were collected in sterile containers and weighed. They were divided into 1 g aliquot and then emulsified in 5 mL of protease inhibitor cocktail (SIGMA, P83401), diluted 1 : 100, and centrifuged for 5 minutes at 400 g , at 4°C, as previously described [ 28 , 29 ]. The supernatant fluid was collected and stored at −80°C until ELISA.…”