Related Experiment Video
Updated: Aug 1, 2025
Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems
Published on: August 23, 2019
Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term
Sandra F Gomes1,2,3, Francisco Jorge Melo1,3, Rita Silva4
1Department of Biomedicine, Unit of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal.
Objectives:
This study assesses the clinical relevance of dipeptidyl peptidase 4 (DPP4) membrane exopeptidase as a biomarker of inflammatory bowel disease (IBD). A spike-and-recovery approach of DPP4 in fecal samples was used to compare two different methods for protein extraction, followed by a stability assessment.
Methods:
Fecal samples of healthy volunteers spiked with known concentrations of recombinant DPP4 were processed using a standard manual extraction protocol and the CALEX® protocol. The two methods were compared by quantification of fecal DPP4 by ELISA, followed by Bland-Altman analysis. For the stability assays DPP4 was extracted from fecal samples and stored under different conditions of temperature and time after collection.
Results:
In general, the levels of spiked DPP4 in stool samples were lower with the manual protocol than in those obtained with the CALEX® method; this trend was corroborated by Bland-Altman analysis. Nonetheless, variability was within the acceptable limits for both protocols. In the stability assessment, no statistically significant differences were found between the results obtained under the different storage conditions.
Conclusions:
Both manual and CALEX® protocols provided equal extraction ability of DPP4 from stool samples. In addition, DPP4 provided flexibility in terms of sample storage enabling the accurate assessment of samples delivered up to a week before analysis.
Insights
Dipeptidyl peptidase 4 (DPP4) is a potential biomarker for inflammatory bowel disease (IBD). Both manual and CALEX® extraction methods effectively quantify fecal DPP4, with stable results even after a week of storage.
Area of Science:
- Biochemistry
- Gastroenterology
- Clinical Diagnostics
Background:
- Inflammatory bowel disease (IBD) diagnosis and monitoring require reliable biomarkers.
- Dipeptidyl peptidase 4 (DPP4), a membrane exopeptidase, is being investigated as a potential IBD biomarker.
- Standardized methods for quantifying fecal DPP4 are crucial for its clinical application.
Purpose of the Study:
- To evaluate the clinical relevance of dipeptidyl peptidase 4 (DPP4) as an inflammatory bowel disease (IBD) biomarker.
- To compare the efficiency and reliability of two distinct protein extraction methods (manual vs. CALEX®) for fecal DPP4 quantification.
- To assess the stability of fecal DPP4 under various storage conditions.
Main Methods:
- A spike-and-recovery approach using recombinant DPP4 in fecal samples.
- Comparison of manual protein extraction versus the CALEX® protocol.
- Quantification of DPP4 via ELISA and statistical analysis using Bland-Altman plots.
- Evaluation of DPP4 stability after storage at different temperatures and time points.
Main Results:
- Both manual and CALEX® protocols demonstrated comparable extraction capabilities for fecal DPP4.
- While the CALEX® method generally yielded slightly higher DPP4 levels, variability was within acceptable limits for both.
- No significant differences in DPP4 levels were observed across various storage conditions, indicating good sample stability.
Conclusions:
- Both manual and CALEX® protocols are suitable for extracting DPP4 from stool samples.
- DPP4 quantification is robust, with samples remaining stable for up to a week before analysis.
- These findings support the potential of DPP4 as a stable and accessible biomarker for IBD.
