Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.
Abstract: To optimize and evaluate methods for the detection of the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces by ELISA. Methods: Healthy horses (n = 28) and horses with intestinal inflammation (n = 10). Methods: Feces were suspended in buffer to create fecal supernatant. Serum and fecal supernatant were analyzed using ELISA kits validated for the detection of MPO and CP in equine serum. Assay validation steps included intra- and interassay variability (coefficient of variation [CV]), dilution linearity, spike recovery, and sample type correlation. Variations in sample handling protocols (centrifugation speed, extraction buffer, and filtration) were evaluated. Results: 17 paired fecal and serum samples were used for initial analysis (10 healthy horses, 7 colitis). Previously reported sample handling protocols resulted in detectable MPO and CP but poor CV, linearity, and spike recovery. There was a linear correlation between serum and fecal samples for CP but not MPO. There was a significant difference between the concentration and CV of alternative sample handling protocols for CP and MPO, with improved CV for CP (2.1% to 18.6%) but not MPO (14.4% to 53.4%). Processing fresh feces with a fecal extraction buffer and filtration of supernatant resulted in the best CV (0.5% to 3.8%) and recovery (45% to 64%) for CP. Detection of MPO was inconsistent regardless of method. Conclusions: There are few reliable diagnostic modalities for inflammation of the equine large colon. Findings support quantification of CP in equine feces using the described ELISA kit and protocol. With additional study to establish reference interval and clinical utility, the fecal inflammatory biomarker CP may allow for noninvasive quantification of intestinal inflammation in horses.
Publication Date: 2024-07-09 PubMed ID: 38984873DOI: 10.2460/ajvr.24.04.0110Google Scholar: Lookup
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Summary
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The research investigates the efficacy of an ELISA-based method for identifying inflammatory biomarkers in horse feces. The results suggest that the ELISA test is effective in quantifying calprotectin (CP), a possible marker for intestinal inflammation in horses.
Research Methodology
- The study involved healthy horses (n = 28) and horses with intestinal inflammation (n = 10).
- The research aimed to detect the inflammatory biomarkers myeloperoxidase (MPO) and calprotectin (CP) in equine feces using enzyme-linked immunosorbent assay (ELISA) kits that have been validated for their detection in equine serum.
- Feces from the horses were suspended in a buffer to create a fecal supernatant, which, together with the serum, was analyzed using the ELISA kit.
- Different aspects of the assay were tested, including intra- and interassay variability, dilution linearity, spike recovery, and sample type correlation.
- Various sample handling protocols involving changes in centrifugation speed, extraction buffer, and filtration were examined.
Key Findings
- The study found a correlation between serum and fecal samples for CP but not for MPO.
- While previous sample handling protocols did result in detectable MPO and CP, they also displayed poor coefficients of variation (CV), linearity, and spike recovery.
- Different sample handling protocols resulted in significant differences in the concentration and CV for both CP and MPO.
- The best results were obtained when fresh feces were processed with a fecal extraction buffer and then the supernatant was filtered. Using this method, the CV and recovery for CP was greatly improved.
- The detection of MPO was found to be inconsistent, regardless of the handling method used.
Conclusion
- The study concludes that currently, reliable testing methods for inflammation in the equine large colon are few.
- The findings of the research suggest that the described ELISA kit and protocol are effective for quantifying CP in equine feces.
- With further study to establish a reference interval and prove clinical utility, the research implies that the fecal inflammatory biomarker CP could be used for noninvasive quantification of intestinal inflammation in horses.
Cite This Article
APA
Bishop RC, Graham SM, Connolly SL, Wilkins PA, McCoy AM.
(2024).
Systematic evaluation supports the use of ELISA for quantification of calprotectin in equine feces, a first step toward noninvasive quantification of intestinal inflammation in horses.
Am J Vet Res, 1-10.
https://doi.org/10.2460/ajvr.24.04.0110 Publication
Researcher Affiliations
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