Competitive enzyme-linked immunosorbent assay to quantify hepcidin in horse serum.

Overview

• This study developed and partially validated a competitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of hepcidin, an important iron-regulating hormone, in horse serum.
• The assay demonstrated acceptable precision and accuracy, providing a tool for better understanding iron regulation in horses.

Background

• Hepcidin (Hepc) is a peptide hormone mainly produced by liver cells (hepatocytes).
• It plays a crucial role as the master regulator of iron homeostasis, controlling iron release into the bloodstream.
• Interest in iron metabolism research has increased since the discovery of hepcidin’s role.
• However, there is currently limited information about hepcidin levels in horse serum, which hinders research and potential clinical applications in equine health.

Objective

• To develop a competitive ELISA specifically designed to quantitatively measure hepcidin levels in horse serum.
• To partially validate the assay by evaluating its precision and accuracy.

Methodology

• The newly developed assay was tested using horse serum samples that represented low, medium, and high concentrations of hepcidin.
• Precision was assessed through intra-assay (within the same assay run) and inter-assay (between different assay runs) variability, expressed as coefficients of variation (CVs).
• Accuracy was evaluated by constructing an 8-point standard curve to ensure the assay can accurately measure known concentrations.
• The assay was then applied to analyze serum from 149 healthy horses and 8 diseased foals to assess real-world performance and gain initial data on hepcidin levels in horses.

Results

• Precision findings:
  • Intra-assay CVs were 6.6% (low), 4.5% (medium), and 3.5% (high) hepcidin concentrations indicating low variability within a single assay.
  • Inter-assay CVs were 9.9% (low), 10.4% (medium), and 8.6% (high) indicating moderate and acceptable variability between different assay runs.
• Accuracy was supported by the 8-point standard curve, showing the assay could reliably quantify hepcidin concentrations.
• The average serum hepcidin concentration across the 149 horses was 62.6 ng/mL with a relatively large standard deviation (46.44 ng/mL), reflecting biological variability or assay sensitivity.
• Data from the 8 diseased foals were not detailed but included in the study application phase.

Conclusions and Implications

• The study successfully developed a competitive ELISA suitable for the quantification of serum hepcidin in horses.
• The partial validation showed that the assay has acceptable precision and accuracy metrics, making it a useful tool for equine iron metabolism research.
• The initial measurement of hepcidin levels in a large sample of horses provides valuable baseline data.
• Further research and additional validation are needed to refine the assay performance and expand the understanding of hepcidin dynamics in both healthy and diseased horses.