Investigation of peptide cross reactivity in equine plasma using two adrenocorticotropic hormone immunoassays.

Overview

• This study investigates how two different immunoassays measure adrenocorticotropic hormone (ACTH) levels in horse plasma, focusing on whether other similar peptides interfere (cross-react) with these measurements.
• The research evaluates the accuracy and cross-reactivity of the Siemens Immulite 2000xpi chemiluminescent assay (CLA) and the Tosoh AIA-900 immunofluorescent assay (IFA) when detecting ACTH and related peptides.

Background

• Adrenocorticotropic hormone (ACTH) is an important biomarker for diagnosing endocrine disorders in horses, such as pituitary pars intermedia dysfunction (PPID).
• Immunoassays are commonly used to measure plasma ACTH concentrations, but other peptides structurally similar to ACTH might cross-react, causing inaccurate readings.
• The two immunoassays tested are widely used but may differ in how they detect ACTH and related peptides, influencing clinical interpretation.

Objective

• To define and quantify cross-reactivity of ACTH-related peptides in equine plasma when assayed by two different ACTH immunoassays.
• Specifically, to assess how the assays respond to exogenous peptides: human ACTH, corticotropin-like intermediate lobe peptide (CLIP), and corticotropin inhibiting peptide (CIP).

Methods

• Equine plasma samples were prepared and spiked with known concentrations of the three different manufactured peptides: human ACTH, CLIP, and CIP.
• Samples were tested in duplicate for each peptide using both:
• Siemens Immulite 2000xpi chemiluminescent assay (CLA)
• Tosoh AIA-900 immunofluorescent assay (IFA)
• Measurement results from both assays were compared against the actual peptide concentrations spiked into plasma.

Key Findings

• Both assays detected human ACTH, but the immunofluorescent assay (IFA) consistently reported higher values than the chemiluminescent assay (CLA):
• IFA measured ACTH at average 132% of the actual concentration.
• CLA measured ACTH at average 104% of the actual concentration.
• For the peptides related to ACTH:
• CLIP was detected by the CLA assay but not by the IFA, with about 29% detection of actual concentration by CLA.
• CIP was detected by the IFA assay but not the CLA, at about 65% of actual concentration in IFA.
• This indicates that the assays have differing specificities, detecting some cross-reactive peptides differently.
• The assays may overestimate actual ACTH levels due to cross-reactivity, potentially affecting clinical diagnosis.

Implications

• Results suggest careful interpretation of ACTH levels in equine plasma is needed because immunoassay readings might be inflated by related peptides.
• Differences between the two assays mean that diagnostic thresholds and clinical decisions may vary depending on the assay used.
• Further research is needed to understand how these findings affect diagnosis and monitoring of equine endocrine diseases like PPID.
• Clinicians should consider assay-specific cross-reactivity when evaluating ACTH results to avoid misdiagnosis.

Conclusion

• The study demonstrates that peptide cross-reactivity occurs in measuring equine plasma ACTH levels with commonly used immunoassays.
• The Siemens CLA and Tosoh IFA assays differ in sensitivity to ACTH and related peptides, potentially leading to overestimation of ACTH concentration.
• Understanding these differences is important to refine diagnostic accuracy and improve clinical care for horses with endocrine disorders.