Evaluation of an Automated Fluorescence Enzyme Immunoassay for Quantification of Equine Insulin and Comparison to Five Other Immunoassays

Rose D. Nolen‐Walston; Jeaneen C. Kulp; Darko Stefanovski; Andrew W. vanEps

Evaluation of an Automated Fluorescence Enzyme Immunoassay for Quantification of Equine Insulin and Comparison to Five Other Immunoassays

2025

Rose D. Nolen‐Walston | Jeaneen C. Kulp | Darko Stefanovski | Andrew W. vanEps

ABSTRACT Background Hyperinsulinemia is an important and treatable risk factor for laminitis in horses. Objectives Evaluate the Tosoh AIA‐360 automated fluorescence enzyme immunoassay for the measurement of serum insulin concentrations in horses, and compare it to five other immunoassays for insulin quantification. Animals One hundred serum samples from 83 horses were submitted for insulin measurement. Methods The Tosoh AIA‐360 was assessed against a reference assay (radioactive immunoassay; RIA). Using the same samples, TOS‐FEIA, ELISA, and three chemiluminescent immunoassays (CLIA) were assessed for correlation and agreement with RIA. Results The TOS‐FEIA showed excellent correlation with RIA (r2 = 0.94, p < 0.0001) and good agreement, with a Bland–Altman constant bias (limits of agreement) of −23.8 μIU/mL (−74.6 to 27.0) and Passing–Bablok fit of y = −8.9 + 0.78x. Mean coefficients of variation were 1.8% for intra‐assay and 5.7% for inter‐assay precision, with mean recovery upon dilution of 104.2%. The assay comparison yielded good or excellent agreement (constant bias, limits of agreement) with RIA in the < 100 μIU/mL cohort for the ELISA (−7.0, −21.4 to 7.4) and the Cobas e CLIA (−31.4, −60.9 to −1.6). Spuriously high results (2 to > 10‐fold of RIA result) were obtained in approximately 10% of results from both Immulite 2000 and 2000XPi CLIA analyzers, rendering the agreement poor. Conclusions and Clinical Importance The TOS‐FEIA had acceptable accuracy and precision for clinical use, including at concentrations of insulin < 100 μIU/mL. The ELISA and one CLIA (Cobas e) showed acceptable accuracy, but the Cobas e demonstrated marked bias compared with RIA. Both Immulite CLIA assays exhibited unacceptable accuracy.

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