Evaluation of singlicate vs duplicate analysis in ligand binding assays: a case study with IFN-γ in mouse and NHP models.

BACKGROUND: The use of duplicate analysis in biomarker assays is a standard practice. While it does not inherently increase variability or contribute to assay failure, it can reveal the high variability associated with manual pipetting, thereby highlighting potential issues within the assy. RESEARCH DESIGN AND METHODS: This study evaluated the reliability of singlicate analysis compared to duplicate analysis for interferon-gamma (IFN-γ) assays using ELISA in mouse and non-human primate (NHP) serum samples. Assay performance was assessed across 50 plates, with results analyzed for minimum required dilution (MRD), standard curve linearity, surrogate sample accuracy, recovery, precision, and variability between analysts, including both an experienced and a novice analyst. RESULTS: There were minimal differences in relative accuracy, and precision between singlicate and duplicate analysis, with CVs less than 5% for both methods. Singlicate and duplicate-generated standard curves were strongly correlated (NHP R(2) = 0.9995, mouse R(2) = 1.0000). Analyst variability had less impact on singlicate analysis, with lower inter-analyst CVs (1.0-6.5%) compared to duplicate analysis (5.0-7.5%). CONCLUSIONS: These findings underscore the robustness of singlicate analysis. A workflow for singlicate assay validation is proposed, demonstrating its potential to streamline biomarker assay development while ensuring regulatory compliance. This study supports the adoption of singlicate analysis as a viable alternative to conventional duplicate methods for biomarker assays.