Antibody-Based Biolayer Interferometry Platform for Rapid Detection of Neutrophil Gelatinase-Associated Lipocalin.

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a critical biomarker for the early diagnosis of acute kidney injury (AKI). The development of novel detection platforms that combine rapid analysis with high sensitivity is essential for improving clinical outcomes. In this study, we established an antibody-based detection system for NGAL using biolayer interferometry (BLI), a label-free optical biosensing technique that monitors real-time interference patterns generated by white light reflected from biomolecular binding events on a biosensor surface. A panel of six anti-NGAL monoclonal antibodies was generated and characterized for its binding properties, identifying candidates with high specificity for NGAL. For robust sensor functionalization, selected monoclonal antibodies were biotinylated and immobilized onto streptavidin-coated biosensor tips, establishing a stable and efficient detection interface. The optimized BLI platform demonstrated a limit of detection (LOD) of 46.1 ng/mL with wild dynamic range of 19 to 40,000 ng/mL. The platform's accuracy was validated using human serum samples, with spike-and-recovery experiments yielding recovery rates of 96.6-104.6%. This demonstrates the capability to accurately quantify NGAL under physiologically relevant conditions with minimal matrix interference. Furthermore, the real-time kinetic measurements enabled rapid analysis, with the entire assay completed in less than half an hour. These findings establish a proof-of-concept for a BLI-based biosensor for NGAL detection, demonstrating sensitivity and specificity that show potential for clinical applications.