Development of a monoclonal antibody-based time-resolved fluorescence immunochromatographic assay strip for rapid and quantitative determination of ubiquitin C-terminal hydrolase L1 in serum.

Traumatic brain injury (TBI) represents a major global health challenge requiring rapid and accurate diagnostic tools. Current detection methods for ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuron-specific biomarker with diagnostic significance at serum concentrations exceeding 327 pg/mL, face limitations in operational complexity and equipment dependency. Herein, we report the development of a monoclonal antibody-based time-resolved fluorescence immunochromatographic assay strip (TRFIA) for rapid and quantitative determination of UCH-L1 in serum. High-affinity antibody pairs were generated via hybridoma technology, and time-resolved fluorescent microspheres (TRFMs) were integrated with lateral flow immunoassay to overcome the limitations of conventional lateral flow platforms. The optimized TRFIA strip demonstrated excellent analytical performance with a linear range of 0.195–12.5 ng/mL (R² = 0.98), a low limit of detection (LOD) of 81.1 pg/ml, and a rapid assay time of 15 min. Intra- and inter-batch coefficients of variation were consistently below 10%, confirming exceptional reproducibility. Specificity analysis revealed minimal cross-reactivity with other TBI-related biomarkers (Tau, S100β, GFAP). Clinical validation using 24 serum samples from mTBI patients and 10 healthy controls demonstrated significant UCH-L1 elevation in TBI cases (AUC = 0.95), confirming the assay’s diagnostic capability. This TRFIA platform offers a portable, cost-effective, and user-friendly solution for point-of-care TBI diagnosis, particularly valuable in resource-limited settings where rapid decision-making is critical. The development represents a significant advancement in TBI diagnostics, providing a complementary tool to enhance diagnostic accuracy while minimizing unnecessary radiation exposure from CT scans.