A-389 Development of a Sensitive Lateral Flow Immunoassay for Simultaneous Detection of GFAP, FABP, and UCH-L1 in Mild Traumatic Brain Injury Management

Abstract Background Traumatic brain injury (TBI) is the most common neurological disorder. Among all cases, 70–85% are categorized as mild TBI (mTBI), also referred to as concussion. Diagnosing mTBI presents a significant challenge due to the lack of visible symptoms, making it difficult to identify accurately. Current diagnostic methods for mTBI primarily depend on patient history, clinical assessments, and advanced neuroimaging techniques such as CT and MRI scans. While these methods are effective, they are often expensive, time-intensive, and impractical for use. As an alternative or complementary approach, the detection of mTBI biomarkers in blood offers a promising diagnostic pathway. Three biomarkers, glial fibrillary acidic protein (GFAP), fatty acid-binding protein (FABP), and ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), have been identified as being highly relevant for mTBI diagnostics. This study aimed to develop a rapid, sensitive, and cost-effective lateral flow immunoassay (LFIA) platform for the simultaneous detection of GFAP, FABP, and UCH-L1, paving the way for point-of-care testing (POCT) applications. Methods A time-resolved fluorescence-based lateral flow immunoassay (TRF-LFIA) was developed for the simultaneous quantitative detection of GFAP, FABP, and UCH-L1 in human clinical samples. The assay employed lyophilized europium nanoparticle (EuNP)-conjugated monoclonal antibodies that specifically target these biomarkers, enhancing sensitivity and reproducibility. The LFIA cartridge was designed with a nitrocellulose membrane featuring three test lines for GFAP, FABP, and UCH-L1 and one control line. The sandwich-style complexes were analyzed using the Exdia TRF-Plus analyzer, an in-house-developed fluorescence reader. Several optimization steps were implemented, including the selection of antibodies using octet and optimization of conjugation of europium and antibody protocol to improve detection sensitivity, reduce background noise, and shorten the detection time. For proof-of-concept validation compare the Exdia mTBI with the commercial product. The detection limit for each biomarker was determined, and cross-reactivity was evaluated. Results The developed Exdia mTBI TRF-LFIA demonstrated high sensitivity, specificity, and reproducibility. The assay achieved a limit of detection (LOD) of 24.5 pg/mL for GFAP, 120 pg/mL for FABP, and 92 pg/mL for UCH-L1, with minimal cross-reactivity between biomarkers. The platform showed excellent linear dynamic detection ranges for all three biomarkers, enabling accurate and reliable quantification. The use of clinical serum samples further validated the performance of the assay, demonstrating high correlation with established diagnostic methods. By combining the quantitative results for GFAP, FABP, and UCH-L1, the LFIA platform enables effective monitoring and management of mTBI. Conclusion The Exdia TRF-LFIA platform offers a rapid, sensitive, and cost-effective solution for the simultaneous detection of mTBI-relevant biomarkers GFAP, FABP, and UCH-L1. With its rapid detection capabilities, wide dynamic range, and minimal sample requirements, this platform has significant potential as a supplemental diagnostic tool for in vitro diagnostics and point-of-care testing. By substantially reducing diagnostic time and medical costs, the proposed platform minimizes treatment delays and enables timely intervention for mTBI patients. This novel technology represents a critical step towards the development of accessible and efficient diagnostic solutions for mild traumatic brain injury management.