The importance of antibody orientation for enhancing sensitivity and selectivity in lateral flow immunoassays

In the field of point-of-care diagnostics, lateral flow assays (LFAs) stand out as highly promising due to their compact size, ease of use, and rapid analysis times. These attributes make LFAs invaluable, especially in urgent situations or resource-limited regions. However, their Achilles' heel has always been their limited sensitivity and selectivity. To address these issues, various innovative approaches, including sample enrichment, assay optimization, and signal amplification, have been developed and are extensively discussed in the literature. Despite these advancements, the importance of antibody orientation is often neglected, even though improper orientation can significantly impair detection performance. This review article first explores well-established traditional methodologies, such as minor physical adjustments and non-specific chemical bond formations. It then shifts focus to the oriented immobilization of antibodies on probe surfaces. This approach aims to enhance sensitivity and selectivity fundamentally by leveraging protein affinities or complementary amino acid sequences. The review summarizes the impact of antibody orientation on the analytical performance of LFAs in terms of sensitivity, specificity, speed, reliability, cost-effectiveness, and stability. Additionally, we introduce recent modifications to assay membrane materials and discuss the current limitations and future prospects of LFAs.