Rational Control of Antibody Orientation for Enhancing Sensitivity and Specificity in Lateral Flow Immunoassays With Clinical Validation

Lateral flow assays (LFAs) are widely used for point-of-care diagnostics, but their sensitivity is often limited by inefficient antibody orientation and poor signal transduction. Herein, we report a universal strategy integrating orientation-controlled antibody conjugation at the immunoprobe with a biotin-streptavidin-modified test line to enhance LFA sensitivity, specificity, and analytical reliability. Using an orientation-controlled conjugation approach (P-6, Scheme 1), antigen accessibility was maximized while nonspecific adsorption and false-positive signals were suppressed. Compared with conventional randomly conjugated probes (P-3, Scheme 1) on unmodified test lines, the combined P-6/biotin-streptavidin configuration achieved limits of detection of 0.0352 ng/mL for cytokeratin 19 fragment (CYFRA21-1) and 0.0808 ng/mL for carcinoembryonic antigen (CEA), corresponding to sensitivity enhancements of approximately 14-19 fold. However, probe rankings get inverted across capture chemistries, indicating the importance of dual-site orientation. The universality of this strategy was further validated using alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), and prostate-specific antigen (PSA). Furthermore, multiplexed LFAs using fluorescent polymer dots enabled simultaneous detection of CYFRA21-1 and CEA with minimal cross-reactivity and were validated in 28 clinical serum samples against the hospital reference method.