COLPLEX: A high-affinity protein adaptor for enhanced sensitivity and multiplex lateral flow immunoassays. application to ebola virus strain detection

To overcome the limitation of lateral flow immunoassays (LFIAs) in multiplexing while preserving high sensitivity and specificity, we present COLPLEX, a high-affinity recognition system based on the ultra-specific interaction between colicin antimicrobial proteins and their cognate immunity proteins. These protein pairs, naturally evolved to exhibit femtomolar binding affinities, are bio-orthogonal and readily accessible through recombinant expression. We integrated the COLPLEX system into a biplex LFIA platform for the simultaneous detection of glycoproteins from Zaire and Sudan Ebola virus variants. Using two distinct, non-cross-reactive colicin-immunity pairs, we immobilized colicins on the nitrocellulose membrane and fused immunity proteins to the capture antibodies. This configuration significantly improved the signal-to-noise ratio, reduced capture antibody consumption, and minimized non-specific binding. Clinical evaluation using sera from Ebola-infected patients (n = 30) and negative controls (n = 40), including healthy donors and patients with other viral haemorrhagic fevers, demonstrated high diagnostic accuracy, with 96.7% sensitivity and 100% specificity, confirming the robustness of COLPLEX for real-world applications. These results highlight COLPLEX as a powerful alternative to streptavidin-biotin system and its potential to revolutionize next-generation LFIA platforms. • A multiplex lateral flow immunoassay powered by COLPLEX ultra-affinity proteins delivers rapid, simultaneous, and high-sensitivity detection of Zaire and Sudan Ebola virus strains • High-affinity colicin-immunity protein interaction was harnessed • Approach outperforms classic direct immunoassays in sensitivity • Clinical evaluation confirms high sensitivity and no non-specific binding