Development of a Sensitive Lateral Flow Immunoassay Using Recombinant Rabbit Single-Chain Variable Fragments Fused to a Nitrocellulose-Binding Protein

Lateral-flow immunoassays (LFIAs) are increasingly used as diagnostic tools for point-of-care testing because of their speed, simplicity, and cost-effectiveness. However, traditional antibody immobilization techniques, which primarily rely on the passive antibody adsorption onto nitrocellulose (NC) membranes, have notable limitations. Accordingly, we developed an innovative LFIA platform that integrates recombinant rabbit single-chain variable fragments (scFvs) genetically fused with NC-binding proteins (NBPs) to enhance immobilization and overall performance. Twenty-one candidate proteins were screened, of which lactoferrin emerged as a superior NBP because of its robust and stable adsorption onto NC membranes, even in the presence of surfactants. Fusion constructs of scFvs and lactoferrin (LF) (designated scFv-LF) were constructed and expressed in ExpiCHO cells. The purified scFv-LF proteins maintained high antigen-binding activity across a wide pH range (2-13) and exhibited improved performance in dot blot assays, enzyme-linked immunosorbent assay (ELISA), and lateral flow formats compared to scFvs alone. A complementarity-determining region (CDR)-grafting strategy was used to produce scFv-LF variants with modified regions that retained the C2R framework. C1R/C2R and B1R/C2R fusion proteins demonstrated enhanced antigen activity and high signal intensities. B1R/C2R scFv-LF fusion achieved a 100-fold lower detection limit for influenza B nucleocapsid protein than its nonfused counterpart. Overall, the scFv-LF fusion platform is a powerful solution for achieving stable and oriented antibody immobilization on NC membranes, substantially enhancing the sensitivity and reproducibility of LFIA systems. This modular approach facilitates the rapid customization of a diverse range of antigens through CDR grafting, paving the way for next-generation diagnostic developments.