Construction and Characterization of Rabbit scFv Antibodies Targeting Fibronectin by Phage Display Technology

Fibronectin (FN) is a multifunctional glycoprotein involved in extracellular matrix remodeling, tumor progression, tissue repair, and host-pathogen interactions. Despite its biological and clinical relevance, FN-binding antibodies derived from non-rodent species remain comparatively less explored. Here, one rabbit was immunized with full-length recombinant human FN, eliciting a robust antibody response confirmed by ELISA and Western blotting. Two rabbit-derived single-chain variable fragment (scFv) phage-display libraries were constructed from peripheral blood mononuclear cells, yielding 5.5 × 10⁷ and 5.0 × 10⁷ transformants for the short- and long-linker formats, respectively. After four rounds of biopanning, phage ELISA confirmed enrichment, and sequence analysis grouped selected clones into five short-linker and four long-linker groups. Seven purified scFvs displayed distinct ELISA binding profiles. Fiber-optic particle plasmon resonance (FOPPR)-based kinetic analysis yielded apparent equilibrium dissociation constants (K_D,app) of 11.21-432.06 nM, with FN-L13 showing the lowest K_D,app. Analytical size-exclusion chromatography indicated predominantly monomeric scFvs. Five scFvs recognized a high-molecular-weight band consistent with fibronectin in mouse plasma by Western blotting. ELISA showed preferential FN binding, with generally low reactivity toward human serum albumin (HSA) and collagens I, III, and IV, although FN-S1 and FN-L1 showed detectable fibrinogen reactivity. These findings support rabbit-derived FN-binding scFvs as research reagents for future FN-related assay development and mechanistic studies.