Isolation and Characterization of a High-Affinity Anti-TSPAN-1 scFv Antibody from a Phage-Display Library for Planarian Neoblast Targeting

Background: Planarian neoblasts are pluripotent adult stem cells responsible for whole-body regeneration, yet antibody-based tools for their specific identification and isolation remain limited. This study aimed to generate a high-affinity single-chain variable fragment (scFv) targeting TSPAN-1, a neoblast surface marker, using phage display technology. Methods: A phage-displayed scFv library (3.86 × 107 CFU) was constructed from mice immunized with the recombinant large extracellular loop (LEL) of TSPAN-1 fused to maltose-binding protein (MBP). Four rounds of bio-panning were performed against immobilized His-LEL. Positive clones were screened by ELISA, colony PCR, and DNA sequencing. Among the characterized clones, scFv-16 showed the highest apparent affinity. This clone was subcloned for prokaryotic expression, and inclusion body proteins were refolded and purified. Results: Friguet analysis gave an apparent affinity constant (Kaff) of 4.20 × 108 M−1 for scFv-16 binding to His-TSPAN-1. This corresponds to a KD of 2.38 nM. Western blotting and immunofluorescence staining supported the specificity of scFv-16 for TSPAN-1. Conclusions: A high-affinity anti-TSPAN-1 scFv was successfully generated, providing a candidate molecular tool for neoblast identification and potential isolation in planarian regeneration studies.