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Peer-reviewedCC BY 4.0International Journal of Molecular Sciences

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

  • Yu-Wei Chiang1,2,3,4
  • Jia-Lin Juang5,6
  • Yi-Yuan Yang7
  • Sy-Jye Leu8
  • Jun-Jen Liu9,10
  • Bor-Yu Tsai11
  • Yan-Chiao Mao12
  • Fang-Yi Yen13
  • Jia-Yun Chiang9
  • Valencia Andriani14
  • Priskila Cherisca Thenaka15
  • Ta-Lun Tan10
2 more authors
  • Ting-Jing Shen9,10
  • Chi-Hsin Lee1,14
15 affiliations
  1. 1Graduate Institute of Biodefense, National Defense Medical University, Taipei 114201, Taiwan
  2. 2Institute of Preventive Medicine, National Defense Medical University, Taipei 114201, Taiwan
  3. 3Department of Occupational Medicine and Clinical Toxicology, Taipei Veterans General Hospital, Taipei 112201, Taiwan
  4. 4Department of Biology and Anatomy, National Defense Medical University, Taipei 114201, Taiwan
  5. 5Zinexts Life Science Corp., New Taipei City 221416, Taiwan
  6. 6JW Diagnostics Inc., New Taipei City 235048, Taiwan
  7. 7Department of New Drug Development, GlycoNex Inc., New Taipei City 221021, Taiwan
  8. 8Department of R&D, Zinexts Life Science Corp., New Taipei City 221416, Taiwan
  9. 9School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, New Taipei City 235603, Taiwan
  10. 10Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, New Taipei City 235603, Taiwan
  11. 11Navi Bio-Therapeutics Inc., Taipei 10351, Taiwan
  12. 12Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung 407219, Taiwan
  13. 13Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
  14. 14iReal Biotechnology Inc., Research and Development Center, New Taipei City 248022, Taiwan
  15. 15Department of Project Management, GlycoNex Inc., New Taipei City 221021, Taiwan
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Abstract

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 (KD,app) of 11.21–432.06 nM, with FN-L13 showing the lowest KD,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.

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Cite

BibTeXRIS
BibTeX
@article{chiang2026construction,
  title = {{Construction and Characterization of Rabbit scFv Antibodies Targeting Fibronectin by Phage Display Technology}},
  author = {Yu-Wei Chiang and Jia-Lin Juang and Yi-Yuan Yang and Sy-Jye Leu and Jun-Jen Liu and Bor-Yu Tsai and Yan-Chiao Mao and Fang-Yi Yen and Jia-Yun Chiang and Valencia Andriani and Priskila Cherisca Thenaka and Ta-Lun Tan and Ting-Jing Shen and Chi-Hsin Lee},
  journal = {International Journal of Molecular Sciences},
  year = {2026},
  month = oct,
  doi = {10.3390/ijms27209074},
  url = {https://www.mdpi.com/1422-0067/27/20/9074},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Construction and Characterization of Rabbit scFv Antibodies Targeting Fibronectin by Phage Display Technology
AU  - Yu-Wei Chiang
AU  - Jia-Lin Juang
AU  - Yi-Yuan Yang
AU  - Sy-Jye Leu
AU  - Jun-Jen Liu
AU  - Bor-Yu Tsai
AU  - Yan-Chiao Mao
AU  - Fang-Yi Yen
AU  - Jia-Yun Chiang
AU  - Valencia Andriani
AU  - Priskila Cherisca Thenaka
AU  - Ta-Lun Tan
AU  - Ting-Jing Shen
AU  - Chi-Hsin Lee
PY  - 2026
DA  - 2026/10/13
JO  - International Journal of Molecular Sciences
DO  - 10.3390/ijms27209074
UR  - https://www.mdpi.com/1422-0067/27/20/9074
AB  - 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 (KD,app) of 11.21–432.06 nM, with FN-L13 showing the lowest KD,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.
SN  - 1422-0067
ER  -