{
  "abstract": "Background/Objectives: Phage display relies on the physical linkage between the antibody fragment displayed on a virion (phenotype) and the phagemid encoding it (genotype), allowing antigen-binding clones enriched by bio-panning to be identified by sequencing of individual colonies. In colony-based screening, an additional practical assumption is that each isolated bacterial colony represents a single phagemid sequence. Single-domain antibodies (sdAbs), including the variable domain of the heavy chain of a heavy-chain-only antibody (VHH), are increasingly used as displayed molecules, yet their library construction and bio-panning protocols have been adopted, largely unmodified, from those established for single-chain variable fragment (scFv) and fragment antigen-binding (Fab). We therefore asked whether the underlying assumption of one phagemid per colony holds for sdAb phage display. Methods: Using two alpaca immune libraries—a VHH library and a VH/VHH library (VH, the variable domain of the heavy chain)—we tracked Sanger sequencing chromatograms through library construction and three rounds of bio-panning, and compared classical single-colony screening with a strategy in which pooled phagemid DNA recovered after bio-panning was retransformed into naïve Escherichia coli (E. coli) before screening. Results: Mixed-peak chromatograms were infrequent in the initial libraries (10–20%) but accounted for 80% and 50% of chromatograms in the final bio-panning round of the VHH and VH/VHH libraries. Classical screening—periplasmic enzyme-linked immunosorbent assay (ELISA) followed by Sanger sequencing—failed: only 5.7% and 6.9% of antigen-binding colonies yielded interpretable single-peak sequences. Retransformation restored clonality, raising the proportion of single-peak chromatograms to ~88% overall, and retransformation of individual mixed colonies showed that single colonies harbored multiple distinct phagemids. Conclusions: Colony-level clonality is not strictly preserved in single-domain VH and VHH phage display—more plausibly through multiple phage infection during amplification—and a simple retransformation step restores reliable identification of antigen-binding clones.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea",
    "Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea",
    "Interdisciplinary Program in Cancer Biology Major, Seoul National University College of Medicine, Seoul 03080, Republic of Korea",
    "Department of Biomedical Science, Seoul National University College of Medicine, Seoul 03080, Republic of Korea",
    "BioMonster Inc., Samsung Cancer Research Building, Seoul National University, Seoul 03080, Republic of Korea"
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 5,
  "authors": [
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Kyeongeun Kim"
    },
    {
      "affs": [
        0,
        1,
        3
      ],
      "name": "Wookyoung Chun"
    },
    {
      "affs": [
        0,
        1,
        3
      ],
      "name": "Hyunji Yang",
      "orcid": "0000-0002-1385-5177"
    },
    {
      "affs": [
        4
      ],
      "name": "Yonghee Lee",
      "orcid": "0000-0001-8095-408X"
    },
    {
      "affs": [
        0,
        1,
        3
      ],
      "name": "Junho Chung"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/5w0spkp8er0mv.bib",
  "cited_by": 0,
  "doi": "10.3390/biom16101523",
  "fulltext_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13702708/?tool=EBI",
  "id": "5w0spkp8er0mv",
  "issns": [
    "2218-273X"
  ],
  "landing_url": "https://www.mdpi.com/2218-273X/16/10/1523",
  "language": "eng",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2741|Monoclonal and Polyclonal Antibodies Research"
    ]
  ],
  "openalex": "W7222954121",
  "pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13702708/pdf/?tool=EBI",
  "pmcid": "PMC13702708",
  "pmid": null,
  "published": "2026-10-01",
  "publisher": "MDPI AG",
  "ris": "https://research.draconitic.com/paper/5w0spkp8er0mv.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:18:59Z",
      "native_id": "10.3390/biom16101523",
      "source": "crossref",
      "url": "https://www.mdpi.com/2218-273X/16/10/1523"
    },
    {
      "first_seen": "2026-10-30T12:21:26Z",
      "native_id": "PMC:PMC13702708",
      "source": "europepmc",
      "url": "https://europepmc.org/article/PMC/PMC13702708"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2741|Monoclonal and Polyclonal Antibodies Research",
      "subfield": "imaging"
    }
  ],
  "title": "Multiple Phagemids Within Single Bacterial Colonies During Construction and Bio-Panning of Single-Domain (VH and VHH) Phage Display Libraries: A Retransformation Strategy for Reliable Clone Screening",
  "title_text": "Multiple Phagemids Within Single Bacterial Colonies During Construction and Bio-Panning of Single-Domain (VH and VHH) Phage Display Libraries: A Retransformation Strategy for Reliable Clone Screening",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:21:26Z",
  "url": "https://research.draconitic.com/paper/5w0spkp8er0mv",
  "venue": "Biomolecules",
  "versions": []
}