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

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.