Hyunji Yang

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.

Background/Objectives: Deep sequencing of antibody phage-display selections commonly uses intracellular double-stranded phagemid DNA (dsDNA), although selected libraries are propagated as virions containing single-stranded phagemid DNA (ssDNA). Whether these two templates yield equivalent quantitative profiles remains uncertain. Methods: We sequenced matched intracellular dsDNA and virion ssDNA from a single-chain variable fragment library at the input and after each of four rounds of bio-panning. Results: A total of 1052 ssDNA-derived and 1093 dsDNA-derived unique heavy-chain complementarity-determining region 3 (HCDR3) clonotypes were analyzed, including 1003 shared clonotypes. The dsDNA and ssDNA profiles were closely concordant before strong enrichment, with Spearman correlations of 0.972–0.978 and Morisita–Horn overlap of 0.994–0.998 through round 2. Once a small number of clonotypes began to dominate the selection, however, the two profiles separated sharply: abundance-weighted overlap fell to 0.501 at round 3 and 0.379 at round 4, whereas rank order was preserved and the rank correlation remained high throughout (ρ ≥ 0.90). This divergence was concentrated among enriching trajectories, in which dominant clonotypes reached substantially higher relative abundance in virion ssDNA than in intracellular dsDNA. Antigen-reactive clonotypes were also preferentially shifted toward the ssDNA-biased fraction, with a median log₁₀(ssDNA/dsDNA) ratio of 0.44 compared with −0.17 for non-reactive clonotypes at round 3 (p = 6.3 × 10⁻⁸) and round 4 (0.41 vs. −0.12, p = 1.1 × 10⁻⁶). Conclusions: dsDNA and ssDNA tell nearly the same story early in bio-panning but can provide markedly different accounts once selection becomes concentrated. In this selection campaign, template choice materially affected interpretation of antibody phage-display sequencing, suggesting that virion ssDNA or paired-template analysis may be preferable to dsDNA alone when late-round abundance is used for candidate prioritization. Because these results come from a single campaign against one antigen, they are descriptive of this dataset and require validation across antigens, scaffolds and display systems.

Background/Objectives: Calsequestrin (CSQ) is a calcium-binding protein that is highly soluble and can serve as a solubility-enhancing fusion tag in recombinant protein expression. Its unique property of calcium-induced precipitation followed by EDTA-mediated resolubilization enables efficient purification. However, the broader application of CSQ-tagged proteins in research have been hampered by the lack of reliable anti-CSQ detection reagents. This study aimed to develop single-domain antibodies (sdAbs) against CSQ for use in diverse immunoassays and cell-based analyses. Methods: Single-domain antibodies were selected from phage-displayed chicken VH libraries generated from CSQ-immunized chickens. After biopanning, CSQ-specific VH sdAb clones were isolated and expressed as VH–human kappa light chain constant region (VH-Cκ) fusion proteins in E. coli. The PE06 clone was chosen for further characterization and conjugated to horseradish peroxidase (HRP) and Alexa Fluor 647 for assay applications. Results: PE06 VH-Cκ fusion protein demonstrated specific binding to CSQ-tagged proteins and enabled reliable detection in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and flow cytometry. These results validated its utility as a chemically defined detection reagent for CSQ fusion proteins expressed in E. coli. Conclusions: This study establishes a CSQ-specific chicken VH sdAb as a versatile detection tool for CSQ-tagged proteins. The approach expands the utility of CSQ as a protein fusion tag and enables the development of recombinant antibodies fused with CSQ, such as scFv-CSQ constructs, for broad application in research and assay systems.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V_H) genes and theλlight chain variable region (V_λ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an “immune library” is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies ofV_H/J_HandV_λ/J_λgene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5–7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as “phage ELISA” to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a “helper” phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V _H and J _H genes, and the somatic gene conversion process usually involves D _H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V _λ light chain, composed of a single V _λ and J _λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.