Research Article
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Published Online: 10 October 2019

Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments

Publication: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Volume 38, Issue Number 5


To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.

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Information & Authors


Published In

cover image Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Volume 38Issue Number 5October 2019
Pages: 190 - 200
PubMed: 31411543


Published online: 10 October 2019
Published in print: October 2019
Published ahead of print: 14 August 2019
Accepted: 11 July 2019
Received: 19 June 2019


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Abdur Rafique
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.
Kiriko Satake
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.
Satoshi Kishimoto
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.
Kamrul Hasan Khan
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.
Dai-ichiro Kato
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima, Japan.


Address correspondence to: Yuji Ito, Department of Chemistry and Bioscience, Graduate School of Science and Engineering, University of Kagoshima, Kagoshima 890-0065, Japan [email protected]

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No competing financial interests exist.

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