title: Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.
abstract: The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065; ARK Resource Co. Ltd., 383-2 Nakahara-machi, Nishi-ku, Kumamoto 861-5271;
authors.forename: Nobuo
authors.initials: N
authors.lastname: Miyazaki
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065;
authors.forename: Norihiko
authors.initials: N
authors.lastname: Kiyose
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577;
authors.forename: Yoko
authors.initials: Y
authors.lastname: Akazawa
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577;
authors.forename: Mizuki
authors.initials: M
authors.lastname: Takashima
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577;
authors.forename: Yosihisa
authors.initials: Y
authors.lastname: Hagihara
authors.affiliation: Department of Cell Science, Institutes for Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295;
authors.forename: Naokazu
authors.initials: N
authors.lastname: Inoue
authors.affiliation: Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumiyama, Otsu, Shiga 520-0811; and.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: MOLCURE Inc., 1-12-11 Taito, Taito-ku, Tokyo, Japan.
authors.forename: Ryu
authors.initials: R
authors.lastname: Ogawa
authors.affiliation: ARK Resource Co. Ltd., 383-2 Nakahara-machi, Nishi-ku, Kumamoto 861-5271;
authors.forename: Seiya
authors.initials: S
authors.lastname: Inoue
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065; yito@sci.kagoshima-u.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000235:genetics / Q000276:immunology*; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology*; D059014:High-Throughput Nucleotide Sequencing; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007963:Leukocytes, Mononuclear / Q000276:immunology
chemical_list: D000906:Antibodies; D000941:Antigens; D007143:Immunoglobulin Heavy Chains
keywords: VHH; alpaca; antibody; domain; folding