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A Novel Strategy for Generation of Monoclonal Antibodies from Single B Cells Using RT-PCR Technique and in Vitro Expression

Authors

  • XiuPing Jiang,

    1. Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
    Current affiliation:
    1. Chubu University, Matsumoto-cho 1200, Kasugai 487–0027, Japan
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  • Hirotatsu Suzuki,

    1. Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
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  • Yuko Hanai,

    1. Laboratory of Molecular and Cellular Regulation, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
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  • Fumitaka Wada,

    1. Laboratory of Molecular and Cellular Regulation, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
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  • Kiyotaka Hitomi,

    1. Laboratory of Molecular and Cellular Regulation, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
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  • Tsuneo Yamane,

    1. Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
    Current affiliation:
    1. Laboratory of Nano Medical Engineering, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2–1, Wako 351–0198, Japan
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  • Hideo Nakano

    Corresponding author
    1. Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan
    • Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464–8601, Japan. Tel: +81 52 789 4142. Fax: +81 52 789 4145
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Abstract

Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called single-cell RT-PCR-linked in vitro expression (SICREX), which enables the high-throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B-cell-selective marker, anti-CD19. The light chain (Lc) and Fd portion of heavy-chain (Hc) genes of each cell are separately amplified by RT-PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full-length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coliin vitro transcription and translation system followed by an enzyme-linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with Kd of 1.6 (± 0.3) × 10−8 against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B-BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high-throughput generation of naturally paired Lc and Hc with antigen-binding activity from various animal sources.

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