title: In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems.
abstract: Rabbit monoclonal antibodies (mAbs) have advantages over mouse antibodies in diagnostics and biotechnological applications owing to higher affinity and specificity.We developed a platform to generate rabbit mAbs by a novel monoclonal antibody generation method named “Single-Cell Reverse Transcription-PCR linked in vitro-Expression (SICREX)” system. In this method,we use single-cell based RT-PCR followed by sequential PCR steps of mAb genes and subsequent cell-free protein synthesis (CFPS) by using linear DNA fragments of mAbs. This platform enables the rapid generation and evaluation of mAbs derived from antigen-specific single B cells in the peripheral blood of immunized animals without mammalian cell cultivation. In this study, the antigen used was a food-borne gram-positive pathogen, Listeria monocytogenes, that is known to cause serious infection. Three active mAbs in CFPS were obtained by constructing the single chain of variable fragment (scFv) form. These scFvs were produced in the cytoplasm of E. coli Shuffle T7 Express strain as an active form and used for further investigation.
authors.affiliation: Knowledge Hub Aichi, Aichi Science and Technology Foundation, 1267-1 Akiai, Yakusa cho, Toyota 470-0356, Japan; Graduate School of Bioagricultural Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Electronic address: teruyo.ojima@gmail.com.
authors.forename: Teruyo
authors.initials: T
authors.lastname: Ojima-Kato
authors.affiliation: Graduate School of Bioagricultural Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Dai
authors.initials: D
authors.lastname: Hashimura
authors.affiliation: Graduate School of Bioagricultural Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Graduate School of Bioagricultural Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Minabe
authors.affiliation: Graduate School of Bioagricultural Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Electronic address: hnakano@agr.nagoya-u.ac.jp.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D008089:Listeria monocytogenes / Q000276:immunology*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal
keywords: Cell-free protein synthesis; Expression in E. coli cytoplasm; Listeria monocytogenes; Rabbit monoclonal antibody; ScFv