title: Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.
abstract: Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.
authors.affiliation: Laboratory of Molecular and Cellular Biology, Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kurosawa
authors.affiliation: Laboratory of Molecular and Cellular Biology, Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Wakata
authors.affiliation: Frontier Research Core for Life Sciences, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Tomonao
authors.initials: T
authors.lastname: Inobe
authors.affiliation: Graduate School of Science and Engineering for Education, University of Toyama, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Haruki
authors.initials: H
authors.lastname: Kitamura
authors.affiliation: Laboratory of Molecular and Cellular Biology, Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Yoshioka
authors.affiliation: Medical &Biological Laboratories Co., Ltd., 15-502 Akaho, Komagane, Nagano, 399-4117, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Matsuzawa
authors.affiliation: Medical &Biological Laboratories Co., Ltd., 15-502 Akaho, Komagane, Nagano, 399-4117, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kishi
authors.affiliation: Laboratory of Molecular and Cellular Biology, Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Isobe
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification*; D006168:Guinea Pigs; D010750:Phosphoproteins / Q000276:immunology*; D013912:Threonine / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D010750:Phosphoproteins; D013912:Threonine