RRC ID 36825
著者 Kurosawa N, Wakata Y, Inobe T, Kitamura H, Yoshioka M, Matsuzawa S, Kishi Y, Isobe M.
タイトル Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.
ジャーナル Sci Rep
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.
巻・号 6
ページ 25174
公開日 2016-4-29
DOI 10.1038/srep25174
PII srep25174
PMID 27125496
PMC PMC4850396
MeSH Animals Antibodies, Monoclonal / immunology* Antibodies, Monoclonal / isolation & purification* Guinea Pigs Phosphoproteins / immunology* Threonine / immunology*
IF 3.998
引用数 8
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
遺伝子材料 Genome Network Project IRAL008F08 (HGY083328)