論文 - 詳細
| 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 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 8 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | Genome Network Project IRAL008F08 (HGY083328) |
| ヒト・動物細胞 | HeLa Saos-2 |