Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies
| Autor: | Yoshihiro Kishi, Tomonao Inobe, Masaharu Isobe, Nobuyuki Kurosawa, Megumi Yoshioka, Shun Matsuzawa, Haruki Kitamura, Yuka Wakata |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Threonine medicine.drug_class Guinea Pigs Plasma cell Monoclonal antibody Article Serine 03 medical and health sciences 0302 clinical medicine Antigen medicine Animals Tyrosine Multidisciplinary biology Antibodies Monoclonal Phosphoproteins Molecular biology 030104 developmental biology medicine.anatomical_structure Biochemistry 030220 oncology & carcinogenesis biology.protein Phosphorylation Antibody |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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