| RRC ID |
36825
|
| Author |
Kurosawa N, Wakata Y, Inobe T, Kitamura H, Yoshioka M, Matsuzawa S, Kishi Y, Isobe M.
|
| Title |
Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.
|
| Journal |
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.
|
| Volume |
6
|
| Pages |
25174
|
| Published |
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
|
| Times Cited |
8
|
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| DNA material |
Genome Network Project IRAL008F08 (HGY083328) |
| Human and Animal Cells |
HeLa
Saos-2 |
