| Autor: |
Nicolas P Andrews, Justin X Boeckman, Colleen F Manning, Joe T Nguyen, Hannah Bechtold, Camelia Dumitras, Belvin Gong, Kimberly Nguyen, Deborah van der List, Karl D Murray, JoAnne Engebrecht, James S Trimmer |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
|
| Zdroj: |
eLife, Vol 8 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
2050-084X |
| DOI: |
10.7554/eLife.43322 |
| Popis: |
Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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