A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain.
Elife
; 82019 01 22.
Article
en En
| MEDLINE
| ID: mdl-30667360
ABSTRACT
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.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Encéfalo
/
Inmunoglobulina G
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Inmunohistoquímica
/
Anticuerpos Monoclonales
Tipo de estudio:
Guideline
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Elife
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos