Please use this identifier to cite or link to this item: https://doi.org/10.1038/s42003-021-01965-x
Title: Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation
Authors: Liew, Oi Wah 
Ling, Samantha SM 
Lilyanna, Shera 
Zhou, Yue 
Wang, Peipei
Chong, Jenny PC
Yan, Xia Ng 
Lim, Angeline ES 
Leong, Eliot RY
Lin, Qifeng 
Lim, Teck Kwang 
Lin, Qingsong
Ng, Enoch MW
Ng, Tuck Wah 
Richards, A Mark 
Keywords: Science & Technology
Life Sciences & Biomedicine
Biology
Multidisciplinary Sciences
Life Sciences & Biomedicine - Other Topics
Science & Technology - Other Topics
Issue Date: 6-Apr-2021
Publisher: NATURE RESEARCH
Citation: Liew, Oi Wah, Ling, Samantha SM, Lilyanna, Shera, Zhou, Yue, Wang, Peipei, Chong, Jenny PC, Yan, Xia Ng, Lim, Angeline ES, Leong, Eliot RY, Lin, Qifeng, Lim, Teck Kwang, Lin, Qingsong, Ng, Enoch MW, Ng, Tuck Wah, Richards, A Mark (2021-04-06). Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation. COMMUNICATIONS BIOLOGY 4 (1). ScholarBank@NUS Repository. https://doi.org/10.1038/s42003-021-01965-x
Abstract: High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13–24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.
Source Title: COMMUNICATIONS BIOLOGY
URI: https://scholarbank.nus.edu.sg/handle/10635/206550
ISSN: 23993642
DOI: 10.1038/s42003-021-01965-x
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