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In vitro evolution of an antibody fragment population to find high affinity hapten binders

Persson, Helena LU ; Wallmark, Henrik; Ljungars, Anne; Hallborn, Johan and Ohlin, Mats LU (2008) In Protein Engineering Design & Selection 21(8). p.485-493
Abstract
Recently, we constructed a focused antibody library tailored to interact with haptens. High functionality of this library was demonstrated, as specific binders could be retrieved to a range of different haptens. In the current study we have developed a mutagenesis and selection strategy in order to further fine-tune the hapten binding properties of these antibody fragments. Testosterone was chosen as model antigen for the investigation. A population, rather than a single clone, originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen concentration and having, or... (More)
Recently, we constructed a focused antibody library tailored to interact with haptens. High functionality of this library was demonstrated, as specific binders could be retrieved to a range of different haptens. In the current study we have developed a mutagenesis and selection strategy in order to further fine-tune the hapten binding properties of these antibody fragments. Testosterone was chosen as model antigen for the investigation. A population, rather than a single clone, originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen concentration and having, or not having, soluble hapten present during the phage capture and elution steps. The different selection procedures resulted in a considerable increase in apparent affinities for several of the selected populations, from which the highest affinity antibody isolated had a K(D) of 2 nM, corresponding to an approximately 200-fold affinity improvement compared with the best clone of the starting population. Importantly, the polyclonal nature of the starting material allowed for the identification of novel unrelated variants that differed in fine-specificity, demonstrating that this approach is valuable for exploring different parts of structure space. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Engineering Design & Selection
volume
21
issue
8
pages
485 - 493
publisher
Oxford University Press
external identifiers
  • wos:000257789400002
  • scopus:47649099663
ISSN
1741-0126
DOI
10.1093/protein/gzn024
language
English
LU publication?
yes
id
f82d2b59-e097-40d8-be3f-c707d8d7ec09 (old id 1056774)
date added to LUP
2008-04-17 11:11:01
date last changed
2017-01-01 06:25:48
@article{f82d2b59-e097-40d8-be3f-c707d8d7ec09,
  abstract     = {Recently, we constructed a focused antibody library tailored to interact with haptens. High functionality of this library was demonstrated, as specific binders could be retrieved to a range of different haptens. In the current study we have developed a mutagenesis and selection strategy in order to further fine-tune the hapten binding properties of these antibody fragments. Testosterone was chosen as model antigen for the investigation. A population, rather than a single clone, originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen concentration and having, or not having, soluble hapten present during the phage capture and elution steps. The different selection procedures resulted in a considerable increase in apparent affinities for several of the selected populations, from which the highest affinity antibody isolated had a K(D) of 2 nM, corresponding to an approximately 200-fold affinity improvement compared with the best clone of the starting population. Importantly, the polyclonal nature of the starting material allowed for the identification of novel unrelated variants that differed in fine-specificity, demonstrating that this approach is valuable for exploring different parts of structure space.},
  author       = {Persson, Helena and Wallmark, Henrik and Ljungars, Anne and Hallborn, Johan and Ohlin, Mats},
  issn         = {1741-0126},
  language     = {eng},
  number       = {8},
  pages        = {485--493},
  publisher    = {Oxford University Press},
  series       = {Protein Engineering Design & Selection},
  title        = {In vitro evolution of an antibody fragment population to find high affinity hapten binders},
  url          = {http://dx.doi.org/10.1093/protein/gzn024},
  volume       = {21},
  year         = {2008},
}