Abstract
We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of αs1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we evaluated two methods that drastically reduced the background binding. An optimized method therefore enabled a panning procedure where the specific (peptide binding) scFv-phages were always dominant. Using 15-mer peptides immobilized on Nunc Immobilizer Streptavidin plates, we successfully generated scFvs specifically against them. The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides.
Keywords: Phage display, scFv, Tomlinson I + J libraries, casein, peptide.
Combinatorial Chemistry & High Throughput Screening
Title: An Efficient Method for Isolating Antibody Fragments Against Small Peptides by Antibody Phage Display
Volume: 13 Issue: 9
Author(s): Zhi Duan and Henrik Siegumfeldt
Affiliation:
Keywords: Phage display, scFv, Tomlinson I + J libraries, casein, peptide.
Abstract: We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of αs1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we evaluated two methods that drastically reduced the background binding. An optimized method therefore enabled a panning procedure where the specific (peptide binding) scFv-phages were always dominant. Using 15-mer peptides immobilized on Nunc Immobilizer Streptavidin plates, we successfully generated scFvs specifically against them. The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides.
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Cite this article as:
Duan Zhi and Siegumfeldt Henrik, An Efficient Method for Isolating Antibody Fragments Against Small Peptides by Antibody Phage Display, Combinatorial Chemistry & High Throughput Screening 2010; 13 (9) . https://dx.doi.org/10.2174/138620710792927376
DOI https://dx.doi.org/10.2174/138620710792927376 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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