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A high affinity digoxin-binding protein displayed on M13 is functionally identical to the native protein
P M Tang1, L A Foltz, W C Mahoney
1Molecular Diagnostics, Research and Development, Boehringer Mannheim Corporation, Indianapolis, Indiana 46250, USA.
The Journal of Biological Chemistry
|April 7, 1995
Summary
Phage display technology accurately measures binding affinities of proteins, like antibodies, without altering their specificity or strength compared to soluble forms. This method is reliable for high-affinity binders.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Phage display is a powerful technique for generating binding molecules with modified affinities.
- The impact of phage display on affinity measurements compared to soluble proteins is not well understood.
Purpose of the Study:
- To investigate whether phage display affects affinity measurements of binding molecules.
- To compare the binding affinity and specificity of phage-displayed proteins to their soluble counterparts.
Main Methods:
- Scatchard analysis was used to determine binding affinities.
- The binding of a native antibody, its single-chain variable fragment (scFv), and its phage-displayed scFv counterpart to digoxin was measured.
- Binding specificity was assessed using cross-reactive compounds like dihydrodigoxin and digitoxin.
Main Results:
- Identical binding affinities for digoxin were observed between the native antibody, soluble scFv, and phage-displayed scFv.
- No significant difference in affinity was detected for phage-displayed scFv compared to soluble forms.
- Binding specificity remained unchanged when tested against cross-reactive compounds.
Conclusions:
- Phage display is a valid method for accurately measuring the binding affinity of proteins.
- The technique preserves the intrinsic binding characteristics of proteins, including high-affinity interactions.
- Phage display can be reliably used for selecting and characterizing high-affinity binding molecules.