Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish s...

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Bibliographic Details
Published in:Protein science 2001-08, Vol.10 (8), p.1522-1528
Main Authors: Lagerkvist, Ann Catrin, Földes‐Papp, Zeno, Persson, Mats A.A., Rigler, Rudolf
Format: Article
Language:English
Subjects:
Antigen-Antibody Complex
Antigens, Viral - immunology
Antigens, Viral - metabolism
Bacteriophages - physiology
combinatorial libraries
fluorescence correlation spectroscopy
Fluorescent Dyes
Hepacivirus - immunology
hepatitis‐C virus
Humans
Immunoglobulin Fragments - genetics
Immunoglobulin Fragments - metabolism
Medicin och hälsovetenskap
monoclonal antibodies
Peptide Library
phage display
Phage‐protein interaction
Solubility
Spectrometry, Fluorescence - methods
Viral Envelope Proteins - genetics
Viral Envelope Proteins - immunology
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Summary:Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis‐C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross‐correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.
ISSN:0961-8368
1469-896X
DOI:10.1110/ps.5701