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Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen
Author(s) -
Lagerkvist Ann Catrin,
FöldesPapp Zeno,
Persson Mats A.A.,
Rigler Rudolf
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.5701
Subject(s) - phage display , antigen , fluorescence correlation spectroscopy , computational biology , antibody , bacteriophage , chemistry , monoclonal antibody , peptide library , microbiology and biotechnology , biology , virology , biochemistry , genetics , escherichia coli , peptide sequence , gene , molecule , organic chemistry
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.