Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.
The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.
Dooley
Helen
H
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK. hdooley@som.umaryland.edu
Flajnik
Martin F
MF
Porter
Andrew J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D022801:Complementarity Determining Regions; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011946:Receptors, Antigen / Q000737:chemistry* / Q000276:immunology; D012754:Sharks / Q000276:immunology*
D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D019151:Peptide Library; D011946:Receptors, Antigen; C092388:nurse shark antigen receptor