Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library

Jinny L. Liu, George P. Anderson, James B. Delehanty, Richard Baumann, Andrew Hayhurst, Ellen R. Goldman

    Research output: Contribution to journalArticle

    72 Scopus citations

    Abstract

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size (∼12 kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >107 unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex100 and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 °C), while conventional antibodies lost all activity in the first 5-10 min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

    Original languageEnglish (US)
    Pages (from-to)1775-1783
    Number of pages9
    JournalMolecular Immunology
    Volume44
    Issue number7
    DOIs
    StatePublished - Mar 1 2007

    Keywords

    • Cholera toxin
    • IgNAR
    • Naïve display library
    • Panning
    • Single domain antibody
    • Spiny dogfish shark

    ASJC Scopus subject areas

    • Immunology
    • Molecular Biology

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    Liu, J. L., Anderson, G. P., Delehanty, J. B., Baumann, R., Hayhurst, A., & Goldman, E. R. (2007). Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library. Molecular Immunology, 44(7), 1775-1783. https://doi.org/10.1016/j.molimm.2006.07.299