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. 2007 Jan;44(4):656-65.
doi: 10.1016/j.molimm.2006.01.010. Epub 2006 Feb 24.

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library

Cui-Ying Shao  1 , Chris J Secombes, Andrew J Porter
Affiliations

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library

Cui-Ying Shao et al. Mol Immunol. 2007 Jan.

Abstract

The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

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