Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.
BACKGROUND
Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences.


RESULTS
A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications.


CONCLUSIONS
We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.
Liu
Jinny L
JL
Center for Bio/Molecular Science and Engineering, code 6920, Naval Research Laboratory, 4555 Overlook Ave, SW, Washington, DC 20375, USA. jinny.liu@nrl.navy.mil
Anderson
George P
GP
Goldman
Ellen R
ER
D000595:Amino Acid Sequence; D000818:Animals; D000992:Antitoxins / Q000032:analysis*; D001665:Binding Sites / Q000276:immunology; D001905:Botulinum Toxins / Q000276:immunology; D022801:Complementarity Determining Regions; D004768:Enterotoxins / Q000276:immunology; D007143:Immunoglobulin Heavy Chains; D007136:Immunoglobulins / Q000737:chemistry* / Q000145:classification*; D019151:Peptide Library; D054730:Protein Interaction Domains and Motifs / Q000276:immunology*; D025941:Protein Interaction Mapping; D011946:Receptors, Antigen / Q000737:chemistry* / Q000145:classification*; D012276:Ricin / Q000276:immunology; D048251:Squalus acanthias
D000992:Antitoxins; D022801:Complementarity Determining Regions; D004768:Enterotoxins; D007143:Immunoglobulin Heavy Chains; D007136:Immunoglobulins; D019151:Peptide Library; D011946:Receptors, Antigen; C092388:nurse shark antigen receptor; C031818:enterotoxin B, staphylococcal; D012276:Ricin; D001905:Botulinum Toxins