Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

BackgroundShark 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.ResultsA 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.ConclusionWe 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.

• Publication year

  2007

• Venue

  BMC Biotechnology

• Publication date

  2007-11-19

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1186/1472-6750-7-78PMID 18021450PMCID 2213646
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Selected soluble sdAbs were purified and characterized with direct binding and thermal stability assays on the Luminex 100, and sandwich assays were developed for sensor applications.

  Confidence 0.95

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• Panning the display library yielded binders against staphylococcal enterotoxin B, ricin, and botulinum toxin A complex toxoid.

  Confidence 0.97

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• Randomized CDR3 primers of three different lengths were used to build a semi-synthetic shark sdAb display library with complexity close to 1e9.

  Confidence 0.98

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review

• botulinum toxin a complex toxoid

  toxin

  An inactivated botulinum toxin A preparation used as one of the panning targets in the library selection.

  Aliases: BoNT/A complex toxoid

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• luminex 100 binding and thermal stability assays

  assay platform

  Assay readouts performed on a Luminex 100 instrument to assess binding and thermal stability of selected sdAbs.

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• randomized cdr3 primers

  method

  Primers designed with varied lengths and randomized sequence content to introduce diversity into CDR3.

  Aliases: randomized CDR3 primers of three different lengths

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• ricin

  toxin

  A plant-derived toxin used as one of the panning targets in the library selection.

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• sandwich assays

  assay

  An assay format in which one sdAb captures antigen and another sdAb serves as the reporter element.

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• semi-synthetic shark sdab display library

  library

  A display library built from naturally occurring shark NAR V sequences with added designed diversity in the CDR3 region.

  Aliases: semi-synthetic NAR V display library

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• soluble sdabs

  antibody

  Purified single-domain antibody variants obtained from selected display-library binders for downstream characterization.

  Aliases: sdAbs

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review
• staphylococcal enterotoxin b

  toxin

  A staphylococcal toxin used as one of the panning targets in the library selection.

  Aliases: SEB

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewKiller Whale (322360f1c1) reviewB (s683577b42) reviewmexicorea (qjvnbu8xg3) reviewjihoonc (k5vuy3tzcm) reviewAK (4715169a40) review

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