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A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery.

Phebe De Keyser1, Valentina Kalichuk1, Thomas Zögg1

  • 1VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, Pleinlaan 2-building E, 1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.

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Summary

This study introduces an improved biopanning strategy using Octet biosensors for high-throughput selection of Nanobodies®. This method accelerates the discovery of target-specific Nanobodies® by reducing panning rounds and material requirements.

Keywords:
Biolayer interferometryBiopanningBiosensorNanobodyPhage display selectionVHH

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Area of Science:

  • Biotechnology
  • Immunology
  • Molecular Biology

Background:

  • Traditional Nanobody® (Nb) selection relies on labor-intensive, time-consuming biopanning methods.
  • Existing methods often require purified antigens and extensive optimization, increasing costs and duration.

Purpose of the Study:

  • To validate an improved biopanning strategy utilizing biosensors for efficient Nanobody® selection.
  • To demonstrate the advantages of Octet biolayer interferometry for high-throughput Nanobody® enrichment.
  • To reduce the time, labor, and material costs associated with Nanobody® discovery.

Main Methods:

  • Phage-displayed Nanobody® libraries were panned against target antigens immobilized on Octet biolayer interferometry sensors.
  • Optimization of association/dissociation times and buffer conditions was performed to enhance specificity and affinity.
  • Downscaling to a 384-well format and using unpurified targets and phage were explored.

Main Results:

  • The Octet panning strategy significantly reduced non-specific and low-affinity binders, decreasing the number of panning rounds required.
  • High-affinity Nanobodies® were enriched in as few as three rounds within five days.
  • Quantification of binder enrichment was achieved directly via interferometry, eliminating the need for phage titration.

Conclusions:

  • Octet biosensor-based panning offers a high-throughput, efficient, and cost-effective alternative to traditional biopanning methods.
  • This improved strategy enables rapid isolation of target-specific Nanobodies® using unpurified reagents and simplified quantification.
  • The validated method streamlines Nanobody® discovery for subsequent screening and characterization.