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Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands. | LitMetric

Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

Methods Mol Biol

Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.

Published: June 2016

AI Article Synopsis

  • - The generation of antibodies against specific parts of folded proteins can be complicated by the presence of irrelevant regions that dominate the immune response and obstruct the desired targets.
  • - Using linear peptides that represent these target regions can be a more effective approach for antibody production, often through techniques that attach these peptides to carrier proteins for better immune response.
  • - The text outlines protocols for creating peptide constructs fused to a verotoxin domain, including steps for expression, purification, and screening of antibodies using advanced sequencing, while emphasizing the importance of carefully selecting peptide sequences based on structural and immunological data.

Article Abstract

Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

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Source
http://dx.doi.org/10.1007/978-1-4939-2999-3_16DOI Listing

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