Isolation, characterization and pentamerization of alpha-cobrotoxin specific single-domain antibodies from a naive phage display library: preliminary findings for antivenom development

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TypeArticle
Journal titleToxicon
Volume49
Issue5
Pages699709; # of pages: 11
SubjectACID; adverse effects; amino acid; Amino Acid Sequence; Amino Acid Substitution; AMINO-ACID; analysis; Animal; Animals; Antibodies; antibody; Antivenins; Blotting,Western; Camelids,New World; Canada; chemistry; Cloning,Molecular; Cobra; Cobra Neurotoxin Proteins; Cobra Venoms; complex; COMPLEXES; DNA; DNA Primers; Enzyme-Linked Immunosorbent Assay; genetics; Human; Humans; Immunization; immunology; interaction; ISOLATION; isolation & purification; Libraries; Mixture; Molecular Sequence Data; peptide; Peptide Library; RESONANCE; SEQUENCE; Sequence Analysis; Sequence Analysis,DNA; Serum; SPECIFICITY; surface; Surface Plasmon Resonance; TARGET
AbstractConventional antivenoms to snakebite generated from the serum of immunized animals, often elicit adverse reactions and have mismatched pharmacokinetic profiles with their target toxins due to antibody/toxin size discrepancies which results in poor neutralization. Furthermore, animal immunization protocols are often lengthy and have batch to batch variability. Recombinant V(H)H-based antivenoms may help overcome these problems. Three V(H)H fragments with specificity to alpha-cobrotoxin, a snake neurotoxin from Naja kaouthia venom, were isolated from a naive llama V(H)H phage-display library. Alpha-cobrotoxin-binding specificity was determined using a phage-displayed V(H)H ELISA format. Sequence analysis shows two of the three clones differ by only two amino acid substitutions, while the third is unique. Surface plasmon resonance analysis determined the K(D) values of the interactions to be 2, 3 and 3 microM. These affinities are too low for alpha-cobrotoxin detection in a standard ELISA format, or for practical use as therapeutic agents. However, improved functional affinity was obtained via antibody pentamerization and alpha-cobrotoxin detection was possible using a pentabody-based ELISA. Development of antivenoms composed of a mixture of antibody fragments, such as V(H)Hs and V(H)H multimers, may help match the pharmacokinetic profiles of complex venoms, improving antivenom biodistribution, and toxin neutralization while reducing adverse effects in humans
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberSTEWART2007
NPARC number9365599
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Record identifierd28e3059-f81c-44c8-8fce-9947dfb7cc4f
Record created2009-07-10
Record modified2016-05-09
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