Isolation, characterization and pentamerization of alpha-cobrotoxin specific single-domain antibodies from a naïve phage display library: preliminary findings for antivenom development.
Conventional 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 naïve 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.
Stewart
Christine S
CS
Department of Environmental Biology, University of Guelph, Guelph, Ont., Canada N1G 2W1.
MacKenzie
C Roger
CR
Hall
J Christopher
JC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D000997:Antivenins / Q000276:immunology*; D015153:Blotting, Western; D002161:Camelids, New World / Q000276:immunology*; D003001:Cloning, Molecular; D003039:Cobra Neurotoxin Proteins / Q000276:immunology*; D017931:DNA Primers; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D020349:Surface Plasmon Resonance
D000906:Antibodies; D000997:Antivenins; D003039:Cobra Neurotoxin Proteins; D017931:DNA Primers; D004546:Elapid Venoms; C521753:Naja kaouthia venom; D019151:Peptide Library; C025154:alpha-cobratoxin