Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1

摘要: The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer two protein chains, each containing single variable five constant do- mains. individual (VNAR) domains bind independently, are candidates for the smallest antibody-based immune recognition units. We have previously produced library VNAR with extensive variability in CDR1 CDR3 loops displayed on surface bacterio- phage. Now, test efficacy this library, further explore dynamics bind- ing we performed selection experiments against infectious disease target, malarial Apical Membrane Antigen-1 (AMA1) from Plasmo- dium falciparum. Two related clones were selected, characterized by long (16- 18-residue) loops. These recombinant VNARs could be harvested at yields approaching 5mg/L mono- meric E. coli periplasm, bound AMA1 nanomolar affinities (KD 2 10 7 M). One clone, designated 12Y-2, was affinity-matured error prone PCR, resulting several variants mutations mapping best these showed 10-fold enhanced affinity over 12Y-2 Plasmodium falciparum strain-specific. Importantly, demon- strated that monovalent co-localized rabbit anti-AMA1 antisera parasites thus may utility diagnostic applications. Proteins 2004;55:187-197.