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

Proteins. 2004 Apr 1;55(1):187-97. doi: 10.1002/prot.20005.


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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Protozoan / chemistry*
  • Antibodies, Protozoan / genetics
  • Antibodies, Protozoan / immunology*
  • Antibody Affinity
  • Antibody Specificity
  • Antigens, Protozoan / immunology*
  • Base Sequence
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology*
  • Membrane Proteins / immunology*
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Library
  • Plasmodium falciparum / immunology*
  • Protein Structure, Tertiary
  • Protozoan Proteins / immunology*
  • Sharks


  • Antibodies, Protozoan
  • Antigens, Protozoan
  • Immunoglobulin Variable Region
  • Membrane Proteins
  • Peptide Library
  • Protozoan Proteins
  • apical membrane antigen I, Plasmodium