Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.
We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.
Coley
A M
AM
Department of Biochemistry, La Trobe University, Bundoora, 3083, Victoria, Australia.
Campanale
N V
NV
Casey
J L
JL
Hodder
A N
AN
Crewther
P E
PE
Anders
R F
RF
Tilley
L M
LM
Foley
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000469:parasitology; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology*; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D004220:Disulfides; D000939:Epitopes; D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D015800:Protozoan Proteins; C090077:apical membrane antigen I, Plasmodium