Analysis of a human monoclonal antibody reactive with multiple Plasmodium falciparum antigen repeat sequences using a solid phase affinity assay.
A solid-phase affinity assay was set up for the determination of the affinity of the interaction between the human monoclonal antibody (mAb) 33G2 and peptides corresponding to repeated sequences in three blood stage antigens of the malaria parasite Plasmodium falciparum. The epitope of this mAb is of interest due to the parasite blocking capacity of the mAb. Previous studies with PEPSCAN have defined the minimal epitope for the mAb as the pentapeptide VTEEI, a sequence frequently found in antigen Pf332. In the previous study, epitopes responsible for the cross-reactivity of the mAb with antigens Pf155/RESA and Pf11.1 were also identified. In the affinity assay described herein, the mAb was coated on a solid phase and binding of a labelled peptide was displaced by homologous or heterologous peptides. The affinity of peptides corresponding to Pf332 increased with increasing length, and the highest affinity was displayed by a dimer (23 amino acids) of a Pf332 repeat (K = 1.9 x 10(8) M-1). Peptide length did not influence the binding of peptides corresponding to the Pf155/RESA and Pf11.1 repeats, which had lower affinities comparable to that of the shortest Pf332 octapeptide (K = 2.2 x 10(4) M-1). Only peptides containing binding sites as defined by PEPSCAN analysis showed a measurable binding. When using peptides as inhibitors in peptide ELISA, binding correlated with the affinity of the peptides, but only the high affinity peptides were inhibitory. In contrast, a poor correlation was found when peptides were used directly for coating in ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)
Ahlborg
N
N
Department of Immunology, Stockholm University, Sweden.
Larsson
A
A
Perlmann
P
P
Berzins
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000913:Antibodies, Protozoan / Q000276:immunology*; D000915:Antibody Affinity; D000953:Antigens, Protozoan / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology*; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D010455:Peptides