Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii.
We have characterized in detail, the epitope of the secreted antigen GP28.5 recognized by the mouse monoclonal antibody TG 17-179 using synthetic peptides and a truncated recombinant fusion protein. The screening of a T. gondii expression library with TG17-179 mAb led to the isolation of cDNAs clones, all encoding for the C-terminal region of GP28.5 and with one encoding for only the five last C-terminal residues. Competitive ELISA with longer peptides revealed that the immunoreactivity was retained for peptides of eight residues or longer, and lost when the peptide was reduced to the six last C-terminal residues or less. Experiments with the octapeptide lacking the carboxy-terminal glutamine residue showed it to be 64-fold less active. Moreover, neither addition of residues in the carboxy-end nor substitution of COOH function changed the immunoreactivity of the epitope. Competition experiments between TG17-179 mAb and sera from infected individuals demonstrates that the epitope defined by a mouse monoclonal probe is also a major epitope for human polyclonal antibodies. These results describe the sequence requirements within a probably linear epitope and give rise to some general question concerning experimental test for epitope mapping.
Cesbron-Delauw
M F
MF
Centre d'Immunologie et de Biologie Parasitaire, Unité Mixte INSERM U167-CNRS 624, Institut Pasteur Lille, France.
Boutillon
C
C
Mercier
C
C
Fourmaux
M P
MP
Murray
A
A
Miquey
F
F
Tartar
A
A
Capron
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D000953:Antigens, Protozoan / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003429:Cross Reactions; D004247:DNA / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015183:Restriction Mapping; D014122:Toxoplasma / Q000276:immunology*; D014123:Toxoplasmosis / Q000276:immunology; D001616:beta-Galactosidase / Q000276:immunology
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D004247:DNA; D001616:beta-Galactosidase