Two different 8 kDa monomers are involved in the oligomeric organization of the native Echinococcus granulosus antigen B.
The present work describes the purification and characterization of antigen B (AgB), the thermostable lipoprotein from E. granulosus. Native AgB was purified to homogeneity by a new strategy involving adsorption on DEAE-Sepharose, followed by immunopurification. The purified antigen was analysed using mapped monoclonal antibodies (MoAbs) and peptide isolation by in situ digestion in gels after SDS-PAGE. Epitope mapping of 7 MoAbs using PEPSCAN, synthetic peptides and competition studies, revealed that six of them defined epitopes which clustered the N-terminal extension of a 8 kDa subunit of AgB, whilst the remaining one reacted against the stretch RGLIAEGE, corresponding to the C-terminus. The epitopes defined by the seven MoAbs were found to be present in all the subunits. Furthermore, the similarities of the peptide finger prints obtained by HPLC analysis and amino acid sequencing of tryptic peptides isolated from the 8, 16 and 24 kDa subunits, indicated that they have most if not all the amino acid sequence in common. We also found evidence that the band representing a component of an apparent molecular weight of 8 kDa in SDS-PAGE, believed to be the smallest subunit of AgB, contained at least two components, which may constitute the building blocks of the higher molecular weight subunits.
González
G
G
Cátedra de Inmunología, Facultad de Química, Universidad de la República, Montevideo, Uruguay.
Nieto
A
A
Fernández
C
C
Orn
A
A
Wernstedt
C
C
Hellman
U
U
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D004446:Echinococcus / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D015801:Helminth Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D008074:Lipoproteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D015801:Helminth Proteins; D008074:Lipoproteins; C089273:antigen B, Echinococcus