B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.
Infect Immun
; 66(2): 717-23, 1998 Feb.
Article
en En
| MEDLINE
| ID: mdl-9453632
ABSTRACT
ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Epítopos de Linfocito B
/
Mycobacterium tuberculosis
/
Antígenos Bacterianos
Límite:
Animals
Idioma:
En
Revista:
Infect Immun
Año:
1998
Tipo del documento:
Article
País de afiliación:
Noruega