Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.
Biosens Bioelectron
; 12(8): 765-78, 1997.
Article
em En
| MEDLINE
| ID: mdl-9421886
ABSTRACT
A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1(22-38), the C-terminal of ET-1, six analogues of ET-1(16-21) each containing a substitution with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, with less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38). Ala substitution in positions 16, 17 and 19 of ET-1(16-21) hardly affected the antibody binding capacity of ET-1(16-21), whereas Ala substitution of Asp18, Ile20 and, in particular, Trp21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(15-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Mapeamento de Epitopos
/
Endotelina-1
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biosens Bioelectron
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Itália