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-115-21 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-122-38, the C-terminal of ET-1, six analogues of ET-116-21 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 pollion of ET-1. Antibodies reacting with ET-1 also bound to ET-116-21 and, with less affinity, to ET-3 but did not cross-react with Big. ET-122-38. Ala substitution in positions 16, 17 and 19 of ET-116-21 hardly affected the antibody binding capacity of ET-116-21, 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-115-21 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. 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-115-21 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-122-38, the C-terminal of ET-1, six analogues of ET-116-21 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-116-21 and, with less affinity, to ET-3 but did not cross-react with Big.ET-122-38. Ala substitution in positions 16, 17 and 19 of ET-116-21 hardly affected the antibody binding capacity of ET-116-21, 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-115-21 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.
DETECTION AND EPITOPE MAPPING OF IMMUNOREACTIVE HUMAN ENDOTHELIN-1 USING ELISA AND A SURFACE PLASMON RESONANCE-BASED BIOSENSOR
MOSCATO, STEFANIA;GUIDI, ALESSANDRA;
1997-01-01
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-115-21 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-122-38, the C-terminal of ET-1, six analogues of ET-116-21 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 pollion of ET-1. Antibodies reacting with ET-1 also bound to ET-116-21 and, with less affinity, to ET-3 but did not cross-react with Big. ET-122-38. Ala substitution in positions 16, 17 and 19 of ET-116-21 hardly affected the antibody binding capacity of ET-116-21, 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-115-21 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. 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-115-21 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-122-38, the C-terminal of ET-1, six analogues of ET-116-21 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-116-21 and, with less affinity, to ET-3 but did not cross-react with Big.ET-122-38. Ala substitution in positions 16, 17 and 19 of ET-116-21 hardly affected the antibody binding capacity of ET-116-21, 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-115-21 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
