Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.
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.
Laricchia-Robbio
L
L
Institute of Mutagenesis and Differentiation, C.N.R., Pisa, Italy.
Moscato
S
S
Guidi
A
A
Viganò
S
S
Rovero
P
P
Revoltella
R P
RP
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D019332:Endothelin-1 / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data
D019332:Endothelin-1