Monoclonal Antibodies to Human Endothelin-1: Characterization and Utilization in Radioimmunoassay and Immunocytochemistry

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Abstract
A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, β-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the C-terminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:106. The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG γI, two were IgM and one of the IgG γ2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.