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

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, beta-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 1:10(6). The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG gamma 1, two were IgM and one of the IgG gamma 2a 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.

A Role for Endothelin-2 and Its Receptors in Breast Tumor Cell Invasion

“…Sections from representative paraffin blocks of tumors were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity using 3% H 2 O 2 . Mature ETs were detected using a rabbit antimouse ET polyclonal antibody (Oncogene Research Products, Cambridge, MA) that recognizes all ET isoforms (18). Sections were also immunostained with antibodies raised against the COOH terminus of the ET receptors (19).…”

Endothelin-1 and Its mRNA in the Wall Layers of Human Arteries Ex Vivo

“…To visualize the distribution of irET-1 peptide, the following primary antibodies were used: rabbit anti-ET-1 antiserum (Peninsula Laboratories Inc) that shows a 7% cross-reactivity for ET-2 and ET-3 and a 17% cross-reactivity for human big ET-1 (proendothelin-1), 17,18 a monoclonal anti-ET-1 antibody (IgG1) 19 (ABR) directed against an epitope encompassing amino acids 8 to 16 of ET-1. The latter antibody has Ͻ10% cross-reactivity for ET-2 and Ͻ10% for ET-3, and no cross-reactivity to nonrelated peptides.…”

Immature endothelial cells initiate endothelin‐mediated constriction of newborn arteries

“…Because the antibody recognizes both ET‐1 and its immediate precursor, Big ET‐1 (Traish et al . ), the immunofluorescence signal is described as (Big)ET‐1 to account for potential contributions from ET‐1 and Big ET‐1. (Big)ET‐1 immunofluorescence was characterized by prominent granular and perinuclear staining in P1 arterial endothelium (Fig.…”

“…The antibody used for immunofluorescence imaging of ET‐1 has a similar affinity for ET‐1 and its immediate precursor, Big ET‐1 (Traish et al . ; Wolf et al . ).…”