Characterizing κ3 opioid receptors with a selective monoclonal antibody

To help characterize κ₃ receptors and establish their relationship to traditional μ and δ receptors, we have generated a κ₃-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing μ, κ₃, and δ opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)-C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of ³H-NalBzoH (κ₃) binding without affecting ³H-DAMGO (μ) or ³H-naltrindole (δ) binding in BE(2)-C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)-C cells by the κ₃ agonist NalBzoH but not the μ agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes κ₃ receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked κ₃ but not morphine (μ) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)-C. These studies demonstrate the selectivity of mAb8D8 for κ₃ receptors and provide additional support for the existence of this unique opioid receptor subtype. © 1996 Wiley-Liss, Inc.