Purification of opioid receptors and preparation and application of monoclonal antibodies to them

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Opioid receptors are classified into three different subtypes, g,&, and K.Opioid receptors are classified into three different subtypes, mu,delta, and kappa. However, little is known of molecular basis of mechanisms of pharmacological actions of opioids through such different subtypes. The present works were focused on the preparation of monoclonal antibodies to opioid receptors and application in order to clarify such molecular mechanisms. Neuroblastoma x glioma hybrid NG108-15 cell membranes were immunized into BALB/C mice for 4 months. The spleen cells were fused with myeloma cells and the culture supernatants in wells containing hybridomas were used for assays in delta-opioid binding experiments. delta-OpR-l showed not only a potent inhibition of delta-opioid agonist binding with a decrease in its Bmax value, but also a blocking of delta-agonist-induced inhibition of adenylate cyclase activity. On the other hand, delta-OpR-3 showed an inhibition of delta-opioid agonist binding with a decrease in its affinity. This antibody was found to stain immunocytochemically delta-opioid receptor expressed in NG108-15 cells. These monoclonal antibodies will be useful for purification of delta-opioid receptors or its expression cloning by immunoscreening methods.