Characterization of a polyclonal antibody to the mu opioid receptor.
Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the mu receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [3H]etorphine (universal opioid) and [3H]dihydromorphine (mu opioid) from rat membranes, but was ineffective against the binding of [3H]ethylketocyclazocine (kappa [3H]D-Ala2,D-Leu5-enkephalin (delta opioid) or [3H]phencyclidine (phencyclidine/sigma receptor ligand). The antibody was able to precipitate the Mr 94,000 component of the 125I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma X Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.
Maneckjee
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Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.
Archer
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Zukin
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D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D003342:Corpus Striatum / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D006822:Hybrid Cells; D009294:Narcotics / Q000378:metabolism; D009447:Neuroblastoma; D011817:Rabbits; D051381:Rats; D011957:Receptors, Opioid / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D017450:Receptors, Opioid, mu; D013574:Synaptosomes / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000032:analysis
D000906:Antibodies; D009294:Narcotics; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu