Identification of kappa opioid receptors in the immune system by indirect immunofluorescence.
A method to visualize the kappa opioid receptor is described that uses a high-affinity fluorescein-conjugated opioid ligand and indirect immunofluorescence with the phycoerythrin fluorophore to amplify the signal. The mouse thymoma cell line R1E/TL8x.1.G1.OUAr.1 (R1EGO), which expresses the kappa 1 but not mu or delta opioid receptors, was used as a positive control for fluorescence labeling. A fluorescein isothiocyanate-conjugated arylacetamide (FITC-AA) compound displaying high affinity for the kappa opioid receptor was synthesized. R1EGO cells were incubated with FITC-AA, in the absence or presence of the kappa-selective opioid antagonist nor-binaltorphimine (nor-BNI) as a competitor. By using fluorescence microscopy and flow cytometry, incubation of R1EGO cells with FITC-AA alone was not sufficient for the detection of specific staining of the kappa opioid receptor. To amplify the FITC-AA fluorescence, the fluorescein served as a hapten for subsequent antibody detection. R1EGO cells were incubated with FITC-AA, followed by biotinylated rabbit anti-fluorescein IgG and extravidin-conjugated R-phycoerythrin. By using this approach, R1EGO cells were stained with phycoerythrin-amplified FITC-AA, and the staining was displaced with nor-BNI. Flow cytometry showed that titrations of both FITC-AA and nor-BNI produced saturable concentration-dependent changes in the median phycoerythrin fluorescence intensity, with optimal staining at 30 microM FITC-AA. Up to 80% of the fluorescence above background was inhibited by nor-BNI. Freshly isolated thymocytes from C57BL/6ByJ mice also showed nor-BNI-sensitive staining with the FITC-AA amplification. This sensitive method of indirect phycoerythrin immunofluorescence can be used to amplify any fluorescein-conjugated opioid ligand for the detection of opioid receptors.
Lawrence
D M
DM
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, NY 14642.
el-Hamouly
W
W
Archer
S
S
Leary
J F
JF
Bidlack
J M
JM
D000081:Acetamides; D000595:Amino Acid Sequence; D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D007107:Immune System / Q000737:chemistry*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009271:Naltrexone / Q000031:analogs & derivatives; D017473:Receptors, Opioid, kappa / Q000032:analysis*; D014407:Tumor Cells, Cultured
D000081:Acetamides; D017473:Receptors, Opioid, kappa; C051844:norbinaltorphimine; D009271:Naltrexone; D016650:Fluorescein-5-isothiocyanate