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 imm unofluorescence with the phycoerythrin fluorophore to amplify the sign al. The mouse thymoma cell line R1E/TL8x.1.G1.OUA(r).1 (R1FGO), which expresses the kappa(1) but not mu or delta opioid receptors, was used as a positive control for fluorescence labeling. A fluorescein isothio cyanate-conjugated arylacetamide (FITC-AA) compound displaying high af finity for the kappa opioid receptor was synthesized. R1EGO cells were incubated with FITC-AA in the absence or presence of the kappa-select ive opioid antagonist nor-binaltorphimine (nor-BNI) as a competitor. B y using fluorescence microscopy and flow cytometry, incubation of R1EG O cells with FITC-AA alone was not sufficient for the detection of spe cific staining of the kappa opioid receptor. To amplify the FITC-AA fl uorescence, the fluorescein served as a hapten for subsequent antibody detection. R1EGO cells were incubated with FITC-AA, followed by bioti nylated rabbit anti-fluorescein IgG and extravidin-conjugated R-phycoe rythrin. By using this approach, R1EGO cells were stained with phycoer ythrin-amplified FITC-AA, and the staining was displaced with nor-BNI. Flow cytometry showed that titrations of both FITC-AA and nor-BNI pro duced saturable concentration-dependent changes in the median phycoery thrin fluorescence intensity, with optimal staining at 30 mu M 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 amp lify any fluorescein-conjugated opioid ligand for the detection of opi oid receptors.