Integrity of tritiated orphanin FQ/nociceptin: implications for establishing a reliable binding assay

In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor the integrity of [(3)H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [(3)H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [(3)H]OFQ/N was significantly compromised in the presence of COS-7 cell membranes. Interestingly, if the peptide was added to COS-7 membranes after they had been exposed to low pH it remained intact, suggesting that the peptide's breakdown during binding is, in part, enzymatically mediated. Although a variety of protease inhibitors were tested, none proved completely effective at protecting the tritiated peptide. The intention of the studies presented here was to evaluate OFQ/N binding components, namely the available [(3)H]OFQ/N ligands, in an effort to standardize the binding conditions for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay.