An immunoassay for Leu-enkephalin based on a C-terminal aequorin-peptide fusion

Recently we demonstrated that the fusion of an octapeptide to the C-terminus of a cysteine-free mutant of aequorin showed no inhibitory effect on the luminescence activity of the photoprotein. This observation is of particular importance when the use of aequorin as a label in the development of immunoassays for peptides whose activity lies in their C-terminal region or the epitope for antibody recognition is at their C-terminus is desired. In the case of opioid peptides, antibodies are directed toward their C-terminus as they differ from each other at this terminus. The goal of this study was to develop an immunoassay for Leu-enkephalin, a mammalian opioid peptide, using a C-terminal aequorin-peptide fusion protein. For that, the N-terminus of Leu-enkephalin was genetically fused to the C-terminus of a cysteine-free mutant of aequorin. It was observed that the C-terminal conjugated aequorin maintained its luminescence activity. An immunoassay for Leu-enkephalin was then developed using the aequorin-Leu-enkephalin fusion protein as a labeled analyte in a competitive as well as in a sequential binding mode. It was demonstrated that aequorin can be used as a label in peptide assays in which it is critical that the peptide's C-terminus be free for activity and/or for antibody recognition.