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-terminu s of a cysteine-free mutant of aequorin showed no inhibitory effect on the luminescence activity of the photoprotein, This observation is of particula r importance when the use of aequorin as a label in the development of immu noassays 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 a s 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, u sing 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-fr ee mutant of aequorin. It was observed that the C-terminal conjugated aequo rin maintained its luminescence activity. An immunoassay for Leu-enkephalin was then developed using the aequorin-Leu-enkephalin fusion protein as a l abeled 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 an d/or for antibody recognition.