Enzyme-linked immunosorbent assay for leucine and methionine enkephalins

Zamboni, Giulia; Jones, Carol

doi:10.1016/0003-2697(83)90652-8

Cited by 10 publications

(4 citation statements)

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“…The detection limit obtained from this assay (3 × 10 -10 M, 3 fmol) is 1 order of magnitude lower compared to that of an enzyme immunoassay (14 nmol/L) and fluorescence assay (360 fmol/injection) reported for Leu-enkephalin. 26,27 Moreover, this detection limit is well within the levels of Leu-enkephalin in biological samples (1-18 pmol/g of brain). 30 It should be noted, however, that, if desired, lower detection limits could be achieved by lowering the concentration of antibody and aequorin-Leu-enkephalin fusion protein employed.…”

Section: Resultssupporting

confidence: 61%

“…A highly sensitive immunoassay for the neuropeptide Leu-enkephalin with antibody recognition at its C-terminus was developed. The detection limit obtained from this assay (3 × 10 -10 M, 3 fmol) is 1 order of magnitude lower compared to that of an enzyme immunoassay (14 nmol/L) and fluorescence assay (360 fmol/injection) reported for Leu-enkephalin. , Moreover, this detection limit is well within the levels of Leu-enkephalin in biological samples (1−18 pmol/g of brain) …”

Section: Resultsmentioning

confidence: 52%

“…Radioimmunoassays (RIA) and enzyme immunoassays have been reported in the literature for quantification of Leu-enkephalin. − Other assays developed separate the peptides using high-performance liquid chromatography (HPLC) followed by either RIA detection or fluorescence detection of postderivatized peptides. − RIA is a highly sensitive technique but has the disadvantages associated with the use of radioactivity. It is important to note that low therapeutic concentrations of the peptides cannot be measured by enzyme immunoassays with colorimetric detection, and they also suffer from background interference.…”

Section: Resultsmentioning

confidence: 99%

“…Radioimmunoassays (RIA) and enzyme immunoassays have been reported in the literature for quantification of Leu-enkephalin. [24][25][26] Other assays developed separate the peptides using high-performance liquid chromatography (HPLC) followed by either RIA detection or fluorescence detection of postderivatized peptides. [27][28][29] RIA is a highly sensitive technique but has the disadvantages associated with the use of radioactivity.…”

Section: Resultsmentioning

confidence: 99%

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An Immunoassay for Leu-enkephalin Based on a C-Terminal Aequorin−Peptide Fusion

Deo

Daunert

2001

Anal. Chem.

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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.

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Section: Resultssupporting

confidence: 61%