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Proceedings Paper

Cloning and expression of an endothelin-1-aequorin fusion protein as bioluminescent a tracer for endothelin-1 immunoassays
Author(s): Mara Mirasoli; Elisa Michelini; Sapna K Deo; Emre Dikici; Aldo Roda; Sylvia Daunert
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Paper Abstract

The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

Paper Details

Date Published: 14 June 2004
PDF: 8 pages
Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); doi: 10.1117/12.529194
Show Author Affiliations
Mara Mirasoli, Univ. di Bologna (Italy)
Elisa Michelini, Univ. di Bologna (Italy)
Sapna K Deo, Univ. of Kentucky (United States)
Emre Dikici, Univ. of Kentucky (United States)
Aldo Roda, Univ. di Bologna (Italy)
Sylvia Daunert, Univ. of Kentucky (United States)

Published in SPIE Proceedings Vol. 5329:
Genetically Engineered and Optical Probes for Biomedical Applications II
Alexander P. Savitsky; Darryl J. Bornhop; Ramesh Raghavachari; Samuel I. Achilefu, Editor(s)

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