Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

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.

[1]  S. Daunert,et al.  Using epitope-aequorin conjugate recognition in immunoassays for complex proteins. , 2001, Analytical biochemistry.

[2]  S. Daunert,et al.  Bioluminescence immunoassay for thyroxine employing genetically engineered mutant aequorins containing unique cysteine residues. , 2001, Analytical chemistry.

[3]  J. Kendall,et al.  Aequorea victoria bioluminescence moves into an exciting new era. , 1998, Trends in biotechnology.

[4]  S. Daunert,et al.  An immunoassay for Leu-enkephalin based on a C-terminal aequorin-peptide fusion. , 2001, Analytical chemistry.

[5]  M. Yanagisawa,et al.  The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression. , 1989, The Journal of biological chemistry.

[6]  S. Inouye,et al.  A C‐terminal proline is required for bioluminescence of the Ca2+‐binding photoprotein, aequorin , 1991, FEBS letters.

[8]  T. Masaki,et al.  The Human Preproendothelin‐1 Gene: Possible Regulation by Endothelial Phosphoinositide Turnover Signaling , 1989, Journal of cardiovascular pharmacology.

[9]  S. Daunert,et al.  Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization. , 2000, Bioconjugate chemistry.

[10]  J. Kenten,et al.  Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Daunert,et al.  Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin. , 2000, Analytical biochemistry.

[12]  Osamu Shimomura,et al.  The crystal structure of the photoprotein aequorin at 2.3 Å resolution , 2000, Nature.

[13]  R. Hunter,et al.  A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: quantitative detection of cytokine cDNA in activated and resting human cells. , 1998, Journal of immunological methods.

[14]  S. Daunert,et al.  Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin , 1998 .

[15]  S. Daunert,et al.  Bioluminescence Binding Assay for Biotin with Attomole Detection Based on Recombinant Aequorin , 1994 .

[16]  M. Ohashi,et al.  Mass spectrometric evidence for a disulfide bond in aequorin regeneration , 1993, FEBS letters.

[17]  S. Daunert,et al.  C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development. , 2001, Bioconjugate chemistry.

[18]  M. Oellerich,et al.  Enzyme-immunoassay: a review. , 1984, Journal of clinical chemistry and clinical biochemistry. Zeitschrift fur klinische Chemie und klinische Biochemie.

[19]  Aldo Roda,et al.  Bioluminescence immunoassay for cortisol using recombinant aequorin as a label. , 2002, Analytical biochemistry.

[20]  S. Inouye,et al.  Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin. , 1990, Biochemical and Biophysical Research Communications - BBRC.

[21]  M. J. Cormier,et al.  Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. , 1985, Biochemical and biophysical research communications.

[22]  E. Schiffrin,et al.  Role of endothelin in human hypertension. , 2003, Canadian journal of physiology and pharmacology.