title: Cysteine-free mutant of aequorin as a photolabel in immunoassay development.
abstract: The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
authors.forename: Suresh
authors.initials: S
authors.lastname: Shrestha
authors.forename: Insook R
authors.initials: IR
authors.lastname: Paeng
authors.forename: Sapna K
authors.initials: SK
authors.lastname: Deo
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
mesh_terms: D000331:Aequorin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D002138:Calibration; D002316:Cardiotonic Agents / Q000032:analysis / Q000097:blood / Q000276:immunology / Q000493:pharmacokinetics; D003429:Cross Reactions; D003545:Cysteine / Q000235:genetics*; D004077:Digoxin / Q000032:analysis* / Q000097:blood / Q000276:immunology / Q000493:pharmacokinetics; D004306:Dose-Response Relationship, Immunologic; D007118:Immunoassay / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D012680:Sensitivity and Specificity
chemical_list: D002316:Cardiotonic Agents; D000331:Aequorin; D004077:Digoxin; D003545:Cysteine