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.

Exploring Bioluminescence Function of the Ca2+‐regulated Photoproteins with Site‐directed Mutagenesis

“…In contrast to the results obtained on aequorin mutants with one cysteine substituted, cysteine-free aequorin displayed increased bioluminescence activity and was independent of reducing agent upon its conversion into active photoprotein as compared to the wild-type photoprotein (42). This cysteine-free aequorin mutant was proposed as a promising label in binding assays (70,71). Replacement of the conserved Cys158 in obelin to Ser or Ala resulted in decrease in bioluminescence activity as well as in slowing down the decay kinetics and the rate of apoobelin conversion into active photoprotein (57).…”

Aequorin mutants with increased thermostability

“…To study synergistic effects of the multiple mutations in this quadruple mutant the single mutants S32T and E156V were also created, and compared to both WT aequorin and a cysteine free (Cys->Ser) aequorin mutant, Mut S [23]. The resulting single mutants retained between 45-88% the original bioluminescent activity of WT aequorin (Table 1, Column 2).…”

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

“…As a signal generator, bioluminescent reporter proteins have been used as an excellent label, which have advantages of low background, large dynamic range, high sensitivity, and no need of substrate or enhancer in mmunoassay systems. Several immunoassays have been developed for determining digoxin using enzymes and mutant aequorin as a label, [13][14][15] however, it is the first time to develop the bioluminescence immunoassay by employing native aequorin for digoxin assay.…”

“…The resulting aequorin conjugates were characterized by their residual activities. [13][14][15][16] All conjugates were kept at 4 °C until the additions of reagents for activity measurements.…”