Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples

Abstract

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.

Sensing glucose based on its affinity for concanavalin A on a glassy carbon electrode modified with a C60 fullerene nanocomposite

“…To apply the method for saliva sample detection, saliva sample was collected from healthy subject in the morning and the method measure the salivary glucose level (SGL) of 0.08 mM (n=3). The result obtained here was in agreement with other methods reported in relevant literature [9,42], indicating that this work holds potential application for an useful noninvasive glucose detection method by monitoring salivary glucose level.…”

“…As is well-known, the salivary sample is convenient for collecting and pretreatment. Salivary glucose level (SGL) has been reported in significant concentrations, which is correlated with blood glucose level (BGL) [8,9]. Hence, numerous efforts have been devoted to develop nonenzymatic glucose sensors with high sensitivity and low detection limit [10].…”

Optical biosensor technologies for molecular diagnostics at the point-of-care

“…Here, the measurement signal is directly altered upon binding of analyte molecules, which allows minimizing or even completely skipping sample preparation procedures. 10 Another advantage of homogeneous assays is their faster transition to equilibrium due to the three dimensional diffusion of both the analyte molecule as well as the capture probe.…”

Molecular Probes for Bioluminescence Imaging

“…The optimized homogeneous immunoassay has a detection limit of 1 10 -10 M of free cortisol. This BLI system proved to be a useful tool to measure the concentration of free cortial in saliva for diagnosis of a veriety of diseases caused by abnormal cortisol levels [28]. So et al recently reported a strategy for imaging discrete mRNAs with a transsplicing ribozyme from Tetrahymena thermophilia.…”