An Immunoassay for Leu-enkephalin Based on a C-Terminal Aequorin−Peptide Fusion

Deo, Sapna K.; Daunert, Sylvia

doi:10.1021/ac001100q

Cited by 22 publications

(20 citation statements)

References 30 publications

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“…The protein pellet was resuspended and filtered through a 0.2 µm filter; the pH was then adjusted to 7.5. The resuspended protein was then purified with a HQ ion exchange chromatography column (18). Purity of the protein was verified with SDS-PAGE, and concentration was determined with the Bradford Assay.…”

Section: Methodsmentioning

confidence: 99%

Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples

et al. 2007

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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.

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Section: Methodsmentioning

confidence: 99%

Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples

et al. 2007

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“…A calibration curve was generated for each conjugation product and for the unconjugated protein by preparing serially diluted solutions in buffer B and measuring the bioluminescent emission as described above. The linear portion of the flash-type emission profiles was utilized to calculate the half-life of the bioluminescent signal for each cortisol-AEQ mutant S conjugate, as previously reported (17,20).…”

Section: Methodsmentioning

confidence: 99%

Bioluminescence Immunoassay for Cortisol Using Recombinant Aequorin as a Label

Mirasoli

Deo

Lewis

et al. 2002

Analytical Biochemistry

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“…13 This novel protein has been applied in fused proteins for various applications of biosensors in solution. [14][15][16][17][18][19][20] However, for many practical biosensor applications, the luminophore needs to be immobilized on surfaces. Consequently, it is important to study the surface properties of the recombinant aequorin.…”

Section: Introductionmentioning

confidence: 99%

Infrared Reflection−Absorption Spectroscopy and Polarization-Modulated Infrared Reflection−Absorption Spectroscopy Studies of the Aequorin Langmuir Monolayer

et al. 2008

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The Langmuir monolayer of aequorin and apoaequorin was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS techniques. The alpha-helices in the aequorin Langmuir monolayer were parallel to the air-water interface at zero surface pressure. When the surface pressure increased to 15 mN.(m-1), the alpha-helices became tilted and the turns became parallel to the air-water interface. As for apoaequorin, the alpha-helices were also parallel to the air-water interface at 0 mN.m(-1). However, the alpha-helix became tilted and the turns became parallel to the air-water interface quickly at 5 mN.m(-1). With further compression of the apoaequorin Langmuir monolayer, the orientation remained the same. The different behaviors of aequorin and apoaequorin at the air-water interface were explained by the fact that aequorin formed dimers at the air-water interface but apoaequorin was a monomer. It is more difficult for a dimer to be tilted by the compression of the Langmuir monolayer.

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An Immunoassay for Leu-enkephalin Based on a C-Terminal Aequorin−Peptide Fusion

Cited by 22 publications

References 30 publications

Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples

Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples

Bioluminescence Immunoassay for Cortisol Using Recombinant Aequorin as a Label

Infrared Reflection−Absorption Spectroscopy and Polarization-Modulated Infrared Reflection−Absorption Spectroscopy Studies of the Aequorin Langmuir Monolayer

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