Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Laura Rowe, Sapna K Deo, Josh Shofner, Mark Ensor, Sylvia Daunert

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 × 10-10 M of free cortisol, with a linear dynamic range spanning from 1 × 10-5 to 1 × 10-9 M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 × 10-7 M to 7.5 × 10-7 M and 1.0 × 10-8 M to 2.5 × 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.

Original languageEnglish
Pages (from-to)1772-1777
Number of pages6
JournalBioconjugate Chemistry
Volume18
Issue number6
DOIs
StatePublished - Nov 1 2007
Externally publishedYes

Fingerprint

Aequorin
Cortisol
Saliva
Immunoassay
Hydrocortisone
Assays
Antibodies
Bioluminescence
Hormones
Luminescent Proteins
Miniaturization
Competitive Binding
Automation
Washing
Lysine
Limit of Detection

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples. / Rowe, Laura; Deo, Sapna K; Shofner, Josh; Ensor, Mark; Daunert, Sylvia.

In: Bioconjugate Chemistry, Vol. 18, No. 6, 01.11.2007, p. 1772-1777.

Research output: Contribution to journalArticle

Rowe, Laura ; Deo, Sapna K ; Shofner, Josh ; Ensor, Mark ; Daunert, Sylvia. / Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples. In: Bioconjugate Chemistry. 2007 ; Vol. 18, No. 6. pp. 1772-1777.
@article{bbf913931fa742e98e1c8dd8986c335a,
title = "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 × 10-10 M of free cortisol, with a linear dynamic range spanning from 1 × 10-5 to 1 × 10-9 M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 × 10-7 M to 7.5 × 10-7 M and 1.0 × 10-8 M to 2.5 × 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.",
author = "Laura Rowe and Deo, {Sapna K} and Josh Shofner and Mark Ensor and Sylvia Daunert",
year = "2007",
month = "11",
day = "1",
doi = "10.1021/bc070039u",
language = "English",
volume = "18",
pages = "1772--1777",
journal = "Bioconjugate Chemistry",
issn = "1043-1802",
publisher = "American Chemical Society",
number = "6",

}

TY - JOUR

T1 - Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

AU - Rowe, Laura

AU - Deo, Sapna K

AU - Shofner, Josh

AU - Ensor, Mark

AU - Daunert, Sylvia

PY - 2007/11/1

Y1 - 2007/11/1

N2 - 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 × 10-10 M of free cortisol, with a linear dynamic range spanning from 1 × 10-5 to 1 × 10-9 M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 × 10-7 M to 7.5 × 10-7 M and 1.0 × 10-8 M to 2.5 × 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.

AB - 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 × 10-10 M of free cortisol, with a linear dynamic range spanning from 1 × 10-5 to 1 × 10-9 M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 × 10-7 M to 7.5 × 10-7 M and 1.0 × 10-8 M to 2.5 × 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.

UR - http://www.scopus.com/inward/record.url?scp=36849048903&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36849048903&partnerID=8YFLogxK

U2 - 10.1021/bc070039u

DO - 10.1021/bc070039u

M3 - Article

VL - 18

SP - 1772

EP - 1777

JO - Bioconjugate Chemistry

JF - Bioconjugate Chemistry

SN - 1043-1802

IS - 6

ER -