TY - JOUR
T1 - Bioluminescence immunoassay for cortisol using recombinant aequorin as a label
AU - Mirasoli, Mara
AU - Deo, Sapna K.
AU - Lewis, Jennifer C.
AU - Roda, Aldo
AU - Daunert, Sylvia
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health (4-63685). S.D. is a Cottrell Scholar and a Lilly Faculty Awardee. S.K.D. thanks the University of Kentucky for an RCTF Fellowship in Biological Chemistry. J.C.L. thanks the National Science Foundation and the University of Kentucky for an NSF-IGERT Fellowship.
PY - 2002/7/15
Y1 - 2002/7/15
N2 - The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4°C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 μl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.
AB - The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4°C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 μl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.
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U2 - 10.1006/abio.2002.5695
DO - 10.1006/abio.2002.5695
M3 - Article
C2 - 12123657
AN - SCOPUS:0037099632
VL - 306
SP - 204
EP - 211
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -