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Salivary Cortisol

Salivary Cortisol Quick Start Research Guide

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Biomarkers

1. How to collect Salivary Cortisol

APPROVED SALIVARY CORTISOL COLLECTION METHODS

Salivary Cortisol Collection Protocol

Collection volume, general considerations, and basic guidelines to maximize salivary cortisol sample integrity. Use this analyte-specific collection protocol to plan you collection methodology and sampling schemes.

Biomarkers

2. How to Assay for Salivary Cortisol

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Order Code5100

Assay Protocol
Rev. 02.12.21
Specifications
Catalog#: 1-3002
Regulatory Status: RUO
Format: 96-well plate
Assay Time: ~ 2 hrs
Sample Volume/Test: 25 µL
Sensitivity: <0.007 ug/dL
Assay Range: 0.012-3.000 ug/dL
Storage Requirements: 2-8°C
Tests Per Kit
Singlet: 76
Duplicate: 38
Target Analyte

Technical Documentation

Assay Kit Overview

Intended Use

The Salimetrics® Cortisol Enzyme Immunoassay Kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of salivary cortisol. It is not intended for diagnostic use. It is intended only for research use in humans and some animals. Salimetrics has not validated this kit for serum or plasma samples.

Introduction

Cortisol (hydrocortisone, Compound F) is the major glucocorticoid produced in the adrenal cortex. Cortisol production has a circadian rhythm, with levels peaking in the early morning and dropping to lowest values at night. Levels rise independently of circadian rhythm in response to stress.

In blood, only about 5-10% of cortisol is in its unbound or biologically active form. The remaining cortisol is bound to serum proteins. Unbound serum cortisol enters saliva via intracellular mechanisms; in saliva, the majority of cortisol remains unbound to protein. Salivary cortisol levels are unaffected by salivary flow rate and are relatively resistant to degradation from enzymes or freeze-thaw cycles. Studies consistently report high correlations between serum and salivary cortisol, indicating that salivary cortisol levels reliably estimate serum cortisol levels.

Normal Diurnal Cortisol Rhythm

Cortisol Assay Principle

This is a competitive immunoassay kit. Cortisol in standards and samples compete with cortisol conjugated to horseradish peroxidase for the antibody binding sites on a microtitre plate. After incubation, unbound components are washed away. Bound cortisol enzyme conjugate is measured by the reaction of the horseradish peroxidase enzyme to the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction with an acidic solution. The optical density is read on a standard plate reader at 450 nm. The amount of cortisol enzyme conjugate detected is inversely proportional to the amount of cortisol present, in the sample.

Assay Protocol
Rev. 02.12.21
Specifications
Catalog#: 1-3102
Regulatory Status: 510(k), CE Mark
Format: 96-well plate
Assay Time: ~ 2 hrs
Sample Volume/Test: 25 µL
Sensitivity: <0.007 ug/dL
Assay Range: 0.012-3.000 ug/dL
Storage Requirements: 2-8°C
Tests Per Kit
Singlet: 76
Duplicate: 38
Target Analyte

Technical Documentation

Biomarkers

3. Technical Summary

Analyte Summary
Analyte: Cortisol
Aliases: hydrocortisone, Compound F
Serum-Saliva Correlation: 0.91
Optimum Collection Volume: 75 μL*
*Add 300 µl to the total volume of all tests for liquid handling
Interfering Factors
Bovine hormones normally present in dairy products can cross-react with anti-cortisol antibodies and cause false results.
Hormone based oral contraceptives and estrogens can cause temporary increase in CBG, potentially lowering cortisol levels
in saliva.
Multiple medications mimic the structure of cortisol and have the potential to influence cortisol levels in saliva.
Assay Summary
Methodology: ELISA
Sensitivity: <0.007 ug/dL
Assay Range: 0.012-3.000 ug/dL
Assay Type: Quantitative
*Salivary Cortisol Example Ranges
Group Number Overall Range (ug/dL)
Children, neonatal 275 ND - 3.417
Children, age 6 months 165 ND - 2.734
Group Number Overall Range (ug/dL)
Normal subjects 19 0.007 - 0.115
Cushing's subjects 21 0.130-2.972
*Note: Examples are to be used as a guide only. Each laboratory should estabilsh its own range.

Background

Cortisol (hydrocortisone, Compound F) is the major glucocorticosteroid hormone produced in the adrenal cortex. Cortisol is actively involved in the regulation of calcium absorption, blood pressure maintenance, anti-inflammatory function, gluconeogenesis, gastric acid, pepsin secretion, and immune function. (1,2,3) Cortisol production has a circadian rhythm. (4) Levels peak in the early morning and drop to the lowest concentration at night. (5) Levels rise independently of circadian rhythm in response to stress. (6) Increased cortisol production is associated with Cushing’s syndrome and adrenal tumors, while decreased cortisol production is associated with adrenal insufficiency (e.g., Addison’s disease) and adrenocorticotropic hormone (ACTH) deficiency. (7) In the blood only 1 to 15% of cortisol is in its unbound or biologically active form. The remaining cortisol is bound to serum proteins. (8) Unbound serum cortisol enters the saliva via intracellular mechanisms, and in saliva the majority of cortisol remains unbound to protein. (9) Salivary cortisol levels are unaffected by salivary flow rate or salivary enzymes. (10) Studies consistently report high correlations between serum and saliva cortisol, indicating that salivary cortisol levels reliably estimate serum cortisol levels. (11,12,13)

References & Salivary Cortisol Research

    1. Migeon, C.J., & Lanes, R.L. (1990). Adrenal cortex: hypo- and hyperfunction. In F. Lifshitz (ed.), Pediatric endocrinology, a clinical guide (2nd ed.), (pp. 333-52). New York: Marcel Dekker.
    2. Drucker, S., New, M.I. (1987).  Disorders of adrenal steroidogenesis. Pediatr Clin North Am, 34(4), 1055-66.
    3. Fischbach, F.T. (1992). The manual of laboratory and diagnostic tests, (4th ed.). Philadelphia: J. B. Lippincott.
    4. Dorn, L.D., Lucke, J.F., Loucks, T.L., Berga, S.L. (2007). Salivary cortisol reflects serum cortisol: analysis of circadian profiles. Ann Clin Biochem, 44(pt 3), 281-84.
    5. Chernow, B., Alexander, H.R., Smallridge, R.C., et al. (1987). Hormonal responses to graded surgical stress. Arch Intern Med, 147(7), 1273-78.
    6. Kreiger, D.T. (1975). Rhythms of ACTH and corticosteroid secretion in health and disease and their experimental modification. J Steroid Biochem, 6(5), 758-91.
    7. Rothfield, B. (1974). Plasma cortisol. In: B. Rothfield (ed.), Nuclear medicine–in vitro (pp. 120-5). Philadelphia: Lippincott.
    8. Robin, P., Predine, J., Milgrom, E. (1977). Assay of unbound cortisol in plasma. J Clin Endocrinol Metab, 46(2), 277-83.
    9. Vining, R.F., McGinley, R.A., Symons, R.G. (1983). Hormones in saliva: mode of entry and consequent implications for clinical interpretation. Clin Chem, 29(10), 1752-56.
    10. Vining, R.F., McGinley, R.A. (1987). The measurement of hormones in saliva: Possibilities and pitfalls. J Steroid Biochem, 27(1-3), 81-94.

  1. Francis, S.J., Walker, R.F., Riad-Fahmy, D., et al. (1987). Assessment of adrenocortical activity in term newborn infants using salivary cortisol determinations. J of Pediatrics, 111, 129-33.
  2. Hiramatsu, R. (1981). Direct assay of cortisol in human saliva by solid phase radioimmunoassay and its clinical applications. Clinica Chimica Acta, 117, 239-249.
  3. Vining, R.F., McGinley, R.A., Maksvytis, J.J., Ho, K.Y. (1983). Salivary cortisol: A better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem, 20(pt 6), 329-35.

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