Application of Competitive ELISA Assay for Serum Cortisol

Paper Info
Page count 4
Word count 1238
Read time 7 min
Topic Health
Type Research Paper
Language 🇺🇸 US


Cortisol is one of the essential steroid hormones, and it has multiple functions in the human body. Traditionally defined as a stress hormone, it is synthesized from cholesterol in the adrenal cortex’s zona fasciculata layer (Thau et al., 2021). In general, the majority of cells have cortisol receptors, and cortisol affects almost every organ system, including immune, nervous, respiratory, cardiovascular, musculoskeletal, reproductive, and integumentary ones (Thau et al., 2021). The main functions of this steroid hormone are the mediation of the stress response, immune function, inflammatory response, and the regulation of metabolism. It fights infection, maintains blood pressure, and regulates blood sugar.

The measurement of cortisol is highly essential to prevent multiple health problems. In general, cortisol levels may change within minutes during a day in response to stress (McEwen, 2019). However, stable high or low levels may indicate severe disorders, such as adrenal fatigue, insufficiency, or incidentaloma, adrenocortical carcinoma, Cushing syndrome, Cushing disease, congenital adrenal hyperplasia, and pituitary tumors (“What is cortisol?,” 2018). Thus, this paper focuses on cortisol measurement, and its purpose is to examine the applicability of the competitive ELISA assay for serum cortisol detection. On the basis of modern peer-reviewed articles, the work initially describes the methods of cortisol measurement and the techniques for its preparation, investigates competitive ELISA, and evaluates its expediency.

Methods to Analyze Cortisol in Human Serum

Cortisol is measured from biological samples, including serum, plasma, urine, and saliva.

The serum is regarded as the most common and suitable for routine examination (Chen et al., 2020). At the same time, there are multiple classifications of methods provided by different researchers. According to El-Farhan et al. (2017), total cortisol in human serum is measured and analyzed through serum control assays divided into immunoassays, mass spectrometers (GC-MS and LC-MS/MS methods), and free cortisol essays (equilibrium dialysis, ultrafiltration, and gel filtration). Nieman (2019) defined several methods of serum cortisol measurement and also mentioned those ones which are not used anymore: Porter-Silber chromogens, fluorometric, radioreceptor, competitive protein-binding, structurally-based assays, radioimmunoassay, and other immunoassays.

According to Hawley et al. (2016), in the present day, a prevalent number of laboratories use automated commercial immunoassays for cortisol measurement and analysis. For their research, scientists applied Abbott Architect, Roche E170 Generation I, Beckman Access, Siemens Centaur XP, and Roche E170 Generation II to analyze samples (Hawley et al., 2016). In general, all methods are highly essential for the early detection of serious diseases connected with extremely high or low cortisol levels in the human body.

Extraction of Sample LLE and SPE

For the measurement of cortisol, biological samples should be initially prepared with the use of various techniques. In order to achieve adequate cortisol levels and selectivity, sample preparation should include several essential steps, such as extraction, clean-up, and pre-concentration (Abujaber et al., 2019). Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are among the most applicable techniques designed for sample preparation (Chen et al., 2020). LLE “employs water-immiscible solvents to extract analytes from aqueous solutions” (Danaceau et al., 2017, p. 1). It is traditionally performed by shaking and subsequently collecting the layer that contains the analytes of scientists’ interest. In turn, for SPE, compounds suspended or dissolved in a liquid matrix are extracted in accordance with their chemical and physical properties (Danaceau et al., 2017).

Despite their efficiency, both LLE and SPE are time-consuming and laborious techniques that require organic solvents (Abujaber et al., 2019). In the present day, these solvents are gradually replaced by environmentally friendly ionic liquids (Bakusic et al., 2019). Nevertheless, SPE and LLE are still applied in practice and scientific research to prepare samples for cortisol measurement.

Competitive ELISA and Comparison with Other Methods

Competitive ELISA is an immunoassay kit for quantitative cortisol determination. In general, it is designed to measure cortisol in serum, plasma, saliva, urine, tissue culture media samples, and dried fecal extracts (“Cortisol Competitive Human ELISA Kit,” n.d.). Free cortisol is measured in urine and saliva, while total cortisol – is in serum, plasma, and extracted samples (Ozgocer et al., 2017). With the use of this assay, cortisol is measured not only for humans but for animals, including cattle. Camels, and dogs, as well (Nejad et al., 2020). As previously mentioned, competitive ELISA belongs to the group of immunoassays, and regardless of certain differences between them, this kit has common characteristics (El-Farhan et al., 2017).

Cortisol and this hormone’s derivatives were historically measured with the use of immunoaffinity-based methods, such as chemiluminescent immunoassay, immunochromatographic test, radioimmunoassay, electrochemiluminescence immunoassay fluorescence assays, immunosensors, or sensors, and ELISA assay (Boolani et al., 2019). However, in the present day, mass spectrometry-based methods, including an LC-MS/MS method, are used to measure cortisol and other steroid hormones (Domenech-Coca et al., 2019). According to a lot of researchers, mass methods outperform immunoassays in sensitivity and specificity (El-Farhan et al., 2017). At the same time, immunoassays are still used massively, as other methods remain cost-inefficient, equipment-requiring, and time-consuming.

Advantages and Disadvantages of Competitive ELISA

First of all, competitive ELISA is a cost-efficient and accessible method of measuring cortisol. According to Gholib and Agil (2019), the efficiency of the ELISA kit is validated through accuracy, parallelism, and precision tests. ELISA standards are frequently used to evaluate the accuracy of new methods (Sanghavi et al., 2016). The provision of accurate results in the measurement of cortisol using a commercial ELISA kit was supported by the research conducted by Naveen et al. (2016) that focused on the examination of yoga’s impact on depression. At the same time, competitive ELISA cannot be regarded as a highly time-sensitive method that allows continuous monitoring of cortisol levels in human serum (Evans et al., 2021). In addition, the results of commercial portable kits should be checked in laboratory settings if precise measurement is required.

Interferences in Immunoassay

In the majority of cases, immunoassay interferences are caused by substances that may change the analyte’s measurable concentration and the reaction between reagent antibodies and analyte on immunoassay. According to Ward et al. (2017), analyte-dependent interferences occur due to cross-reacting substances, heterophile antibodies, human anti-animal antibodies, autoantibodies, and a high dose hook effect. With cortisol, there may be cross-reactivity due to small structural differences that will inevitably corrupt the accuracy of results. That is why the results of immunoassays should be checked in laboratory settings with the use of other methods, especially in the case of ambiguous results.


Cortisol is the steroid hormone highly essential for human health that affects almost all body systems and has multiple functions. Too high or too low cortisol levels may cause severe diseases, and that is why it is essential to measure this hormone. There are multiple methods of cortisol measurement in serum, saliva, and other samples, and immunoassays remain the most common in laboratory settings. Competitive ELISA is an accessible immunoassay kit used for free and total cortisol quantitative determination. However, despite its considerable affordability and accuracy, the ELISA assay requires development to be less time-consuming, more reliable, and applicable for monitoring in order to be competitive with mass methods and other assays.

In general, there are multiple peer-reviewed articles dedicated to cortisol and the methods of its measurement. However, there is a lack of modern research that specifically addresses, describes, and evaluates competitive ELISA. In general, this work may be regarded as a basis of further scientific activity that aims to investigate how ELISA assay may be developed in order to become the most efficient and affordable cortisol measurement technique.


Abujaber, F., Ricardo, A. I. C., Ríos. A., Bernardo, F. J. G., & Martín-Doimeadios, R. C. R. (2019). Ionic liquid dispersive liquid-liquid microextraction combined with LC-UV-Vis for the fast and simultaneous determination of cortisone and cortisol in human saliva samples. Journal of Pharmaceutical and Biomedical Analysis, 165, 141-146. Web.

Bakusic, J., De Nys, S., Creta, M., Godderis, L., & Duca, R. C. (2019). Study of temporal variability of salivary cortisol and cortisone by LC-MS/MS using a new atmospheric pressure ionization source. Scientific Reports, 9, 1-12. Web.

Boolani, A., Channaveerappa, D., Dupree, E. J., Jayathirtha, M., Aslebagh, R., Grobe, S., Wilkinson, T., & Darie, C. C. (2019). Trends in analysis of cortisol and its derivatives. In A. G. Woods & C. C. Darie (Eds.), Advancements of Mass Spectrometry in Biomedical Research (pp. 649-664). Springer.

Chen, B., Lyu, H., Xu, X., & Wang, C. (2020). Simultaneous quantification of cortisol and cortisone in serums and saliva from depressive patients by supported liquid extraction coupled to HPLC–MS/MSs. Acta Chromatographica, 32(4), 269-275. Web.

Cortisol Competitive Human ELISA Kit. (n.d.). ThermoFisher Scientific. Web.

Danaceau, J. P., Haynes, K., & Chambers, E. E. (2017). A comprehensive comparison of solid phase extraction (SPE) vs. solid liquid extraction (SLE) vs. liquid liquid extraction (LLE) sample prep techniques in bioanalysis and forensic toxicology analyses [PDF document]. Waters Corporation. Web.

Domenech-Coca, C., Mariné-Casadó, R., Caimari, A., Arola, L., Maria del Bas, J., Bladé, C., & Rodriguez-Naranjo, M. I. (2019). Dual liquid-liquid extraction followed by LC-MS/MS method for the simultaneous quantification of melatonin, cortisol, triiodothyronine, thyroxine and testosterone levels in serum: Applications to a photoperiod study in rats. Journal of Chromatography B, 1108, 11-16. Web.

El-Farhan, N., Rees, D. A., & Evans, C. (2017). Measuring cortisol in serum, urine and saliva – are our assays good enough? Annals of Clinical Biochemistry, 54(3), 308-322. Web.

Evans, G. W. H., Bhuiyan, W. T., Pang, S., Warren, B., Makris, K., Coleman, S., Hassan, S., & Niu, X. (2021). A portable droplet microfluidic device for cortisol measurements using a competitive heterogeneous assay. Analyst, 146, 4535-4544. Web.

Gholib, G., & Agil, M. (2019). The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle. F1000 Research, 8, 1-19. Web.

Hawley, J. M., Owen, L. J., Lockhart, S. J., Monaghan, P. J., Armston, A., Chadwick, C. A., Wilshaw, H., Freire, M., Perry, L., & Keevil, B. G. (2016). Serum cortisol: An up-to-date assessment of routine assay performance. Clinical Chemistry, 62(9), 1220-1229. Web.

McEwen, B. S. (2019). What is the confusion with cortisol? Chronic Stress, 3, 1-3. Web.

Naveen, G. H., Varambally, S., Thirthalli, J., Rao, M., Christopher, R., & Gangadhar, B. N. (2016). Serum cortisol and BDNF in patients with major depression—effect of yoga. International Review of Psychiatry, 28(3), 273–278. Web.

Nejad, J. G., Park, K-. H., Forghani, F., Lee, H-.G., Lee, J-. S.& Sung, K-. I. (2020). Measuring hair and blood cortisol in sheep and dairy cattle using RIA and ELISA assay: A comparison. Biological Rhythm Research, 51(5), 813-823. Web.

Nieman, L. K. (2019). Measurement of cortisol in serum and saliva. UpToDate. Web.

Ozgocer, T., Yildiz, S., & Uçar, C. (2017). Development and validation of an enzyme-linked immunosorbent assay for detection of cortisol in human saliva. Journal of Immunoassay and Immunochemistry, 38(2), 147-164. Web.

Sanghavi, B. J., Moore, J. A., Chávez, J. L., Hagen, J. A., Kelley-Loughnane, N., Chou, C-. F., & Swami, N. S. (2016). Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device. Biosensors and Bioelectronics, 78, 244-252. Web.

Thau, L., Gandhi, J., & Sharma, S. (2021). Physiology, cortisol. StatPearls. Web.

Ward, G., Simpson, A., Boscato, L., & Hickman, P. (2017). The investigation of interferences in immunoassay. Clinical Biochemistry, 1-22.

What is cortisol? (2018). Hormone Health Network. Web.

Cite this paper


EssaysInCollege. (2022, November 5). Application of Competitive ELISA Assay for Serum Cortisol. Retrieved from


EssaysInCollege. (2022, November 5). Application of Competitive ELISA Assay for Serum Cortisol.

Work Cited

"Application of Competitive ELISA Assay for Serum Cortisol." EssaysInCollege, 5 Nov. 2022,


EssaysInCollege. (2022) 'Application of Competitive ELISA Assay for Serum Cortisol'. 5 November.


EssaysInCollege. 2022. "Application of Competitive ELISA Assay for Serum Cortisol." November 5, 2022.

1. EssaysInCollege. "Application of Competitive ELISA Assay for Serum Cortisol." November 5, 2022.


EssaysInCollege. "Application of Competitive ELISA Assay for Serum Cortisol." November 5, 2022.