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Some steps in the development of an immunochromatographic test to quantify cortisol in human salivary fluid.

Elizaveta V. Panfilova
Institute of Biochemistry and Physiology of Plants and Microorganisms.

Abstract

In scientific research, cortisol is widely used as a marker of stress. Fenol A. et al. [1] measured cortisol levels in conjunction with the presence of stress and periodontal disease in prison settings. The authors found that the level of cortisol in the salivary fluid serves as a reliable marker of the stress state of the body and correlates with cases of periodontal disease. In his dissertation work, Brown C.W. [2] studied the effect of cortisol on the state of anxiety mental disorder in adolescents, provides recommendations for the use of cortisol as a marker for the diagnosis of depression. Bedini S. et al. [3] conducted studies on the increase in salivary cortisol among ambulance dispatchers. All of these studies have found a link between stress and increased levels of cortisol in the body. Due to its high speed and ease of analysis, immunochromatography has a potential advantage for detecting stress-dependent conditions, however, the development of a quantitative version of LFIA is a complex and complicated task.
In this work, we carried out one of the stages in the development of an immunochromatographic test for the quantitative determination of cortisol in biological samples - we obtained a calibration curve for the dependence of the intensity of staining on an immunochromatographic test strip on the concentration of cortisol in the analyzed liquid. Solutions of the cortisol-BSA complex with a cortisol content of 0.001, 0.01, 0.1, 1, 10, 31.62, 100 ng / ml and 1, 3.162, 10 mg / ml were used as calibration solutions. The content of cortisol in the complex was determined spectrophotometrically. A solution of the cortisol-BSA complex at a concentration of 1.05 mg / ml and secondary polyclonal antibodies at a concentration of 200 μg/ml were applied to a nitrocellulose membrane. After assembling all test components and drying the test strips in at 37 ° C, 60 μL of the calibration solution was preincubated with 10 μL of a solution of colloidal gold conjugate with (d = 25 nm) with antibodies to cortisol, then applied to the sample pad of the test. Within 15 minutes, staining of one or two (depending on the concentration of cortisol in the analyzed fluid) spots on the membrane occurred.
The membranes were dried and scanned. The stained spots were digitized using the Image J. program. Based on the data obtained, a calibration curve was constructed to determine the concentration of cortisol in the analyzed liquid by the intensity of staining the spot on the test strip.

Research support
This research was supported by the Russian Science Foundation (project no. 19-73-00202).

References
1. Fenol A. et al. association of stress, salivary cortisol level, and periodontitis among the inmates of a central prison in Kerala // Dent. Res. J. 2017. № 3. V. 4. P. 288-292.
2. Brown C.W. Salivary cortisol as a Measure of stress reactivity in adolescents with psychiatric disorders. 2016. Yale Medicine Thesis Digital Library. 2041. http://elischolar.library.yale.edu/ymtdl/2041
3. Bedini S. Stress and salivatory cortisol in emergency medical dispatchers: a randomized shifts control trial // Plos One. 2017. doi.org/10.1371/journal.pone.0177094



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Elizaveta Panfilova
Institute of Biochemistry and Physiolody of Plants and Microorganisms
Russia

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