Title : Blue tetrazolium as a fast and efficient assay for salivary cortisol in 96-well format
Abstract:
Background: Frequent or disproportionately elevated psychological distress can be a sign of mood disorders, such as Major Depressive Disorder and anxiety disorders. Due to its availability and non-invasive nature, salivary cortisol content is often used as an indicator of psychological stress. The gold standard methods of measuring salivary cortisol are ELISA and LC/MS. Both methods are time consuming and expensive. Previous publications have tested and verified a method of measuring salivary cortisol using blue tetrazolium in cuvettes that is simpler and more affordable, but is impractical for larger scale projects. Here, we modified this assay for a 96-well plate to keep the benefits while enabling it for more practical applications.
Methods: Initial reagent concentrations and ratios were based off of the work published by Ahmed et al. (2024). One part saliva was combined with four parts blue tetrazolium and four parts tetramethylammonium hydroxide for each test. Increasing final well volumes were tested. OD values were measured using the BioTek Epoch plate reader and Gen5 software. Salivary cortisol test samples were collected in the morning and evening from a single subject, and measured first with ELISA. Variables tested included absorption wavelength, light sensitivity in the development process, development time, final well volume, and the viability of reagents to be stored in a methanol solution at room temperature or in colder temperatures.
Results: Results from initial tests found that OD values peaked between 490nm and 500nm, demonstrating the biggest potential range for differentiating concentrations. There was no noticeable difference between incubating in the dark compared to in a normally lit lab environment. OD values increased proportionally with final well volume until 300ul, where the increase tapered. When controlling for well volume, there was a difference in OD between replicates incubated in separated wells, and replicates incubated together and then aliquoted, indicating that reaction order is significant for this method. There were no observed differences between freshly made reagents in solution and one week old stock solutions of blue tetrazolium kept in -20C and tetramethylammonium hydroxide kept in -4C, but the stock solutions kept at room temperature for one week produced consistently lower ODs compared to both the fresh and refrigerated solutions. Overall, the samples measured with this method showed similar measured concentrations when compared to ELISA results.
Conclusion: Our pilot results show promise in using blue tetrazolium to measure salivary cortisol in a 96-well format. Given salivary cortisol’s widespread use as a measure of HPA axis activity, this method opens doors for more accessible testing of cortisol markers in HPA-related mental health disorders.
