Heat Treatment of Saliva to Reduce Infectious Disease Contamination Does Not Impact the Analysis of Melatonin by Radioimmunoassay.

Abstract

The determination of melatonin levels in saliva represents one of the key methods for assessing the timing of the central circadian clock in humans, both in research and clinical settings. Melatonin levels in saliva are typically determined in a laboratory setting by RIA or enzyme-linked immunosorbent assay and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presented a serious challenge to the routine, safe assessment of melatonin in saliva samples. However, SARS-CoV-2 present in biological fluids can be inactivated by exposure to temperatures of at least 55-60°C for 30 min and the aim of this study was to assess the validity of applying a pretreatment heating step to saliva samples being prepared for melatonin determination using the Novolytix Radioimmunoassay (RK-DSM2). 40 archived saliva samples collected under a Dim Light Melatonin Onset sampling protocol were thawed and aliquoted into three identical groups-Controls (no pretreatment), 56°C pre-assay heat-treatment (30 min), and 70°C pre-assay heat-treatment (30 min). Melatonin concentrations in samples that were heated to 56°C for 30 min before assaying showed close agreement with the untreated controls, with the Pearson's correlation coefficient between the two sets of samples of 0.99 (p < 0.0001) and the slope of the Deming regression analysis close to 1.0 (Y = 1.04X + 0.168). When saliva samples were pretreated to 70°C for 30 min before assaying, the subsequent melatonin determinations were still strongly correlated with the untreated controls (Pearsons correlation coefficient = 0.97 (p < 0.0001), however melatonin concentrations were consistently overestimated when compared to the untreated controls with Deming regression slope of Y = 1.26X + 0.241. These results indicate that a 56°C pretreatment step is suitable for inclusion in standard operating protocols for melatonin determinations using the Novolytix RIA, as a way of effectively minimizing the potential for accidental pathogen exposure while handling saliva samples.