Salivette cotton roll

Unstimulated mixed saliva was collected from all patients between 9:00 and 11:00 a.m., with a Salivette cotton roll (Sarstedt, Nümbrecht, Germany) as described previously [15 (link)]. One hour before sampling, ASD and TD patients refrained from eating, drinking, mouth rinsing, and teeth brushing. Salivette rolls were chewed for 5 min and then placed in special centrifugation tubes. Each sample was refrigerated or stored on ice until arrival at the laboratory, which was no longer than 45 min after the sample had been collected. All the samples were first weighed and then centrifuged at 4500× g for 10 min at 4 °C. The supernatant from whole saliva samples obtained by passive flow and saliva samples from the Salivettes were transferred to 1.5 mL Eppendorf tubes and stored at −80 °C for a maximum of three months until analysis. In all cases, the minimum volume of saliva obtained was 1 mL.

Saliva samples were collected using the Salivette^® cotton roll (Sarstedt, Nümbrecht, Germany) [25 (link),29 (link)] at around 5:00 AM on days 2, 5, 8, 13, 18, 20, 21, 22 and 30 (Table 1). To avoid contamination, the participants were instructed to refrain from consuming food and drinks other than water and from brushing teeth for 60 min and 10 min, respectively before sample collection. Collected samples were immediately stored in a freezer at −20 °C and sent to the National Center for Child Health and Development (Tokyo, Japan) at the end of the camp to be kept at −80 °C until analysis. OT concentrations were measured using a commercial ELISA kit (Assay Designs; Ann Arbor, MI, USA) and standardized by total protein content per milliliter and expressed in picograms per milligram protein (pg/mg protein) [28 (link)]. Missing information on height and weight (n = 1) was replaced with the average of three participants with the same age, and missing information on OT level on day 2 (n = 2) and day 18 (n = 2) was replaced with subsequently measured OT level of the same participants.