**3.3 Quantitative determination of salivary biomarkers**

The concentrations of salivary biomarkers have frequently been determined by enzymelinked immunosorbent assay (ELISA). ELISA is nowadays one of major molecular determination techniques. It is much easier in treatment and cheaper in running cost than other molecular determination techniques, such as radioimmunoassay (RIA), fluorescent immunoassay (FIA), and the high performance liquid chromatography (HPLC). Several products which are designed for determination of "salivary" biomarkers, not for serum and urine, are placing on the market (e.g., Salivary Secretory IgA Indirect Enzyme Immunoassay Kit, Salimetrics, LLC., USA). By using such an ELISA kit in which all specimens and

to prepare the same size and volume of cotton for the repetitive sampling otherwise the volume of saliva collection would become as an unintended confounder. The small cotton

Experimenter should pay attention in handling the sample saliva to avoid contingent infection of virus or other possible diseases. All experimenters should ware disposable gloves and glasses. The saliva samples should be kept in the freezer below -20 Celsius by the

The natural secretion of abovementioned biomarkers has a diurnal change: the highest level is in the morning and gradually decreases afterwards to the lowest level in the night time (it should be noted that only alpha-amylase act controversy; Strahler, 2010). Therefore saliva sampling should be conducted depending on the objective of a study: (1) it should be corrected in the afternoon where the secretion of biomarkers is expected to be stable in the case of laboratory stressor studies by which a level of transient increase of these biomarkers are focused, and (2) in the morning or just after awakening in the case of chronic stress study since basal secretion level is expected to show a remarkable difference among subjects in the morning (e.g. Steptoe, 2005). Because of its remarkable diurnal change, repetitive sampling by a distinct time point of a day is recommended even in a chronic stress study.

Postprandial effects on the secretion of biomarkers are considerable. In addition pH of saliva could affect the quantitative determination of biomarkers. Subjects should not take any food or drink except for water at least an hour prior to the experiment. Hard exercise should be avoided prior to the experiment since it could make biomarkers temporary elevate. Moreover it is strongly recommended to take 5 to 10 minutes of an initial rest period before conducting experiment. If subjects were not familiar with or nervous to an experiment, most of biomarkers would increase by such a negative feeling or strain. Subjects who take any medications, especially oral contraceptive, suffer from any disease, are pregnant, or other cases in which physiological states were considered to be unusual should be excluded. Age and gender should be balanced in groups of between-subjects study since it is known to be

Subjects must be well informed about the objective and method of the study. Any experimental design targeting on human mental or physical stress should be approved by a

The concentrations of salivary biomarkers have frequently been determined by enzymelinked immunosorbent assay (ELISA). ELISA is nowadays one of major molecular determination techniques. It is much easier in treatment and cheaper in running cost than other molecular determination techniques, such as radioimmunoassay (RIA), fluorescent immunoassay (FIA), and the high performance liquid chromatography (HPLC). Several products which are designed for determination of "salivary" biomarkers, not for serum and urine, are placing on the market (e.g., Salivary Secretory IgA Indirect Enzyme Immunoassay Kit, Salimetrics, LLC., USA). By using such an ELISA kit in which all specimens and

should be placed under the subjects' tongue so as to collect the fresh saliva.

day of the quantitative determination of biomarkers.

confounders, especially for the study assessing hormones.

**3.3 Quantitative determination of salivary biomarkers** 

local ethics committee or equivalent organization.

**3.2 Possible confounders** 

materials are included, experimenter can easily assay various biomarkers with a minimum knowledge of biochemical analysis.

The principle of ELISA is based on the antigen-antibody reaction which is for capturing a target substance and the enzyme reaction which is for detecting the mass of a target substance via optical density of reaction produced color. The brief description of ELISA (competitive method) is as follows: (1) Thaw saliva samples kept in a biological freezer by moving them into a biological refrigerator (4 Celsius). (2) Centrifuge each saliva samples for 10 minutes at 1500 rpm to precipitate mucins or other solid contents. (3) Add each saliva sample (or known samples for references) into antibody-coated 96-well micro-plate. (4) Add a constant amount of "enzyme conjugate" which is the target biomarker (antigen) combined with horseradish peroxidase (HRP) into the micro-plate and incubate for an hour. In this step, antigen-antibody reaction is occurred competitively between original target in the saliva sample and that in the enzyme conjugate. (5) Wash the micro-plate to flush unbind target. (6) Add tetramethylbenzidine (TMB) solution to induce enzyme reaction with enzyme conjugate which is captured by antigen coated on the bottom of each well of the micro-plate in the step (4). The amount of the bind enzyme conjugate, which is there as a result of competitive reaction process, can be detected as the strength of optimal color (450nm) cased by enzyme reaction. Therefore this optimal density is inversely proportional to the concentration of target containing in the original saliva sample. (7) Finally, the target concentration in each sample is determined by referencing the optimal density of the reference samples. All analysis procedures take roughly about 3 to 5 hours for one microplate. Correct handling of samples and specimens with well calibrated micropipette is critical for all steps.
