Monday, 24 August 2015

Salimetrics One World: Profiles of International Leaders in Salivary Bioscience: Dr Arwel Jones, Research Fellow, Institute for Health, The University of Lincoln, UK

Each month we will feature an expert from the Salimetrics Saliva Research Community. We will bring together University Researchers around the World in order to encourage the sharing of ideas. 

We want to encourage Collaborative Research and to maximise Grant Applications / Awards in these challenging economic times. We have made it possible for you to communicate directly with the "Expert" featured.

Dr Arwel Jones, University of Lincoln
Biography

Dr Jones is a research fellow at the Institute for Health of the University of Lincoln. Following an entrance scholarship to Aberystwyth University in 2007, Arwel graduated with a First Class BSc (Hons) degree in Biology and Sport Science in 2010.  In July of that year, he commenced his PhD entitled "Effects of bovine colostrum on immune responses to prolonged exercise and upper respiratory illness in active males" with a Knowledge Economy Skills Scholarship.This was a collaborative project between The Golden Dairy and Aberystwyth University. 

The scholarship was part-funded by the European Social Fund through the European Union's Convergence Programme (West Wales and the Valleys) and administered by the Welsh Government. On completion of his PhD in 2013, Arwel was appointed as a postdoctoral researcher, a position that was jointly funded by Hywel Dda University Health Board and the Department of Sport and Exercise Science at Aberystwyth University. Here, he was involved in projects examining immune and inflammatory responses in patients with chronic obstructive pulmonary disease (COPD) at the Clinical Research Centre of Prince Phillip Hospital (Llanelli) and acted as a module coordinator within the Sport and Exercise Science degree scheme at Aberystwyth University. Since April 2014, Arwel has been based at the Lincoln Institute for Health.

One of the primary aims of his current position is to develop a joint research agenda between the Lincolnshire NHS Trusts, which provide primary, secondary and community care services, and the Lincoln Institute for Health, which provides an access platform to a wide range of research expertise. This will involve identifying the research priorities of COPD within the Lincolnshire NHS Trusts and develop fully specified research questions to direct collaborative research activities.


Interview with Arwel

1. Can you tell us about the major themes in your research program?
Broadly speaking my main research interests are exercise immunology and COPD.
Specifically, my studies investigate the acute and chronic effects of nutrition on immune responses to prolonged, strenuous exercise and how this relates to incidence of upper respiratory tract symptoms. I am interested in evaluating the positive effects of exercise (e.g. moderate intensity) on immunity and inflammation in clinical populations, particularly those diagnosed with COPD. My studies also aim to identify potential biomarkers in COPD, in order to better understand why some patients are prone to acute exacerbations, which are events that account for most of the morbidity, mortality and costs associated with COPD.

2. If you had to pick 1 publication in the past 5 years as the "best of your best", what would it be and why?
I would pick this one:
Jones AW, Cameron SJ, Thatcher R, Beecroft MS, Mur LA, Davison G (2014). Effects of bovine colostrum supplementation on upper respiratory illness in active males. Brain, Behavior and Immunity, 39, 194-203.

This was the first study on salivary microbiome in athletes, showing increased bacterial load during winter months, which was limited with bovine colostrum supplementation. I would say it is my best/favourite, not only for its novel findings, but also as it was the first study from my PhD to be published.

 3. How did you get interested in using saliva in your research?
I was most enthused by the exercise physiology and immunology content of my Biology and Sports Science Undergraduate degree hence in my final year I wanted to complete a research project dissertation in the area of exercise immunology. Collection of saliva was a non-invasive method to allow me to measure immune responses to exercise. The project (under the guidance of my supervisor Dr Glen Davison) determined the effects of a nutritional intervention on the responses of salivary secretory IgA and salivary lysozyme to intermittent exercise.

4. Which salivary analytes are you working with?
Most of my projects to date have involved IgA and other salivary antimicrobial peptides including lactoferrin and lysozyme. I always measure salivary transferrin (marker of blood contamination) if the analyte of interest is also present in the circulation. I am very interested in identifying what other immune parameters can be measured in the oral activity. We recently published a short-communication on assessing immune cell function from oral samples (Davison and Jones, 2015; Oral neutrophil responses to acute prolonged exercise may not be representative of blood neutrophil responses; Applied Physiology, Nutrition and Metabolism, 40, 298-301). Current work also involves assessing the effects of both nutrition and exercise on salivary microbiome and reactivation of latent viruses.

5. How has working with saliva changed the direction of your research plans?
There is no single marker available to predict the effect of an intervention (e.g. exercise, nutrition) on all aspects of immune function. Saliva collection provides a, simple, non-invasive route by which to measure the mucosal components of host defence.

6. What analyte is not measured in saliva now that you would hope could be measured in the future?
I would say there is an array of assays available to use saliva for measuring most parameters nowadays. However, I probably speak on the behalf of the field of exercise immunology by saying that we need further research to verify which analytes (present or new) are valid, reliable and robust predictors of upper respiratory tract infection in athletes. Given its proximity to where most pathogens enter the human body, saliva is likely to play an important role.  

7. What advice would give young investigators who might be considering working with saliva in their research?
Although saliva is often considered to be more acceptable to study participants than repeated blood samplings, I think some people (most often those new to the area) can underestimate the number of factors (e.g. time of day, fasted/fed, hydration status, collection method) that can influence your target analytes and hence the need to control for them as much as possible within your study. We have a standardised method for the collection and processing of saliva in our laboratory but we also always ensure research participants fully understand what is expected (e.g. minimal movement during unstimulated passive drool). Keeping an accurate record of the duration of each collection and saliva volume also allows you interpret your results on the basis of absolute concentration and relative saliva flow rate. Last but very much not least, recognition of blood contamination and its potential confounding effect on your saliva samples. We try to reduce the risk of contamination by having strict control procedures in place (e.g. avoid teeth brushing on sample day, rinse mouth with water 10 min prior to sample collection) but, unfortunately, some individuals can be prone to providing saliva samples that have been contaminated with blood. As a result of samples testing positive for blood contamination, we’ve previously had to remove up to 25% of participants from our dataset. I dread to think what it would be if we didn’t have our preventive procedures in place! Therefore, in addition to the conventional dropout rate that you may experience when running a research study, you also need to be wary of participants lost for blood contamination. Any study measuring analytes present in both saliva and blood should be wary of this.

8. Tell us something about you (a hobby or special interest) that we would be surprised to know?
Most of my free time is spent with family. That may not be very surprising but you may be surprised to hear that I complete this blog on the due date of our new arrival. No sign yet! I’d probably provide an interesting case study over the coming weeks for many of the analytes offered by Salimetrics, particularly cortisol and melatonin!

To contact Arwel e mail: ArJones@lincoln.ac.uk

To feature in this series, contact: europe@salimetrics.com

Friday, 21 August 2015

If you are starting University this year, studying Psychology, Sport Science, Endocrinology, Reproductive Biology or involved in Biomarker Testing, here Salimetrics publishes "An Introduction to Saliva Research"

Salimetrics publishes "An Introduction to Saliva Research"

One of the most important issues with Salivary Research is to ensure you collect the saliva at the right time of day and use the correct procedure, Salimetrics can supply you with the necessary tools to achieve this, call us, e mail us for assistance BEFORE you begin the Process!


Introduction to saliva research
In 1992 Dr. Irwin Mandel addressed the first New York Academy of Sciences meeting on the topic of Saliva as a Diagnostic Fluid, where he referred to the concept of using saliva as “a mirror of the body.”  At that time, the number of researchers who shared Dr. Mandel’s enthusiasm for saliva testing was relatively small, and there were some who felt that saliva could not serve as a reliable testing fluid.  After nearly 20 years of subsequent work in this field, however, we are now witnessing a sharp rise in the acceptance of saliva as a valuable testing medium alongside, or as an alternative to, traditional testing fluids like blood and urine.  This adoption of saliva has been especially evident in psychology and related fields, where researchers were among the first to recognize the advantages of multiple, non-invasive collections that saliva testing has to offer.

Saliva composition
Whole saliva is a complex mixture of water, ions and locally secreted organic compounds. Measurements of the locally secreted compounds in saliva, such as secretory immunoglobulin A (SIgA) and alpha-amylase (sAA), provide useful information on the biologically active portion of those analytes. In addition to locally secreted compounds, drugs, drug by-products, hormones and some proteins pass into saliva from the capillaries that surround the salivary glands. For neutral steroids that are able to passively diffuse across the salivary acinar cells, such as cortisol and testosterone, saliva concentrations correlate very highly with serum levels. This makes saliva testing a safer, non-invasive alternative to blood sampling.

What can be measured?
Cortisol is the major glucocorticoid hormone produced in the adrenal cortex and is actively involved in the regulation of many physiological systems. Salivary cortisol has been studied extensively in research on human behaviour, emotions, and development; examples include studies involving anxiety, depression, PTSD, and behavioural disorders (Schlotz, 2006; Wessa, 2006; Gordis, 2006; Dorn 2009)

Due to their common origin as products of the HPA axis, DHEA and DHEA-S are often examined in conjunction with cortisol.  Examples of studies that have measured salivary DHEA/DHEA-S have involved disruptive behaviour (Dorn, 2009) and depression (Assies, 2004). Given the important neuro-protective effects that have been recognized for DHEA and DHEA-S (Manninger, 2009), it seems likely that these two steroids will continue to be scrutinized in relation to brain health and mental disorders.  

Psychology-related studies involving salivary steroids controlled by the HPG axis (estradiol, estrone, estriol, progesterone, and testosterone) have also grown significantly. Many studies of the sex hormones have focused on their roles in sexual function and fertility, and in these fields blood measures of these hormones are often still preferred. However, the sex hormones also have well-established links to emotions, behaviour, and development; and it is in these research areas that investigators have been quicker to exploit the advantages of salivary hormone testing (Stanton, 2009; Welling, 2007).

In addition to the steroid hormones, other small, neutral molecules such as melatonin also diffuse readily from blood into saliva.  Salivary melatonin has excellent correlation with serum values, and a growing number of studies have been exploring it in connection with various disorders that may be related to light cycles and sleep (Novakova, 2011; de Almeida, 2011).

The digestive enzyme alpha-amylase (sAA), which is one of the major proteins secreted by the salivary glands, has been one of the analytes most heavily studied in connection with the nervous control of protein secretion.  By its use as a marker of autonomic activity, research has revealed that sAA measurements are related to a variety of behavioural, social, health, and cognitive phenomena in human subjects (Nater & Rohleder, 2009). Many psychobiological studies now routinely include measurements of both cortisol and sAA in order to observe activity in the HPA axis and the ANS, respectively (Gordis, 2006; Vigil, 2010).

Another major protein found in saliva is secretory immunoglobulin A (SIgA).  This important component of the immune system is different from most other salivary proteins in that it is neither synthesized by the salivary glands, nor related to IgA levels in the circulation.  Rather, IgA that originates from B lymphocyte cells adjacent to the salivary glands is bound and actively transported through the salivary cells, then released into saliva as SIgA.  (See figure 1.)  Although the details of the control of secretion of SIgA into saliva are still not totally clear, it is believed that both the synthesis of IgA by the immune cells and its transportation into saliva are affected by ANS signals (Bishop, 2009). Consequently, SIgA is another biomarker whose levels in saliva have been found to change in response to various types of stress and mood states (Kugler, 1992).

Methodology
Investigators who intend to use saliva testing should understand that the methods used to collect and handle saliva samples can have a direct effect on their results.

Saliva can be collected by a number of methods; the most appropriate will depend on the analytes of interest and the age of the participants. For the Passive drool method, participants allow saliva to pool in the mouth before passing it through a small straw into a cryovial; this can be used for all analytes. When measuring sAA, cortisol, CRP, S-IgA or testosterone, participants unwilling or unable to drool into a vial may find an oral swab easier to use. A small, absorbent and non-toxic insert is placed in the mouth for 1 to 2 minutes before being placed in a storage tube. Child, infant and animal swabs are also available – designed to be held under the tongue to reduce choking hazard.

ELISA assays require very small volumes of saliva (10-100µL). While it is advisable to collect an extra 300µL to cover for liquid handling losses, in most cases collecting 0.5mL of saliva by the passive drool method, or 1 to 2 minutes of collection with an oral swab, is sufficient.

As steroid hormones are non-polar molecules, they have a tendency to be attracted to some types of plastic. Therefore it is important to select tubes or vials made from high-grade polypropylene to avoid retention of the analyte.

In preparation for testing, participants should avoid alcohol for 12 h prior to collection and food 1 h prior to collection; it may also be pertinent to record physical activity levels 24 h prior. Participants should rinse out their mouths with water 10 minutes prior to collection to remove any food residues. 

Some analytes, such as cortisol, fluctuate markedly during a 24 h period according to a regular diurnal cycle, meaning the time of day that samples are collected should be considered; while others are affected by stimuli such as stress, which may need to be controlled. As S-IgA, DHEA-S and sAA are affected by flow rate, the time taken to collect the sample should be recorded and the quantity weighed; results can then be expressed as secretion rates (µg/min) rather than concentrations (mg/l).

Bacteria are present in saliva, and unless steps are taken to slow or stop their action, they can cause degradation of the analytes. Temperature stability varies between analytes; however, researchers are advised to freeze samples as soon as possible after collection. Samples can be stored at 4°C for a maximum of 4 hours, before freezing at -80°C for long term storage. Repeated freeze-thaw cycles should also be avoided, when investigating multiple analytes it is advisable to divide samples into smaller aliquots before freezing.

On commencing analysis, all samples should be thawed, vortexed and centrifuged to help break up any mucus and mix the samples. This will leave a clear solution, free from any unwanted particles, ready for use in the immunoassay.

Most immunoassays share to basic steps:
1.     Pre-prepared antibodies (highly specific against the antigen/analyte of interest) are used to capture molecules of the antigen present in the sample, binding them to the microplate
2.     A measurable label (conjugate enzyme) which attaches to the analyte is added, indicating the analytes presence by colour change

Once appropriate volumes of saliva sample and conjugate have been added to the microplate, and stated incubation periods and wash cycles observed, colour change can be read by a microplate reader. The amount of colour, or optical density, can then be compared using computer software to the range of known standard concentrations supplied with the kit; allowing the conversion of  optical density values into concentrations of the analyte of interest. 

What questions can be answered?
As demonstrated by the selected highlights from the literature given above, salivary biomarkers can be used to answer a wide range of questions within psychology. Perhaps most pertinent for those who are new to saliva testing, it can offer an objective and fully quantitative result to assess psychological disorders, such as stress, anxiety and depression. Testing can be incorporated into longitudinal study designs to investigate the progression of these disorders, overcoming some of the issues that surround the use of questionnaires and participant recall. While cross-sectional designs can be employed to identify potential physiological ‘risk factors’ may be particular conditions.

Saliva testing is also frequently used to assess the physiological response to psychological stimuli, both in lab and field-based scenarios. Concentrations of salivary biomarkers change within minutes in response to psychological stimuli, allowing researchers to analyse a participant’s response to even the most acute stimuli.

The simple and safe nature of sample collection lends itself to studies that require measurements to be taken ‘in the field’, for example those examining sleep patterns or the cortisol-awkening response. It also represents a less stressful alternative to blood sampling, which may be of importance for those evaluating stress, anxiety and depression.

What’s new?
An additional related topic receiving considerable attention is the relationship between systemic inflammation and brain health. It has been demonstrated that physical and mental stress lead to increased levels of circulating pro-inflammatory cytokines, such as IL-6, IL-1b, and TNF-a, which in turn are thought to interact with the brain; contributing to the development of mental disorders such as major depression (Debnath, 2011).
Salivary cytokines also appear to be influenced by the same outflow of nervous signals that affect systemic levels, and a small number of papers have observed that these salivary markers vary in connection with stress and various other mental conditions (Keller, 2010; Sjögren, 2006). This area of research is relatively new, and the details of the control of these salivary markers are not yet well understood; studies to date have found that the correlation between salivary and serum levels is only modest (Sjögren 2006). Further work is therefore needed to assess more fully the significance and general utility of these salivary markers of inflammation for psychobiological research.
Hair has long been analysed for exogenous compounds, specifically drugs of abuse, providing a useful tool in detecting long-term drug exposure. More recently, evidence has shown that hormones become trapped in hair as it grows, opening the potential for cortisol analysis as a quantitative measure of chronic stress (Gow, 2010). While emerging evidence is encouraging (Kirschbaum, 2009; Thomson, 2010), further work is required to validate the use this method in psychology.

References
Assies , J., Visser, I., Nicolson, N.A., Eggelte, T.A., Wekking, E.M., Huyser, J., Lieverse, R., & Schene, A.H. (2004).  Elevated salivary dehydroepiandrosterone-sulfate but normal cortisol levels in medicated depressed patients: Preliminary findings.  Psychiatry Res, 128(2), 117-22.
Bishop, N.C. & Gleeson, M. (2009). Acute and chronic effect of exercise on markers of mucosal immunity.  Front Biosci, 14, 4444-56.
De Almeida, E.A., Di Mascio, P., Harumi, T., Spence, D.W., Moscovitch, A., Hardeland, R., Cardinali, D.P., et al. (2011).  Measurement of melatonin in body fluids: Standards, protocols and procedures.  Childs Nerv Syst, 27, 879-91.
Debnath, M., Doyle, K.M., Langan, C., McDonald, C., Leonard, B., & Cannon, D.M. (2011).  Recent advances in psychoneuroimmunology: Inflammation in psychiatric disorders.  Transl Neurosci, 2(2), 121-37.
Dorn, L.D., Kolko, D.J., Susman, E.J., Huang, B., Stein, H., Music, E., & Bukstein, O.G. (2009).  Salivary gonadal and adrenal hormone differences in boys and girls with and without disruptive behavior disorders: Contextual variants.  Biol Psychol, 81(1), 31-39.
Gordis, E.B., Granger, D.A., Susman, E.J., & Trickett, P.K. (2006).  Asymmetry between salivary cortisol and α-amylase reactivity to stress: Relation to aggressive behavior in adolescents.  Psychoneuroendocrinology, 31(8), 976-87.
Gow, R., Thomson, S., Rieder, M., Van Uum, S., & Koren, G. (2010). An assessment of cortisol analysis in hair and its clinical applications. Forensic Sci Int., 196, 32-37
Keller, P.S., El-Sheikh, M., Vaugh, B., & Granger, D.A. (2010).  Relations between mucosal immunity and children’s mental health: The role of child sex.  Physiol Behav, 101(5), 705-12.
Kirschbaum, C., Tietze, A., Skoluda, N., & Dettenborn, L. (2009). Hair as a retrospective calendar of cortisol production – Increased cortisol incorporation into hair in third trimester pregnancy. Psychoneuroendocrinology, 34, 32-37.
Kugler, J., Hess, M., & Haake, D. (1992).  Secretion of salivary Immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol and catecholamines in saliva.  J Clin Immunol, 12(1), 45-49.
Maninger, N., Wolkowitz, O.M., Reus, V.I., Epel, E.S., & Mellon, S.H. (2009).  Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS).  Front Neuroendocrinol, 30(1), 65-91.
Nater, U.M. & Rohleder, N. (2009).  Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: Current state of research.  Psychoneuroendocrinology, 35(10), 1565-72.
Nováková, M., Paclt, I., Ptáček, R., Kuželová, H., Hájek, I., & Sumová, A. (2011).  Salivary melatonin rhythm as a marker of the circadian system in healthy children and those with attention-deficit/hyperactivity disorder.  Chronobiol Int, 28(7), 630-7.
Schlotz, W., Hellhammer, J., Schulz, P., & Stone, A.A. (2006).  Perceived work overload and chronic worrying predict weekend-weekday differences in the cortisol awakening response.  Psychosom Med, 66(2), 207-14.
Sjögren, E., Leanderson, P., Kristenson, M., & Ernerudh, J. (2006).  Interleukin-6 levels in relation to psychosocial factors: Studies on serum, saliva, and in vitro production by blood mononuclear cells.  Brain Behav Immun, 20(3), 270-78.
Stanton, S.J. & Schultheiss, O.C. (2009).  The hormonal correlates of implicit power motivation.  J Res Pers, 43(5), 942-49.
Thomson, S., Koren, G., Fraser, L.A., Rieder, M., Friedman, T.C., & Van Uum, S.H. (2010). Hair analysis provides a historical record of cortisol levels in Cushing’s syndrome. Ex Clin Endocrinol Diabetes, 118, 133-38
Vigil, J.M., Geary, D.C., Granger, D.A., & Flinn, M.V. (2010).  Sex differences in salivary cortisol, alpha-amylase, and psychological functioning following Hurricane Katrina.  Child Dev, 81(4), 1228-40.
Welling, L.L., Jones, B.C., DeBruine, L.M., Conway, C.A., Smith, M.J., Little, A.C., et al. (2007).  Raised salivary testosterone in women is associated with increased attraction to masculine faces.  Horm Behav, 52(2), 156-61.
Wessa, M., Rohleder, N., Kirschbaum, C., & Flor, H. (2006).  Altered cortisol awakening response in posttraumatic stress disorder.  Psychoneuroendocrinology, 31(2), 209-15.

Link to our full range of Assays

By the way we also offer Hair Cortisol Testing!
Need to know more, e mail europe@salimetrics.com

Tuesday, 18 August 2015

If you are starting University this year, studying Psychology, Sport Science, Endocrinology, Reproductive Biology or involved in Biomarker Testing this glossary is for you, let Salimetrics teach you the ABC of ELISA Testing

 The ABC of ELISA










If you are starting University this year, studying Psychology, Sport Science, Endocrinology, Reproductive Biology or involved in Biomarker Testing this glossary is for you

In today’s blog post we would like to introduce and explain to you common terms used in Salimetrics kit inserts. This little glossary will help to understand the principle behind the assays, support you with your experimental design and will aid your analysis and interpretation of the results. Please get in touch if you think there are terms missing that you would like to have explained in more detail. 

Just send an email with your suggestion and/or question to: europe@salimetrics.com

17α Hydroxyprogesterone (17OHP): a steroid hormone produced in the adrenal glands and the gonads. 17OHP can be used as a marker to assess adrenal function. 


Absorbance (or optical density): a measure of the transmission of an optical medium for a given wavelength. The higher the optical density, the lower the transmittance is. Most Salimetrics assays are read at 450nm. 

Alpha Amylase: a digestive enzyme that breaks down dietary starch. Also a marker for the sympathetic nervous system. 

Androstenedione: a steroid produced in the adrenal gland and gonads. It is a common precursor of male and female sex hormones and therefore a good marker for androgen synthesis. 

Antibody: a protein produced by B cells as primary immune defence. The principle behind the ELISA technique is that antibodies capture antigens, which are the molecules of interest.  

Assay Diluent: the assay diluent contains phosphate buffered saline, a pH indicator and preservatives. The pH indicator in the assay diluent alerts the user to samples with high or low pH values by turning the diluent either yellow (acidic) or purple (alkaline). 

Blood contamination: samples visibly contaminated with blood should be recollected as they can interfere with the assay. Salimetrics recommend that samples be screened for possible blood contamination using a reliable screening tool such as the Salimetrics Blood Contamination kit. 

Coefficient of Variation (CV%): is the percentage variation in mean; the standard deviation being considered as the total variation in the mean. 

Competitive immunoassay: the antigen in the unknown saliva sample competes with labelled antigen to bind to the pre-coated antibodies. The amount of labelled antigen bound to the antibody site is then measured. The response is inversely proportional to the concentration of antigen in the unknown. 

Controls: each kit contains vials of high and low levels of the molecule in interest in a saliva-like matrix to be used as control values. The control ranges established are to be used as a guide only and laboratories should establish its' own range. 

Cortisol: is the major glucocorticoid produced in the adrenal cortex in a circadian rhythm. Levels of cortisol rise independently of this rhythm in response to stress. 

Cotinine: is a metabolite of nicotine, found in tobacco. Cotinine is a reliable marker for the exposure to tobacco smoke. 

C-Reactive Protein: is an acute-phase protein which is found increased in certain inflammatory disorders. CRP is widely used as a biomarker of inflammation in the body. 

Cross reactivity: the possibility of an antibody raised against one antigen, also reacting against another, similar antigen, e.g. cortisol and dexamethasone. 

Chromogenic Substrate: peptides that react with proteolytic enzymes under the formation of colour. 

Conversion Factor:  is a multiplier for converting the concentration indicated in Salimetrics Kits to SI units (see table below). * affected by salivary flow rate

Dehydroepiandrosterone (DHEA): is an important endogenous steroid hormone which is co-released along with cortisol in response to ACTH from the pituitary gland. 

Dehydroepiandrosterone-sulfate (DHEA(S): a steroid hormone produced primarily in the adrenal cortex. Unlike DHEA, DHEA-S does not normally exhibit any diurnal pattern of secretion. 

Duplicate Analysis: saliva is assayed in two different wells on the microtiter plate, allowing a quality control check to ensure that the assay is giving the same reading in both wells. If the CV% is above 10%, the samples in question should be re-tested. 

Enzymea type of protein which acts as a catalyst in specific biochemical reactions.

Enzyme-linked immunosorbent Assay (ELISA): is an immunological technique to detect and quantify the presence of a molecule based on antibody-antigen interactions. 

Estradiol: a steroid hormone primarily produced by the ovarian follicles from testosterone. Research has focused predominantly on reproductive issues such as conception, ovulation, infertility and menopause. 

Estriol: is a female sex steroid hormone largely associated with pregnancy and foetal development. 

Estrone: a naturally occurring steroidal hormone. Research is often focused on pregnancy, reproduction and menopause. 

Freeze Thaw Cycle: the process of freezing and thawing a sample. Multiple freeze-thaw cycles for saliva samples should be avoided as they can affect analyte concentration.

Horseradish Peroxidase: an enzyme used to label antigens and antibodies. 

Human Tissue Authority: an authority for the regulation of organisations that remove, store and use tissue for research, medical treatment, post-mortem examination, teaching and display in public. 

Inter-assay Precision: expression of the plate-to-plate consistency that is calculated from the mean values for the high and low controls on each plate. 

Interleukin-1 Beta: a key pro-inflammatory cytokine that is released after infection, injury or antigenic challenge. 

Interleukin-6: a pleiotropic cytokine involved in a multitude of inflammatory responses with roles in immune regulation and acute and chronic inflammatory diseases. 

Intra-assay Precision: the average value calculated from individual CV’s for all duplicates, even if the total number of samples requires the use of multiple assay plates. 

Kinetic Assay: the assay measures the enzymatic activity of a molecule, rather than the concentration. The increase in absorbance is directly proportional to the molecules enzymatic activity. 

Linearity: the ability of an assay to return values that are directly proportional to the concentration of the target analyte in the sample. 

Mean: average of all measurements belonging to one sample

Melatonin: a hormone derived from serotonin and secreted by the pineal gland. Melatonin helps to regulate the sleep-wake cycle. 

Microtiter plate: is a flat plate with multiple wells used as individual test tubes. 

Monoclonal antibody: monospecific antibodies which are made by identical immune cells that are all clones of a unique parent cell. 

Nonspecific binding wells: are specific wells on a microtiter plate that do not have any antibody coated to it. They don’t have to be included in sample analysis, but provide a measure to assess analysis quality. 

Optical density (or absorbance): a measure of the transmission of an optical medium for a given wavelength. The higher the optical density, the lower the transmittance is. Most Salimetrics assays are read at 450nm.

Oxytocin: a peptide hormone secreted by the posterior pituitary gland. It is also called the "love hormone" due to its role in pair bonding, sexual relationships and maternal behaviour. 

Passive Drool Collection: is a collection method where participants allow the saliva to pool in their mouths before transferring to a cryovial using straw-like collection materials. For detailed collection instructions, please have a look at our saliva collection booklet. 

Plate Reader: are laboratory instruments designed to analyse biological and chemical substances in microtiter plates. 

Polyclonal antibody: are a collection of immunoglobulin molecules reacting against a specific antigen, each identifying a different epitope

Progesterone: a steroid hormone playing a role in the female menstrual cycle, pregnancy and embryogenesis. 

Recovery: a test used to determine whether analyte detection can be affected by the difference between diluent used for preparation and the experimental sample matrix. 

Room Temperature: all Salimetrics assays have been validated at room temperature (20°C-23.3°C). It is important to bring all reagents to room temperature before starting the analysis.  

Salimetrics Children Swab: is an inert polymer cylindrical swab with a volume capacity of 2ml. The children swab is longer than the oral swab and can be used for children from 6 months to 6 years. For detailed collection instructions, please have a look at our saliva collection booklet.

Salimetrics Infant Swab: is an inert polymer cylindrical swab with a volume capacity of 1ml. The infant swab is longer and thinner than the oral swab and can be used for children under 6 months. For detailed collection instructions, please have a look at our saliva collection booklet.

Salimetrics Oral Swab: is an inert polymer cylindrical swab with a volume capacity of 2ml. The oral swab can be used from children aged 6 and above. For detailed collection instructions, please have a look at our saliva collection booklet.

Salivary Flow rate: can affect the concentration of some of the analytes and has to be taken into account when testing for alpha-amylase, DHEA(S) and SIgA. 

Secretory Immunoglobulin A: is the dominant immunoglobulin in external secretions that bathe mucosal surfaces and is often characterized as a component of the immune systems first line of defence against bacteria and microorganisms.  

Sensitivity: the lower limit of sensitivity is determined by interpolating the mean optical density minus 2 standard deviations of a number of replicates at the zero concentration level. 

Saliva-Serum Correlation: a measure of how well concentrations of certain analytes can be reflected in saliva when compared to serum.

Singlet: saliva is assayed in only one well on the microtiter plate, therefore no CV% can be established. 

Spit Camp: a workshop aimed at researchers to assist with training in the integration of salivary measures into scientific studies. More information can be found here: www.salimetrics.com/spit-camp/.


Spit Report: a service from Salimetrics to stay informed with the latest in saliva research and products: www.salimetrics.com/spit-report/.


Spit Tips: provide you a source to improve and perfect your saliva research. The tips offer advice on any saliva-related issue, such as analyte selection, saliva collection and assay technique (www.salimetrics.com/spit-tips/). 

Standards: Different known concentrations of the analyte of interest in order to establish a standard curve which is used for calculating analyte concentrations in the unknown samples. 

Standard Deviation: indicates how much variation from the average exists. 

Substrate: a substance on which an enzyme acts. 

Sulphuric Acid: a highly corrosive acid used to stop the enzymatic reaction before measuring the absorbance. 

Swab Collection: a form of saliva collection using a swab consisting of inert polymer material. Salimetrics offers infant, children and oral swabs. For more information visit: www.salimetrics.com/collection-supplies 

Testosterone: is an anabolic steroid hormone synthesized from Androstenedione in the Leydig cells of the testes of males and, in smaller quantities, in the ovaries of females. 

Tetramethylbenzidine (TMB): a chromogenic substrate used as visualising reagent in ELISA’s. 

Transferrins: are iron-binding blood plasma glycoproteins which can be used to detect blood contamination in saliva samples.  

Unknown Samples (Unk): a term used for a saliva sample with unknown concentration of the analyte of interest. 

Wash Buffer: consists usually of phosphate buffered saline containing detergents and preservatives and is used to wash any unbound antibodies from the microtiter plate.

Zero Wells: also known as blank wells; have antibody for the specific antigen coated to them, but only assay diluent will be added and therefore serve as zero values.



AnalyteConcentration (Kit)MW (g/mol or Da)Multiply bySI units
17 OH Progesteronepg/mL3303.026pmol/L
Alpha Amylase*U/ml554000.017nKat/L
Androstenedionepg/mL2863.492pmol/L
C-Reactive Proteinpg/mL251000.040pmol/L
Cortisolug/dL36227.589pmol/L
Cotininepg/mL1765.675pmol/L
DHEApg/mL2883.467pmol/L
DHEA(S)*pg/mL3712.695pmol/L
Estradiolpg/mL2723.671pmol/L
Estriolpg/mL2883.468pmol/L
Estronepg/mL2703.699pmol/L
IL-6pg/mL225000.044pmol/L
IL-1bpg/mL307480.033pmol/L
Melatoninpg/mL2334.296pmol/L
Progesteronepg/mL3143.180pmol/L
Secretory IgA*pg/mL3850000.003pmol/L
Testosteronepg/mL2883.467pmol/L
Transferrinmg/dl760000.132umol/L


Link to our Saliva Assays
Link to our Saliva Collection Devices

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