Thursday, 29 September 2016

SalivaBio’s Saliva Collection Aid Receives PCT Patent Approval, full press release link below





SalivaBio, LLC, developers of innovative, easy to use saliva collection devices, and Johns Hopkins University announced today that they have received final approval from the United States Patent Office for their Saliva Collection Aid (SCA). The Saliva Collection Aid simplifies the collection of passive drool saliva biospecimens for salivary bioscience research, thereby reducing participant burden and increasing compliance.




Tuesday, 20 September 2016

Salimetrics One World: Profiles of International Leaders in Salivary Bioscience: Dr James Jackson, Associate Principle Lecturer, School of Social and Health Sciences, Leeds Trinity University, UK


Dr James Jackson (Centre)

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"


Biography
James is a Chartered Psychologist and an Associate Fellow of the British Psychology Society. Having previously undertaken a degree in Biological Sciences from Heriot-Watt University, James also holds a BSc (hons) degree in Psychology from the University of Sheffield, an MSc in Industrial Psychology from the University of Hull, and a PhD (Psychology) from the University of Hull. His doctorate considered the effects of tinnitus on concentration and task performance.

Research Interests
The key aspect informing my teaching and my research is the concept of individual appraisal; why do some individuals cope with a given situation while others do not? I believe in the relevance of the Biopsychosocial Model, and in the interaction between body, mind and environment. Different people - with different personalities, different backgrounds and different skills - can and do respond differently in given situations. If we can gain further understanding as to why some people cope better than others, we can begin to consider whether others can be taught or trained to cope better with a stressful situation or a challenging condition.

Interview with James:

1. Can you tell us about the major themes in your research program? 

As someone with tinnitus (i.e. ringing in the ears) myself, I am very keen to learn more about why some people habituate to the condition whereas others are extremely distressed. My fascination with the condition is that it is so subjective and so personal. It is not the tinnitus itself, but personality and mood and environment. It is all about subjective appraisal. Can I cope with this? Will it get worse? These are the thoughts that determine the severity of tinnitus distress.

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’ve had a smattering of papers across a range of topics, but the one I suggest would be:

Jackson, J. G., Coyne, I. J. & Clough, P. J. (2014). A preliminary investigation of potential cognitive performance decrements in non-help-seeking tinnitus sufferers. International Journal of Audiology, 53 (2), 88-93.

Here, I push the idea that It is not just tinnitus that causes concentration difficulties, but the distress caused by tinnitus which does so. In essence, you notice tinnitus, you pay attention to it, it seems to get worse, you worry, you pay more attention to it using up finite cognitive resources, etc... It is a positive feedback loop, but it can be broken. – i.e. with intervention and effective coping strategies, it becomes possible to restore cognitive performance.

3. How did you get interested in using saliva in your research? 

My teaching! I’m a firm believer that teaching informs research just as research informs teaching. My final year elective module is the Biopsychology of Stress and Health, and while I could tell you how that happened, it’s a long (and boring) story. But here, my interest in tinnitus and my teaching about cortisol (and measurement thereof), and the Cortisol Awakening Response collided. Eventually, self-taught, I sat there thinking “Why hasn’t anyone done this yet?” Nobody ever did, so I decided to do it.

4. Which salivary analytes are you working with? 

So far, cortisol only.

5. How has working with saliva changed the direction of your research plans?

Definitely. I have drifted into becoming more of a methodology type, and I see so many ways in which patterns of cortisol secretion can be used elsewhere. This is what I did in the first place, being one of the first to apply salivary cortisol measurement to tinnitus. I will stick with tinnitus – my first and foremost interest – but I remain open to any opportunity to use salivary cortisol measurements as an objective measure of chronic stress.

6. What analyte is not measured in saliva now that you would hope could be measured in the future?

It has to be a reliable measure of oxytocin – there would be hugely interesting possibilities for looking at the effects of social support and trust on stress and pain perception.

7. What advice would give young investigators who might be considering working with saliva in their research? 

Do it!! You will have to be very careful, and you need to get your methodology just right if you want to be published. You will also need to ensure your participants know just how important these timings are – and that they won’t get to brush their teeth first thing in the morning, or have that glass of wine the night before. It can be costly, but there are many small grants out there to get young investigators going. Prove your concept with a pilot, then advance.

8. Tell us something about you (a hobby or special interest) that we would be surprised to know? 

Though rusty, my sign language (BSL) isn’t too bad. That and my love of coffee. Shame I can’t sign and hold a cup of coffee at the same time.

James Contact Details:

Dr James Jackson (C.Psychol., AFBPsS., FHEA.)
Associate Principal Lecturer in Psychology
Programme Leader MSc Psychology (conversion)
School of Social and Health Sciences
Leeds Trinity University

Monday, 19 September 2016

Starting a University Degree this year....Diagnostic Biochemistry, Psychology, Sport Science, Cognitive Sciences, Endocrinology, Veterinary, Salimetrics publish an Introduction to Immunoassay, Talk to us about "Spit Camp Training Days" then plan your Research

AN INTRODUCTION TO IMMUNOASSAY

This information is presented to benefit both students, lecturers and those working in Biochemistry Laboratories. Our Assays are commonly used within Psychology, Psychiatry, Sport and Exercise Sciences, Endocrinology, Behaviour, Cognitive Sciences or Scientists working within Diagnostic areas such as Hospital Biochemistry, Forensics, or Private Healthcare Laboratories

Immunoassay is an important laboratory technique that can be used to measure concentrations of many different substances found in saliva, blood, or other body fluids. It does this by making use of antibodies, which are special proteins made in the body in response to invading micro-organisms or other foreign substances.

Most immunoassays share two basic steps:

1. Antibodies prepared beforehand against a specific antigen are used to capture molecules of the antigen present in samples. Alternatively, antigens may be used to capture antibodies present in samples. The substance of interest that is captured is known as the analyte.

2. A means of measuring the concentration of the captured analyte must be included. This is most often done by introducing some type of measurable label that directly or indirectly indicates the presence of the analyte.

Most modern assays employ a labelling design known as enzyme immunoassay, which uses enzymes that have been coupled to antibodies or antigens (the enzyme conjugate). The conjugated enzymes act on compounds known as substrates to modify them chemically. Salimetrics uses TMB (3,3’,5,5’-tetramethylbenzidine) as the substrate in its immunoassay kits. TMB is often the preferred colorimetric substrate because it gives the highest color intensity and low background values.1 Salimetrics uses horseradish peroxidase (HRP) as the conjugated enzyme in most of its immunoassay kits. It is one of the most favored enzymes used in immunoassay because it acts on the substrate at a high rate, which increases the amount of color produced.2

In addition to colorimetric assays, there are also chemiluminescent immunoassays (CLIA or ChLIA), which use substrates that yield products that emit chemically-produced light.3 Chemiluminescent substrates are expensive, however, and they require a specialized instrument for measuring the light emitted. For most purposes, and particularly with competitive enzyme immunoassays (EIA, see below), colorimetric measurement can give sufficiently low detection limits,4 and accurate results.

IMMUNOMETRIC ASSAYS

Numerous schemes have been developed that use antibodies to capture and measure analytes. The details of some can be complex, but most are designed around two basic strategies. Perhaps the easiest to understand is the immunometric assay (Figure 3):

1. Antibodies immobilized onto a plastic surface (most often a 96-well microtiter plate) are used to capture the target antigen present in the sample.

2. A second antibody linked to an enzyme (the conjugate) is then added. It binds to a different location on the target antigen.

3. Plate wells are washed to remove unbound components.

4. Substrate is added. Bound enzyme present reacts with the substrate, yielding color.

5. The enzymatic reaction is stopped in order to establish a consistent time period for all wells. After stopping, the color is measured.



Figure 3. Immunometric assay to measure antigen.

In an immunometric assay the color generated is directly proportional to the amount of analyte present (Figure 4).




Figure 4. Immunometric assay signal response.

Immunometric assays are also commonly used to measure antibodies as the analyte (Figure 5). In this case a capture antigen is fixed to the plastic surface, and the target antibody binds to it. An antibody-enzyme conjugate that binds to the target antibody is then added, the plate is washed, and the conjugate reacts with the substrate to produce the color.



Figure 5. Immunometric assay to measure antibodies.


Because the analyte in an immunometric assay is surrounded on two sides, the procedure is often referred to as a sandwich assay. The acronym ELISA (Enzyme Linked Immuno Sorbent Assay) is also often associated with sandwich assays, but some authorities prefer to use the term in a more general sense for all sorts of microtiter plate immunoassays that involve enzymatic labels.5 The acronym IEMA is used more formally to refer to the Immunoenzymometric assay.

COMPETITIVE ASSAYS

Immunometric assays work well when the analyte is a molecule large enough to bind two separate antibodies at one time. Drugs and many hormones are small molecules, however, and they require a different assay design, known as a competitive assay (Figures 6,7). As in the immunometric assay, a capture antibody is used, and it may be attached beforehand to the surfaces of the plastic test wells. It is available only in limited amounts, however, and no secondary signal antibody used. The steps are as follows:

1. Antigen (analyte) and antigen molecules conjugated to an enzyme compete for the available antibody sites.

2. Plate wells are washed to remove all unbound analyte and conjugate.

3. Substrate is added. Bound enzyme present reacts with substrate, yielding color.

4. After stopping the reaction, color is measured.



Figure 6. Competitive assay, high concentration of analyte.

If the concentration of the analyte in the sample is high (Figure 6), relatively less conjugate will bind to the antibodies in the plate well, and the color generated will be low. If the concentration of the analyte in the sample is low (Figure 7), relatively more conjugate will bind, and the color generated will be high. The color in a competitive assay is therefore inversely proportional to the amount of analyte present in the sample (Figure 8).



Figure 7. Competitive assay, low concentration of analyte.


Figure 8. Competitive assay signal response.


INTERPRETING EIA RESULTS


IN ORDER TO DETERMINE THE CONCENTRATIONS OF ANALYTE IN SAMPLES, SEVERAL KNOWN CONCENTRATIONS OF ANALYTE MUST BE ANALYZED AS PART OF EACH TEST PLATE AS STANDARDS (OR CALIBRATORS). RESULTS FROM THESE WELLS ARE USED TO ESTABLISH A CALIBRATION CURVE, FROM WHICH THE RESULTS FOR THE UNKNOWN SAMPLES CAN BE FOUND. THE AMOUNT OF COLOR, OR OPTICAL DENSITY (OD), MEASURED FOR EACH SAMPLE AND STANDARD (B) IS DIVIDED BY THE OD OF A SAMPLE WITH NO ANALYTE PRESENT (B0), AND THE RATIOS (B/B0) PLOTTED AGAINST THE CONCENTRATION OF ANALYTE (ON A LOGARITHMIC SCALE). THE RESULT IS AN INVERSE S-SHAPED CURVE (FIGURE 9). THE PROCESS OF FITTING A CURVE TO THE DATA POINTS FROM THE STANDARDS IS MOST ACCURATELY ACCOMPLISHED BY USING SPECIALIZED COMPUTER SOFTWARE IN CONJUNCTION WITH THE PLATE READER. SALIMETRICS ADVISES USING A 4-PARAMETER SIGMOID MINUS CURVE FIT.



Figure 9. Typical Cortisol standard curve, 4-parameter sigmoid minus curve fit.


Some competitive assays are done with plates not pre-coated with capture antibody. In these assays the capture antibody is added in solution to each test well at the same time as the other components, rather than being immobilized to the plate ahead of time. During incubation, the antibodies bind to the analyte or conjugate, and also attach to the surface of the test well. Afterwards, the plates are washed as usual, and substrate is added for the color generation phase.

Another assay variation is the indirect competitive assay, used by Salimetrics in the kit for SIgA(Figure 10):

1. Samples and standards are initially incubated in separate tubes with a constant amount of antibody-enzyme conjugate. The conjugate binds to the analyte (SIgA).

2. The amount of conjugate left unbound is inversely proportional to the concentration of SIgA that is present in the samples or standards.

3. The amount of unbound conjugate remaining is determined by taking a sample from the first tube and transferring it to a microtiter plate with SIgA immobilized in the wells. Unbound conjugate from step 1 adheres to the SIgA in the well.

4. Conjugate previously bound to SIgA in step 1 is not free to attach to the well, and is washed away, leaving the unbound conjugate from step 1 adhering to the wells.

5. Substrate is added. Bound enzyme present reacts with the substrate, yielding color.
After stopping the reaction, color is measured.

6. The color generated corresponds to the amount of unbound conjugate from step 1, and it is therefore inversely proportional to the concentration of the analyte.



Figure 10. Indirect Competitive Assay for SIgA.

1. David Wild, ed., The Immunoassay Handbook, 2nd ed. (New York, 2001), p. 162.

2. Wild, p. 162.

3. Wild, p. 165.

4. James P. Gosling, Immunoassays: A Practical Approach (Oxford, 2000), p. 93.

5. Gosling, p. 14.

6. Gosling, p. 13.

So in this Post we have covered the Assay Principles, You may however wish to attend one of our Spit Camp Saliva Training Days, Details Here


If you need further advice: europe@salimetrics.com