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The Salimetrics Salivary Cortisol ELISA Kit was specifically designed to standardize the quantitative determination of free cortisol concentrations in saliva samples for research and biomedical laboratories. Using a small sample volume, this assay kit has an extended range that spans the expected cortisol levels found in human saliva. The average inter- and intra-assay precision coefficients of variation are low with no deleterious matrix effects often found in saliva which are characterized through dilution- and spike-recovery validation procedures. The Salimetrics cortisol assay kit has also been formatted to minimize cross reactivity for related steroids. Salimetrics salivary assay kits are expertly designed, developed and validated to ensure accuracy in saliva and proven to deliver precision results for biomarkers in saliva .
Salivary cortisol levels are unaffected by salivary flow rate and are relatively resistant to degradation from enzymes or freeze-thaw cycles. Studies consistently report high correlations between serum and salivary cortisol, indicating that salivary cortisol levels reliably estimate serum cortisol levels. Cortisol production has a circadian rhythm, with levels peaking in the early morning and dropping to lowest values at night. However, levels rise independently of circadian rhythm in response to stress.
|Catalog number:||1-3002 (5PK 1-3002-5)|
|Testing protocol:||Download PDF|
|Calibrator range:||0.012-3.000 ug/dL|
|Saliva volume/test:||25 µL|
|Incubation time:||1.5 hours|
|Tests per kit:||76 (singlet)|
|Correlation with serum:||0.91|
|Controls included in kit?||Yes|
|MSDS sheets:||Available upon request.|
Cortisol ELISA Intended Use
The Salimetrics® Cortisol Enzyme Immunoassay Kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of salivary cortisol. It is not intended for diagnostic use. It is intended only for research use in humans and some animals. Salimetrics has not validated this kit for serum or plasma samples.
Cortisol (hydrocortisone, Compound F) is the major glucocorticoid produced in the adrenal cortex. Cortisol production has a circadian rhythm, with levels peaking in the early morning and dropping to lowest values at night. Levels rise independently of circadian rhythm in response to stress.
In blood, only about 5-10% of cortisol is in its unbound or biologically active form. The remaining cortisol is bound to serum proteins. Unbound serum cortisol enters saliva via intracellular mechanisms; in saliva, the majority of cortisol remains unbound to protein. Salivary cortisol levels are unaffected by salivary flow rate and are relatively resistant to degradation from enzymes or freeze-thaw cycles. Studies consistently report high correlations between serum and salivary cortisol, indicating that salivary cortisol levels reliably estimate serum cortisol levels.
Cortisol ELISA PrincipleThis is a competitive immunoassay kit. Cortisol in standards and samples compete with cortisol conjugated to horseradish peroxidase for the antibody binding sites on a microtitre plate. After incubation, unbound components are washed away. Bound cortisol enzyme conjugate is measured by the reaction of the horseradish peroxidase enzyme to the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction with an acidic solution. The optical density is read on a standard plate reader at 450 nm. The amount of cortisol enzyme conjugate detected is inversely proportional to the amount of cortisol present, in the sample.
Do you have freeze/thaw data for each analyte?
The effects of freeze thaw on most biological measures, regardless of biospecimen type, can be dramatic. Analytes in oral fluid are not distinct or different in this way. As a general rule, multiple freeze-thaws should be avoided. The most practical way to address this concern is by aliqouting samples after collection. Some analytes are more resistant to freeze thaw than others. We recommend that investigators consult the literature for the analytes of interest. If there is freeze thaw data for a specific biological measure in traditional biospecimens, it is reasonable to assume this would also be true for saliva.
Do you have data for the circadian/diurnal pattern for each analyte?
No, but the literature is rather extensive on this subject for several salivary analytes; especially for salivary alpha-amylase and cortisol. We do not track that information internally.
Will Salimetrics kits will work with my plate reader?
Salimetrics kits are not plate reader specific. Any plate reader should work with our kits. Each specific kit insert gives the recommended filter(s) and curve fit in the calculation section; Salimetrics recommends a 4 parameter curve fit for most of our kits.
How do I set up my plate reader to read dual wavelengths or secondary filter ‘correction’?
First you have to have a plate reader that can read at duel wavelengths, which most can. Then you set up the plate reader program (protocol) to use the 450nm filter as the primary filter (405nm for alpha-amylase). The secondary filter is then added as specified in the kit instructions. The plate reader program should then automatically subtract the OD readings from the secondary filter from the OD readings of the primary filter and these would then be your ‘corrected’ OD readings, which would be used in the calculation of your data. (The absorbance readings form a bell shaped curve with the peak at 450nm. What subtracting the secondary OD reading does is subtract any interference (or background ‘noise’) outside the desired wavelength of 450nm.) You can just read at 450nm without correction if your plate reader is a single wavelength reader or you don’t have the proper secondary filter - correction is desirable for the most accurate results, but not necessary to complete the assay.
How do I set up plate washer for Salimetrics assays?
Salimetrics is familiar with Biotek plate washers as we recommend them. If you have a Biotek plate washer, we can coach you via our technical support team. If you don’t have a Biotek washer we can provide guidance, but you may need to contact your sales representative for specific details.
What are the important performance characteristics of an assay that should be used when determining the best or appropriate assay for my lab?
Performance characteristics for each Salimetrics kit can be found in the kit inserts/protocols online. One of the most important performance characteristics is the specificity of the antibody. You should not see a lot of cross-reactivity with similar compounds to the one you are measuring as this could indicate that you will not get an accurate measurement of the desired analyte.
Can I leave the kits out overnight to warm to room temperature so I can start the assay as soon as I get to work?
We have not yet tested our assays under conditions that do not follow the standard kit protocol. If you are unable to follow the assay procedure as written, it is recommended that you validate your results by running a pilot.
I would like to know if the Salimetrics Secretory IgA saliva kit measures total-IgA in saliva (alpha-chain-specific) or secretory IgA (dimeric) only?
The Salimetrics SIgA kit will pick up all forms of IgA, but not the free secretory piece alone. The concentration of IgA in saliva is minimal compared to the very high concentrations of SIgA. The Salimetrics SIgA kit measures both secretory IgA and monomeric IgA; it uses a polyclonal antibody and it is not specific for secretory IgA. It measures monomeric IgA (as found in serum) which accounts for approximately 15% of total IgA in saliva and diffuses into saliva from serum. The other 85% of IgA in saliva is true secretory IgA.
Cited from; Brandtzaeg P (2007) Do salivary antibodies reliably reflect both mucosal and systemic immunity? Ann NY Acad Sci 1098: 288-311. Furthermore, according to Dr. Gleeson; both monomeric and secretory IgA in saliva will contribute to mucosal immunity.
Other good references from Dr. Gleeson;Acute and chronic effects of exercise on markers of mucosal immunity. Nicolette C Bishop, Michael Gleeson
Frontiers in Bioscience (impact factor: 3.52). 02/2009; 14:4444-56; 19273362
A. K. Blannin, P. J. Robson, N. P. Walsh, A. M. Clark, L. Glennon and M. Gleeson:The effect of exercising to exhaustion at different intensities on saliva immunoglobulin A, protein and electrolyte secretion.Int J Sports Med 19, 546-552 (1998)
Do we need hoods in our lab to run your kits?
No, they are not necessary. However, this raises safety concerns. Saliva is a source of infectious disease. If you are running samples from patients with known infectious diseases, running samples under a biosaftey hood would be recommended. Sometimes samples held at room temperature can develop a strong odor and technicians running these assays may prefer to run such samples under a hood.
Does sodium azide interfere with Salimetrics assays?
It is a known fact that sodium azide is an inhibitor of horseradish peroxidase, which is used in most Salimetrics immunoassay kits. No Salimetrics kits contain sodium azide except alpha-amylase in which sodium azide, at 0.01%, is added as a preservative. (The alpha-amylase kit is not an immunoassay and does not contain horseradish peroxidase.) According to some salivary assay manufacturers, concentrations of sodium azide < 0.02% do not interfere with peroxidase. Salimetrics has not conducted studies to verify this claim.
WARNING: Sodium Azide is highly toxic to humans and very dangerous for the environment!
Can I use a plate cover for amylase assays?
Salimetrics does not recommend using plate covers for alpha-amylase because it would likely interfere with the plate reads.
Can I change the assay or collection procedure?
Failure to follow kit procedure and recommendations for saliva collection and sample handling may result in false values. If you are unable to follow the assay procedure as written, it is recommended that you validate your results by running a pilot.
Which kit from your company is the most appropriate for my research applications?
On our website we have information that connects each of our assay kits to a range of applications. Given the volume of publications using salivary analytes the amount of information generated is not easy for Salimetrics to maintain internally.
We recommend that you (a) use Pubmed and Psychlit to conduct a literature search. We know that the quality of the literature is higher, with respect to assay technology, starting about 1998. Publications before that time, should be reviewed carefully and (b) contact us via our “ask and expert”. Salimetrics scientists are actively participating in creating this literature and have a wealth of information we are happy to share.
How many kits do I need to analyze my # of samples?
The number of tests that can be conducted for each assay type is noted in each assay’s kit insert (generally 38 samples in duplicate per kit). Divide the number of samples by the number samples that can be done on one plate. If you are operating in an experienced lab we suggest adding 5% to this figure to accommodate for repeats. If you are just getting started, or have less-experienced operators running the assays, it is wise to include 10% in addition to the number you calculate to enable repeat testing if needed.
Can Salimetrics kits be used to measure analytes in specimens from other species / veterinary animals? (mice, rats, deer, bear, pigs, horses, dogs, fish, birds, etc.)
Yes, in theory as long as the molecular structure of the analyte is conserved across species, then the assay would perform. Salimetrics cortisol assay has been used with horses, dogs, deer, antelope, fish, sea lions for example. However, the levels of salivary analytes can be very different between species, and some species may not have certain analytes in their saliva. Careful pilot testing should be conducted to verify basic assay performance in non-human species.
What are the different types of immunoassays and which Salimetrics assays use which formats?
The assay formats differ for different analytes. In the kit insert, the SOP for running that specific protocol is outlined. It’s a good idea to review these SOPs on-line before you order. In this way you can be sure that you have the right equipment and know how to perform the procedures as outlined. An illustrated explanation of different types of immunoassay kits can be found under training and resources article “Introduction to Immunoassays.”
How essential is it to the researcher to have the same lot number of kits?
It is always best to use the same lot of kits and saliva collection supplies when feasible. Salimetrics monitors kit and collection supply lot variation and does not approve lots that are not within specifications.
Can I measure alpha amylase in babies?
Although salivary alph-amylase (sAA) can be sometimes be detected at birth the linkage between the ANS and salivary gland is not mature; thus sAA may not reflect the activity of the ANS in newborns. Salivary alpha-amylase can be detected in the saliva of infants as young as 3 months old. Over the first several months the amount of sAA produced in infants increases until it reaches the levels seen in adults at during the toddler period:
Davis, E. P., Granger, D. A.;Developmental differences in infant salivary alpha-amylase and cortisol responses to stress; Psychoneuroendocrinology (In press, 2009) (DOI:10.1016/j.psyneuen.2009.02.001)
There is also a Salimetrics document that provides this reference at LINK!
Can I run more than one strip at a time with the alpha amylase assay?
Yes, read this Technical Bulletin that gives more information about running alpha amylase assays.
I don’t have a Microtiter plate 37°C incubator/rotator; can I still run the alpha amylase assays in my laboratory?
Yes, Salimetrics recommends using a microtiter plate incubator/shaker, but other amylase substrate heating options are available - you can use an incubator that heats with air or a water bath.NOTE: It takes a long time to heat the substrate with either of these options; an hour or more – be sure to cover the substrate if you use an open air incubator to prevent evaporation of the substrate.
You can use the plate shaker in the plate reader to shake the plates. I would caution you that some plate shakers shake to vigorously and liquid may spill into your plate reader. Most of the newer models of plate readers have several settings to adjust the speed of shaking. You should test the shaking with the appropriate volume of water in the wells to see if spilling does occur in order to avoid problems.
Should I run my assays in duplicate or singlet?
This is a study design question. There are times when singlet testing is more acceptable, but generally duplicate testing is the ‘gold’ standard. Ideally, duplicate vs singlet testing should be performed to demonstrate no significant difference between the two. This could be also be shown by testing some samples in duplicate and some in singlet. Please make an appointment to talk with one of our experts by phone at 800.790.2258 or 760.448.5397
What kit validation test procedures should I run in my laboratory?
Salimetrics kits have been validated at Salimetrics during development. See Validation Criteria on our SalivaLab Services page. You should contact your laboratory accrediting body to see if further validation is necessary in your laboratory.
Can I order expired kit components or get a replacement for an expired component?
Salimetrics does not replace expired kit components. These kits are QC’d with the components that come with the kit and most the components are kit specific. Generally we do not have replacement components available because they are kit lot/batch specific and they have also expired. The best option if you kit has passed it’s expiration date is to purchase a new kit.
Define functional and analytical sensitivity.
Analytical sensitivity or minimum detection limit of an assay, calculated from the mean plus two standard deviations of multiple replicate measurements of the zero calibrator within an assay run (intra-assay), defines the lowest concentration of an analyte that can be distinguished from zero (also called Limit of Detection, LOD or the Lower Limit of Sensitivity, LLS).
In contrast, the functional sensitivity limit, represented as the lowest analyte concentration with an inter-assay coefficient of variation of 20%, provides a better indication of an assay’s actual performance (accuracy) when measuring samples with low concentrations of the analyte.
Why does Salimetrics recommend doing salivary alpha-amylase in singlet?
Alpha-amylase testing is an enzyme (kinetic) reaction which we have miniaturized onto a 96 well plate to increase testing throughput. This protocol is not an enzyme immunoassay (EIA) and there is no standard curve. Other than making the sample dilution, there are only 2 pipetting steps in the assay and no wash step.
If your laboratory technicians are experienced or using robotic pipetting equipment, we recommend singlet testing due to the low number of pipetting steps. However, we always recommend you do a percentage of samples in duplicate as a double check.
If you are pipetting and constructing dilutions by hand with manual pipettes we recommend that this assay is done in duplicate. Because each pipetting step increases the chance of variation, we recommend duplicate testing for EIA protocols only. Current alpha-amylase studies in the literature have reported significant findings using singlet testing.