Browse Assay Kits
Salimetrics salivary assay kits are expertly designed, developed and validated to ensure accuracy in saliva and proven to deliver precision results for saliva biomarkers.
The Salimetrics Salivary Alpha-Amylase Assay Kit is specifically designed to standardize the detection of alpha-amylase in saliva samples for research and biomedical laboratories. α-Amylase has been identified as a biomarker that shows potential to be a surrogate marker of autonomic (sympathetic) nervous system activation. Secretion of α-amylase from the salivary glands is controlled by autonomic nervous signals and a substantial amount of literature reveals that salivary α-amylase is a correlate of sympathetic activity under conditions of stress. Studies show that levels of salivary α-amylase increase under a variety of physically (i.e., exercise, heat and cold) and psychologically (i.e., written examinations) stressful conditions in human subjects. Studies also show that α-amylase levels do not often correlate with cortisol during stress, suggesting that individual differences in α-amylase represent a response to a stress signal independent of the LHPA axis.
Alpha-amylase is also believed to play a key role in the establishment and maintenance of the oral microflora to form dental plaques. Although its release from the salivary cells is greatly increased in response to taste or chewing motions of the jaw, salivary α-amylase levels are not related to α-amylase levels in blood, which are derived from pancreatic secretion. In addition, alpha-amylase is present in low levels in non-stimulated saliva between meals, due to its important anti-bacterial role in the oral cavity.
Using a small sample volume, this direct saliva assay kit has an extended range which spans the expected alpha-amylase 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. This alpha-amylase assay kit has also been formatted to minimize cross reactivity for related proteins.
|Catalog number:||1-1902 (5PK 1-1902-5)|
|Assay Protocol:||Download Rev: 04.08.16|
|Tech Bulletin:||Running Multiple Strips|
|Format:||96-well plate (kinetic reaction)|
|Saliva volume/test:||10 µL of saliva then 8 µL of X200 dilution|
|Incubation time:||3 minutes|
|Tests per kit:||94 (singlet)|
|Correlation with serum:||N/A|
|Controls included in kit?||Yes|
|MSDS sheets:||Available upon request.|
α-Amylase Assay Kit, Kinetic Enzyme Intended Use
The Salimetrics® α-Amylase Kinetic Enzyme Assay Kit is specifically designed and validated for the kinetic measurement of salivary α-amylase activity. 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.
Technical advances that make the assessment of biomarkers in saliva possible have enabled researchers to non-invasively study biosocial processes related to stress in naturalistic contexts. Secretion of alpha-amylase from the salivary glands is controlled by autonomic nervous signals, and substantial literature reveals that salivary alpha-amylase is a correlate of sympathetic activity under conditions of stress. Studies show that levels of salivary alpha-amylase increase under a variety of physically (i.e., exercise, heat and cold) and psychologically (i.e., written examinations) stressful conditions in human subjects. Interestingly, studies show that cortisol levels often do not correlate with α-amylase during stress, suggesting that individual differences in alpha-amylase represent a response to a stress signal independent of the LHPA axis.
Early studies on salivary alpha-amylase showed that its concentrations are predictive of plasma catecholamine levels, particularly norepinephrine (NE), and are highly correlated with NE changes in response to stress. However, more recent studies call this relationship into question. The literature does show that stress-related increases in salivary alpha-amylase can be inhibited by the adrenergic blocker propranolol and also that beta-adrenergic agonists are capable of stimulating alpha-amylase release without increasing salivary flow. This link suggests that the same stimuli that increase autonomic (sympathetic) arousal may activate sympathetic input to the salivary glands. The salivary alpha-amylase response to stress is complex, however, and it appears also to involve the parasympathetic system to a lesser degree. A recent article has emphasized the contribution of the parasympathetic system to salivary alpha-amylase secretion, pointing out in particular that autonomic reflex activity from the oral cavity, which can increase the parasympathetic signaling to the salivary glands, may have the potential to obscure the effects of central SNS activity. However, a subsequent study has found that salivary alpha-amylase responses significantly predict responses to the TSST for norepinephrine (NE) but not for epinephrine (E). The relationship between salivary alpha-amylaseand NE was stronger than the relationship between NE and E responses, indicating the predictive power of salivary alpha-amylase is well within the expected range for different SNS markers.
Although further work is necessary to understand better the underlying physiological factors that influence salivary alpha-amylase secretion, studies have already shown that salivary α-amylase measurements may be employed as a non-invasive measure of autonomic nervous system activation and are related to a variety of behavioral, social, health, and cognitive phenomena in human subjects.
Enzymatic Alpha-Amylase Test PrincipleThis method for alpha amylase activity determination utilizes a chromagenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. (17) The enzymatic action of α-amylase on this substrate yields 2-chloro-p-nitrophenol, which can be spectrophotometrically measured at 405 nm. The amount of α-amylase activity present in the sample is directly proportional to the increase in absorbance at 405 nm. For ease of use, the reaction is read in a 96-well microtiter plate with controls provided.
Salivary α-Amylase Example Ranges*
*To be used as a guide only. Each laboratory should establish its own range.
- Granger, D.A., Kivlighan, K.T., El-Sheikh, M., Gordis, E., & Stroud, L.R. (2007). Salivary alpha-amylase in biobehavioral research: Recent developments and applications. Ann N Y Acad Sci, 1098, 122-44.
- Chrousos, G.P. & Gold, P.W. (1992). The concepts of stress and stress system disorders: Overview of physical and behavioral homeostasis. JAMA, 267(9), 1244-52. Erratum in JAMA (1992), 268(2), 200.
- Kirschbaum, C., Read, G.F., & Hellhammer, D.H. (1994). Assessment of hormones and drugs in saliva in biobehavioral research. Göttingen: Hogrefe & Huber.
- Scannapieco, F.A., Torres, G., & Levine, M.J. (1993). Salivary α-amylase: Role in dental plaque and caries formation. Crit Rev Oral Biol Med, 4(3/4), 301-7.
- Rogers, J.D., Palmer, R.J., Jr., Kolenbrander, P.E., & Scannapieco, F.A. (2001). Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation. Infect Immun, 69(11), 7046-56.
- Chatterton, R.T., Jr., Vogelsong, K.M., Lu, Y.C., Ellman, A.B., & Hudgens, G.A. (1996). Salivary alpha-amylase as a measure of endogenous adrenergic activity. Clin Physiol, 16(4), 433-48.
- Nater, U.M., & Rohleder, N. (2009). Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: Current state of research. Psychoneuroendocrinology, 34(4), 486-96.
- Speirs, R.L., Herring, J., Cooper, W.D., Hardy, C.C., & Hind, C.R. (1974). The influence of sympathetic activity and isoprenaline on the secretion of amylase from the human parotid gland. Arch Oral Biol, 19(9), 747-52.
- van Stegeren, A., Rohleder, N., Everaerd, W., & Wolf, O.T. (2006). Salivary alpha amylase as marker for adrenergic activity during stress: Effect of betablockade. Psychoneuroendocrinology, 31(1), 137-41.
- Gallacher, D.V. & Petersen, O.H. (1983). Stimulus-secretion coupling in mammalian salivary glands. Int Rev Physiol, 28, 1-52.
- Rohleder, N., Wolf, J.M., Maldonado, E.F., & Kirschbaum, C. (2006). The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate. Psychophysiology, 43(6), 645-52.
- Bosch, J.A., Veerman, E.C., de Geus, E.J., & Proctor, G.B. (2011). α-Amylase as a reliable and convenient measure of sympathetic activity: Don’t start salivating just yet! Psychoneuroendocrinology, 36(4), 449-53.
- Thoma, M.V., Kirschbaum, C., Wolf, J.M., & Rohleder, N. (2012). Acute stress responses in salivary alpha-amylase predict increases of plasma norepinephrine. Biol Psychol, 91(3), 342–48.
- Granger, D. A., Kivlighan, K. T., Blair, C., El-Sheikh, M., Mize, J., Lisonbee, J.A., Buckhalt, J. A., et al. (2006). Integrating the measurement of salivary alpha-amylase into studies of child health, development, and social relationships. J Soc Pers Relat, 23(2), 267-90.
- Segal, S.K., & Cahill, L. (2009). Endogenous noradrenergic activation and memory for emotional material in men and women. Psychoneuroendocrinology, 34(9), 1263-71.
- Susman, E.J., Dockray, S., Granger, D.A., Blades, K.T., Randazzo, W., Heaton, J.A., & Dorn, L.D. (2010). Cortisol and alpha amylase reactivity and timing of puberty: Vulnerabilites for antisocial behaviour in young adolescents. Psychoneuroendocrinology, 35(4), 557-69.
- Wallenfels, K., Foldi, P., Niermann, H., Bender, H., Linder, D. (1978). The enzymic synthesis, by transglucosylation of a homologous series of glycosidically substituted malto-oligosaccharides, and their use as amylase substrates. Carbohyd Res, 61(1), 359-68.
- Weiner, D., Levy, Y., Khankin, E.V., Reznick, A.Z. (2008). Inhibition of salivary amylase activity by cigarette smoke aldehydes. J Physiol Pharmacol, 59(Suppl 6), 727-37.
- Klein, L.C., Bennett, J.M., Whetzel, C.A., Granger, D.A., & Ritter, F.E. (2010). Caffeine and stress alter salivary α-amylase activity in young men. Human Psychopharmacol, 25(5), 359-67.
- Nater, U.M., Rohleder, N., Scholtz, W., Ehlert, U., & Kirschbaum, C. (2007). Determinants of the diurnal course of salivary alpha-amylase. Psychoneuroendocrinology, 32(4), 392-401.
- Mackie, D.A. & Pangborn, R.M. (1990). Mastication and its influence on human salivary flow and alpha-amylase secretion. Physiol Behav, 47(3), 593-95.
- Lo Piparo, E., Scheib, H., Frei, N., Williamson, G., Grigorov, M., & Chou, C.J. (2008). Flavonoids for controlling starch digestion: Structural requirements for inhibiting human α-amylase. J Med Chem, 51(12), 3555-61.
- Hara, K., Ohara, M., Hayashi, I., Hino, T., Nishimura, R., Iwasaki, Y., Ogawa, T., et al. (2012). The green tea polyphenol epigallocatechin gallate precipitates salivary proteins including alpha-amylase: Biochemical implications for oral health. Eur J Oral Sci, 120(2), 132-39.
- Beltzer, E.K., Fortunato, C.K., Guaderrama, M.M., Peckins, M.K., Garramone, B.M., & Granger, D.A. ( 2010). Salivary flow and alpha-amylase: Collection technique, duration, and oral fluid type. Physiol Behav, 101(2), 289-96.
- Harmon, A.G., Towe-Goodman, N.R., Fortunato, C.K., & Granger, D.A. (2008). Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: A preliminary note of caution. Horm Behav, 54(5), 592-96.
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 and uric acid in which sodium azide is added as a preservative. (The alpha-amylase & uric acid kit 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. 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.