Alpha-Amylase (or α-Amylase) is a digestive enzyme that hydrolyses alpha-1,4 bonds of large polysaccharides such as starch and glycogen, yielding the smaller by-products of glucose and maltose. (1) Alpha amylase is synthesized in the acinar cells of the saliva glands and stored in secretory granules inside the cells. (2) Its release from the salivary cells is greatly increased in response to taste or chewing motions of the jaw. (3,4,5) Salivary α-amylase levels are not related to α-amylase levels in blood, which are derived from pancreatic secretion. (1) In addition to its digestive function, alpha-amylase also plays an important anti-bacterial role in the oral cavity; for this reason it is present in lower levels in non-stimulated saliva between meals. (6,7) Salivary α-amylase exhibits a diurnal rhythm, with a pronounced decrease within 60 minutes after awakening and a steady increase of activity during the course of the day. (8) Alpha-amylase production in the saliva glands increases in response to psychological and physical stress through interactions with the autonomic nervous system, and it has been found to be a useful as a marker of activity in the autonomic nervous system. (9,10,11) Salivary alpha-amylase levels have been used as a biomarker of ANS activity in various fields of biobehavioral research. (12,13,14,15)
Salimetrics does not recommend using plate covers for alpha-amylase because it would likely interfere with the plate reads.
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!
Yes, read this Technical Bulletin that gives more information about running alpha amylase assays.
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.
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.
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.
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.
With modern search tools online, we no longer maintain records of this type. We suggest that you use Pubmed or Psychlit to search the literature or you can Ask An Expert and we will be happy to assist you in your search.
If the analyte you are interested in is not noted in our website, please contact Dr. Granger at [email protected] to find out what developments are in the pipeline.
Salimetrics does not release products for sale if the quality control (QC) testing shows any issues. Here are some probable causes:
1) Can timing of adding reagents be off? For instance with a multichannel you can pipette the conjugate and TMB so many times before you refill, but you can pipette the stop twice as fast because it is a smaller volume. If you do this, it shortens the time the bottom rows have with TMB.
2) Can your washer be uneven and sheering off antibody in the bottom corner? Aspirate and check the amount of fluid left. It should be even in all wells and no wells should be completely dry.
3) Are you mixing faster than recommended? Or slower?
4) Are all reagents completely at room temperature? A bottle of assay diluent takes 2 hours to come to RT. You can pour some off into a smaller tube to warm up quicker for the zero and nsb.
5) Are you leaving the plate come to room temperature BEFORE opening the bag? (Otherwise moisture due to humidity may form in the wells and this is particularly a problem in this high humidity weather)
6) Are your multichannel pipettes dispensing the same amount each time reliably? We discard the first and last dispenses as they are not as reliable.
7) Are you incubating with TMB in the dark? (We no longer recommend aluminum foil.)
8) Are you testing one plate at a time? For example, do not put the standards on 5 plates then go back and fill in with samples. This delays the addition of conjugate to the plates.
9) Clean your plate reader filter. Dust from the lab can collect on the filter.
10) Are you adding assay diluent to the zero in sequence after the standards and not the last thing?
11) Never put the multichannel pipette tips into the wells as you can drag down standard from the wells above it causing lower readings in other wells.
12) Thoroughly blot all wells just before adding TMB but do not let the plate dry out.