In the bloodstream the majority of steroid hormones are bound either to non-specific proteins such as albumin or to specific proteins such as corticosteroid binding globulin (CBG) or sex hormone binding globulin (SHBG). These protein molecules are generally too large to diffuse from the blood stream into the saliva glands. Consequently, only the 1-15% of circulating hormone molecules that are found in an unbound state are free to diffuse from blood into saliva. Steroid hormone concentrations in saliva are therefore much lower than those in blood. If the barrier between the bloodstream and the oral mucosa is compromised by inflammation or microinjury such that there is leakage of blood or plasma into saliva, then there is a possibility that the higher levels of hormones found in the blood stream will contaminate saliva samples, causing falsely high readings. (1)
Visual inspection of saliva samples is not adequate to detect blood contamination, since microinjuries to blood vessel membranes may allow hormone molecules and proteins to pass through while still retaining the much larger red blood cells. Dipstick tests for blood, which look for the presence of hemoglobin, are also not a reliable means of screening saliva samples, due to the presence of peroxidases in saliva, which can generate false positive results. (2,3)
The Salimetrics Salivary Blood Contamination Enzyme Immunoassay quantitatively measures transferrin, a large protein (mol wt 76,000) present in abundance in blood that is normally present in only trace amounts in saliva. Higher levels of transferrin measured in saliva by this assay indicate the presence of blood contamination and serve as a warning to investigators that samples should be excluded from subsequent quantitative assays for salivary analytes and statistical analyses. (2,3,4) Saliva samples collected from populations that have little or no dental care, or known oral health problems, are especially appropriate for screening with the Blood Contamination Assay Kit.
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.