Measuring SIgA in Saliva
Unlike the various other salivary anti-microbial proteins that are synthesized directly in the saliva glands, or serum-derived analytes such as the steroid hormones that enter saliva by passive diffusion, Secretory IgA (SIgA) is a product of the adaptive immune system. Plasma B cells formed in the bone marrow migrate and form clusters around the secretory structures and ducts of most salivary glands. These immune cells make specific and non-specific antibodies to invading pathogens, including immunoglobulin A (IgA), which is regarded as an important first line of defense against such invaders. The plasma cells secrete a dimeric form of IgA into the space around the salivary glands. A special Polymeric IgA Receptor (pIgR) protein then binds the IgA molecules at the outer membrane of the salivary cells and actively transports them through the cells towards the inner chambers of the gland. Once these complexes reach the inner membrane, the pIgR is cleaved away, except for a portion known as the secretory component, which remains attached to the IgA dimer. The resulting molecule, known as Secretory IgA (SIgA), is then released into the salivary product. The secretory component helps protect the SIgA molecule from degradation by enzymes that are found in saliva. (1)
Because the pathway of entry for SIgA into saliva is different from that of other salivary analytes, it is important that researchers understand how the mechanism may affect SIgA levels in saliva. The rate at which SIgA is secreted into saliva depends both on the secretion rate of dimeric IgA by the plasma cells and on the speed of synthesis and availability of the pIgR . Both processes are affected by hormonal regulation and by nervous signals. (2) The details of the acute and longer-term control over secretion rates in humans are not well understood, but animal studies have shown that nervous stimulation results in increased delivery of SIgA to saliva. (3,4) It is known that stimulation by sour taste or chewing increases the secretion rate (μg/min) for SIgA in humans. At the same time, however, saliva flow is also increased, and this effect is greater than the increase in secretion rate. Absolute levels of SIgA (μg/mL) therefore usually decrease in stimulated saliva. (1,5,6)
The levels of IgA secreted in glandular saliva are also related to the numbers of plasma cells associated with each gland. The submandibular glands, for example, have approximately twice as many plasma cells per unit weight of gland tissue when compared to the parotid glands, and they secrete roughly twice as much SIgA into their saliva. (1) The minor glands have even higher numbers of plasma cells, possibly because of their close proximity to the mucosal surfaces of the mouth and exposure to antigens. The labial glands secrete saliva that has four times the level of SIgA compared to parotid saliva, and they are an important source of SIgA in the mouth in spite of the relatively small volumes of saliva that they produce. (7)
Advice on Saliva Collection Techniques for SIgA Measurement
Because SIgA levels in saliva can be affected both by the type of saliva collected and by the degree of stimulation present, it is important to maintain consistency in the procedure used to collect saliva samples. Salimetrics recommends that unstimulated whole saliva collected by the passive drool technique is the best type of saliva to use for most general studies, especially when analytes such as SIgA that are sensitive to flow rate are being assayed. It is advisable not to allow donors to chew on gum or paraffin or to use sour flavor to increase saliva flow because of the dilution effect on SIgA. In addition, chewing also may increase the release of serum-derived monomeric IgA from gingival crevices into the saliva, especially when donors have periodontal disease. Injuries in the mouth, which may be common in athletes involved in contact sports, or recent oral surgery can also increase the presence of serum-derived IgA and other blood components in saliva. This can affect assay results for a number of analytes. Screening donors for oral health and injuries is therefore advised.
If your saliva donors will be more comfortable using an absorbent device to collect saliva samples, the Salimetrics Oral Swab (SOS) can be successfully used in studies that measure SIgA. Due the effect of mouth location on SIgA levels, it is important that the SOS be placed in a consistent location. We advise using it underneath the tongue on the floor of the mouth, where it will absorb the whole saliva that pools there. When used in this manner, we find that the results for SIgA are similar to those obtained from whole saliva collected by passive drool. Under certain conditions, however, there is a possibility that the SOS might collect specific glandular saliva, rather than whole saliva. This could affect assay results for analytes such as SIgA. Researchers should be aware of this potential and decide on their collection strategy accordingly.
Cotton-based collection materials negatively influence SIgA measurements and we advise that they should be avoided.
Correcting SIgA Levels for Flow Rate
Even when donors are at rest, there will be some degree of salivary stimulation present, and saliva flow rates will vary significantly from one participant to another. It is therefore important to control for saliva flow when expressing SIgA results. Some studies have reported SIgA data in relation to total salivary protein, but Brandtzaeg has explained that this method is not advisable due to the fact that concentrations of the other salivary proteins are themselves highly variable. (1) Comparison of SIgA to albumin levels is also not appropriate since most albumin in saliva is derived from serum leakage
through the gingival crevices and therefore tied to the presence of periodontal disease. Salimetrics advises that the best method for reporting SIgA data is to express the results as a secretion rate. The length of time needed to collect a given volume of saliva should be noted in order to determine the flow rate (mL/min). The assay results (μg/mL) are then multiplied by the flow rate (mL/min) to obtain the secretion rate (μg/min).
If the SOS is used to collect samples, it will be necessary to note the length of time that the swab is in the mouth and to measure the volume of saliva recove
red from the swab in order to estimate the flow rate. Since the maximum volume of saliva that can be absorbed by the SOS is approximately 2 mL, it is important to remove the swab before it becomes saturated, in order for the estimate of flow rate to be accurate.
Researchers have recently begun to use triangular-shaped microsponges on a shaft (Sorbettes) for saliva collection from infants. Salimetrics has determined that these hydrocellulose devices do not affect the levels of SIgA measured in the saliva. However, the capacity of the Sorbette is only 200-300 μL, and it can be difficult to determine how quickly the sample is absorbed. For this reason, we feel that it is not possible to estimate reliably the flow rate of saliva collected with this device. In addition, due to their very small size, Sorbettes may have added potential to collect specific glandular saliva rather than whole saliva. For these reasons, we advise that the use of Sorbettes may not be appropriate for studies that measure SIgA in infants. Salimetrics is currently introducing a new Children’s Swab that will collect larger volumes of saliva, which should make possible more accurate assessment of SIgA levels in children under the age of 6.