A Novel Point-of-Care Method for Measuring Human Salivary Immunoglobulin A
Kelsi Irvine*, David Vollmer and Xuesheng Han
4Life Research, LLC, Sandy, UT;
Abstract
Purpose: The purpose of this study was to examine the potential of a novel method for quantifying salivary immunoglobulin A (sIgA) using a point of care device.
Method: This novel method included the use of a non-invasive oral fluid collector, lateral flow strips, and a cube reader device to collect and evaluate sIgA. Volunteers were recruited to collect saliva for analysis over three separate evaluations. These evaluations examined the precision, accuracy, and robustness of the method.
Result: Results indicated that this novel method could provide a reasonably reliable and fast option to monitor sIgA levels at an accessible price point.
Conclusion: This method could be a useful tool to improve individuals' ability to self-monitor important health biomarkers and gain insight into the status of their oral immunity with minimal effort and at a low cost compared to current standard methods.
Abbreviations:
LFD- Lateral Flow Device
OFC- Oral Fluid Collector
sIgA- Salivary Immunoglobulin A
ELISA – Enzyme-Linked Immunosorbent Assay
Introduction
Point of care devices for both personal and clinical use are becoming more effective, affordable, and widely available. As new tests come to the market, individuals and medical professionals become better equipped to measure and track biomarkers of health that previously would have been impractical or required use of a clinical lab. In recent years, The FDA has encouraged further development of mobile medical apps that improve health care and provide consumers and health care professionals with valuable health information1.
Collecting saliva for diagnostic testing instead of blood or urine would provide many testing advantages for individuals and clinicians. Saliva collection is less invasive than blood or urine collection and is more stable in long term storage. However, there are some challenges in using saliva as a preferred biological fluid including lower level of analytes and biomarkers compared to urine and blood, and confounding factors, including time of collection, that can further influence the abundance of analytes in a sample2. Currently, there are limited tools that utilize saliva to measure and quantify biomarkers2. However, it would be highly advantageous to increase research in this area and expand our understanding of salivary analytes that can be quantified.
Salivary immunoglobulin A (IgA) is an antibody found in saliva, tears, and human GI tract. It plays a crucial role in human immune system health by attaching itself to potential health threats and neutralizing their activity. IgA levels found in saliva can represent the immune system's status at a given time3. This biomarker is an example of a potential analyte that, if more easily quantified, could give valuable insight into individuals' health and wellness.
In past studies identifying and quantifying sIgA, the ELISA immunoassay has been the traditional method of quantifying concentrations of sIgA4,5. The ELISA test can be expensive, time-consuming, and requires sending the sample to a third-party lab, all of which could prevent individuals from regularly monitoring this biomarker. A rapid point-of-care method has previously been developed which utilizes Oral Fluid Collectors (OFC), Lateral Flow Devices (LFD), and a cube reader to provide users with a numerical approximation of biomarker concentrations such as salivary cortisol and IgA. These advancements provide the potential for individuals to test and keep track of their own health data. However, more needs to be done to qualify this method, and to give users and medical professionals context to understand what these measurements indicate.
In the qualification of the cube reader, developers examined the relationship between measurements both from the standard ELISA method and from a rapid method lateral flow device. They determined the relationship was suitable for use in athletic settings. These findings were presented in a conference paper at the 2011 conference for The International Society of Exercise Immunology (ISEI) (see supplementary material, Dunbar et al., 2011). Still, further investigation is needed to expand the scope of use for this instrument.
Rapid method testing of salivary biomarkers such as IgA has been recently studied in athletes from British football clubs. In addition to the development of a rapid reader for lateral flow devices, the oral fluid collector has been introduced to make saliva collection more convenient and accurate for analysis of biomarkers such as testosterone, cortisol, IgA, and IgG. The design and effectiveness of analyte recovery of the OFC was described at the ISEI professional conference. The evaluation to validate use of OFCs, presented at this conference, found that this method allows for analyte recovery over 85%.
Researchers are finding innovative ways to use data collected from these rapid tests. In one study, researchers determined participation in a training camp preceding high stakes international matches hindered mucosal immunity in players who attended6. Monitoring biomarkers such as sIgA could help individuals better understand how daily activities impact their immune system, and what they can do to improve their health overall.
Further research is needed to establish ranges of normal or expected IgA levels in healthy people and understand confounding variables that may alter readings. One study examined salivary cortisol and IgA levels in soccer players at different time points before a game. They found that there was a significant spike in both salivary cortisol and IgA levels immediately before the match. Researchers suggested that based on this finding, salivary biomarkers that can be linked to stress, such as IgA and cortisol, should be measured the day before an athletic performance7.
There has been a strong interest in utilizing this technology to better understand how nutraceutical products influence the immune system and to give consumers a valuable new tool. Previously, the cube reader was used in a separate, preliminary study to examine the impact of a multivitamin on immune biomarkers. Cube reader measurements showed an overall increase in sIgA over the course of 12 weeks8. This method can further help in a research capacity, or even help individuals better understand how they can boost their mucosal immune biomarkers.
This exploratory study examined the potential of new technology that allows users to quantify sIgA concentrations in saliva using a non-invasive oral fluid collector (OFC). We evaluated limitations of the instrument as well as robustness, precision, and accuracy in comparison to the current standard for evaluating sIgA concentrations (ELISA Immunoassay).
Methods
Two studies were conducted to evaluate this new methodology. The first study, hereafter called study A, focused on variability related to time of day of sample collection, fasted vs fed states of participants and test result accuracy. Study A had eight participants. The second study, called study B, evaluated variability from user error in sample collection, CR reading, and storage techniques. Study B had five participants. A third assessment, called the “Salivary IgA Normal Range Evaluation”, was conducted separately to evaluate typical sIgA to provide a preliminary reference range for sIgA. In assessment C, 39 participants were evaluated.
Study A
Eight healthy volunteers were recruited. Volunteers did not meet the exclusion criteria (presence of mouth infection). Participants were instructed to rinse their mouths prior to testing at all visits and refrain from eating or drinking, except water, for at least 3 hours prior to fasted visits. If participants brushed their teeth, they were instructed to finish brushing at least 30 minutes before the visit. During sample collection, participants filled out a form to confirm compliance with instructions. Failure to comply would lead to exclusion of data recorded during that visit.
Participants attended six visits over 3 days (two visits per day). Samples were collected in the late morning, just before lunch (fasted state), and in the early afternoon after participants ate (fed state). During the first visit (morning, fasted state), researchers collected three samples of sIgA were collected from each participant, 5 minutes apart. In all subsequent visits, one sIgA sample was collected from each participant.
During each visit, participants were given instructions on how to collect their own saliva sample using the OFC. Instructions were included on the sample collection form and explained by the study coordinator during the visit. Participants removed the SOMA OFC swab from the bag; swab was placed in the mouth, on top of the tongue and mouth was closed (participants were instructed not to suck). Participants kept the swab in their mouth and continued to collect until the volume adequacy indicator on the swab turned royal blue in color. Collection was to stop immediately after the swab indicator turned blue. The instructions were taken directly from the manual provided by SOMA Biosciences (SOMA Bioscience, 2017).
After collection, participants gave samples taken during the visit to a study coordinator. After the fluid had been collected properly, the swab was placed in the SOMA Buffer solution for 2 minutes and mixed gently. In order to measure both morning and afternoon samples together, measurement of salivary IgA was performed at 5 PM at the end of each day. Study coordinators measured IgA using the LFD, and the cube reader, allowing the LFD to develop for 10 minutes. According to the SOMA Biosciences’ manual, a SOMA LFD (lateral flow device), an immunochromatographic strip test, is used to quantify the concentration of the target biomolecules. Two to Three drops of the buffer/saliva mixture are placed on the sample pad of the LFD. The sample flows through the device, picking up dried conjugates on the way. As the mixture flows, gold labelled anti-secrecy sIgA analytes are captured in the window and show up as a red line. If sIgA molecules are in the mixture, they bind to gold labelled anti secretory IgA antibody and prevent them from binding to the test line. The more sIgA in the mixture, the less intense the line will appear. The concentration of sIgA is inversely proportional to the intensity of the red line. The LFD is allowed to incubate for ten minutes; the cube reader then measures the intensity of the red line and converts that into corresponding sIgA concentrations (mcg/ml) in the original oral fluid sample6. The cube reader is calibrated to the LFD lot prior to each test. Samples were processed according to methods described in the SOMA Biosciences’ manual. Results were recorded on an excel spreadsheet.
All samples collected on Monday were sent to a third party, Salimetrics, in their buffer solution to be analyzed using an indirect competitive ELISA method. The four samples taken on Monday, in their respective buffer solutions, were placed in a 4°C environment immediately after measurement and then sent to Salimetrics. Salimetrics analyzed each sample using an indirect competitive ELISA method. ELISA results were compared to the previous reading of the same samples using the cube reader and LFD after 10 minutes of incubation. Only samples taken on Monday were sent to Salimetrics to be analyzed using ELISA, all other samples were analyzed using the cube reader method only. The limit of detection for the cube reader method is 25 µg/mL, and the limit of detection for the ELISA method is 2.5 µg/mL.
Cube reader precision and incubation time were also evaluated in Study A. Eight samples, one from each participant, taken during the first visit, were measured three times, one after the other using the same cube reader and same LFD. Incubation time was also evaluated using eight saliva samples, one from each participant taken during the first visit. The saliva sample was dropped into one LFD and then measured after 10 minutes. The same LFD was then measured 5 minutes later at the 15-minute incubation period.
Informed Consent Statement: All study participants were properly informed of the study. Informed Consent was obtained from all study participants before the study started.
Study B
Five of the eight volunteers from Study A were recruited to participate in a second study examining the efficacy of different collection techniques. Three volunteers from study A elected not to participate in study B. Participants were required to attend one 1 hour-long visit and were fasted for at least 3 hours prior. Before the visit, volunteers were instructed to refrain from eating or drinking, except water, from breakfast until their visit. If participants brushed their teeth, they were asked to finish at least 30 minutes prior to the visit.
During the hour-long visit, 3 saliva samples were collected from each participant.
CONTROL Sample 1 procedure (Sample Immediate): Participants removed the SOMA OFC swab from the bag; swab was placed in the mouth, on top of the tongue and mouth was closed (participants were instructed not to suck). Participants kept the swab in their mouth and continued to collect until the volume adequacy indicator on the swab turned royal blue in color. Collection was to stop immediately after the swab indicator turned blue. Samples were then analyzed using the cube reader shortly after collection. This is the method recommended by SOMA for saliva collection.
Sample 2 procedure (Sample 1 Min): Participants removed the SOMA OFC swab from the bag; swab was placed in the mouth, on top of the tongue and mouth was closed (participants were instructed not to suck). Participants kept the swab in their mouth and continued to collect until the volume adequacy indicator on the swab turned royal blue in color. Collection was to stop 1 minute after the swab indicator turned blue. Samples were then analyzed using the cube reader shortly after collection.
Sample 3 procedure (Sample Sucking): Participants removed the SOMA OFC swab from the bag, swab was placed in the mouth, on top of the tongue and mouth was closed, participants were instructed to suck on the swab during collection. Participants kept the swab in their mouth and continued to collect until the volume adequacy indicator on the swab turned royal blue in color. Collection was to stop immediately after the swab indicator turned blue. Samples were then analyzed using the cube reader shortly after collection.
The order of sample collection was randomized for each participant. Participants waited 30 minutes between the collection of each sample. Each volunteer received a form to fill out during testing. This form asked them to report compliance with fasting instructions and record sample IDs as they performed the tests. Instructions were included on each form for the participants to follow. Each form was customized for each participant to ensure randomization of sample collection order.
LFD variability and buffer solution stability were also evaluated during Study B. Five samples, one taken from each participant, were measured three times using three different LFDs. Saliva samples were dropped into each LFD and then measured after the 10-minute incubation period. To determine stability of samples after storage, one sample taken from each participant during study B was measured once using one LFD, then stored at 4°C for 9-10 days. After 9-10 days in the refrigerator, the buffer solution was warmed to room temperature and then measured once using one LFD.
Informed Consent Statement: All study participants were properly informed of the study. Informed Consent was obtained from all study participants before the study started.
Salivary IgA Normal Range Evaluation
The normal range evaluation was conducted after studies A and B with 39 participants, none of whom participated in studies A or B. Participants with mouth infections were excluded from the study. Originally, there were 43 volunteers, four were excluded due to noncompliance. Healthy men and women were recruited per inclusion and exclusion criteria via invitation through their company email (4Life Research LLC and Biomedical Research Laboratory LLC). The invitation email contained instructions concerning the sample collection visit.
The purpose of this evaluation was to collect additional data points from a wider range of participants to estimate a “normal range” for salivary IgA. Collection protocol followed the instructions from the manual from SOMA Biosciences. Participants were to collect saliva in the manner intended by the manufacturer; no modifications to protocol were made in this evaluation. During the visit, each participant collected their own saliva by following instructions on the sample collection form and instructions given verbally by the study coordinators. Participants were asked a series of questions to determine if they complied with the following conditions, fasted for at least 3 hours, not brushed their teeth for at least 30 minutes. Four participant samples were excluded due to noncompliance.
After volunteers filled out and followed instructions of the sample collection form, samples were given to the study coordinator. The sample and buffer solution were mixed gently for 2 minutes. Samples were left at room temperature and were analyzed in singlet within 1 hour of the sample collection. Samples were processed according to methods described in the SOMA Biosciences’ manual (SOMA Bioscience, 2017). Results were recorded on an excel spreadsheet
Informed Consent Statement: All study participants were properly informed of the study. Informed Consent was obtained from all study participants before the study started.
Results
The target of many of the measurements taken across studies A and B was to evaluate the variability in measurements due to the precision of the instruments, the robustness of the methodology, and biological variance. Table 1 divides each measurement into one of these three categories.
Table 1: Listed P values of all measures of precision, robustness, and biological variance.
|
Measures of Precision |
n |
P Value |
| Cube Reader Variability | 8 | 0.9999 |
| LFD Variability | 5 | 0.9289 |
|
Measures of Robustness |
||
|
Saliva Collection (Indicator Time) |
5 | 0.2174 |
| Saliva Collection (Sucking vs not) | 5 | 0.7705 |
| Incubation Time | 8 | 0.0002* |
| Buffer Stability | 5 | 0.0241* |
| Measures of Biological Variance | ||
| Intra-Day | 8 | 0.8401 |
| Inter-Day Fasted | 8 | 0.4510 |
| Inter-Day Fed | 8 | 0.1133 |
| Average Inter-Day Fasted Vs Fed | 8 | 0.0104* |
*Statistically significant values at p<0.05.
Study A
A one-sided t-test was used to analyze the difference between all fasted and fed measurements. It was found that there was a significant difference between average sIgA readings taken in fasted vs fed subjects, (p-value= 0.0104) (Table 1).
All subjects were evaluated in both fasted and fed states. In a fasted state, all 8 subjects had saliva collected over three five-minute intervals, except for subject 2, who only had two viable samples. Subjects’ sIgA was also monitored across three days. Samples were collected both in the morning (fasted) and in the afternoon (fed). All three sets of data were analyzed using ANOVA (single factor) and none of the p-values indicated a significant difference between time intervals both inter- and intra-day regardless of subjects being fasted or fed (Table 1).
Variability in the cube reader was evaluated across 3 readings of the same LFD. Readings were taken one after the other, with no time delay (p-value = 0.9999). Incubation time was also evaluated; there was a significant difference between average LFD readings allowed to incubate for the recommended 10 minutes vs 15 minutes (p-value: 0.0002).
Study B
Subjects were asked to collect samples according to manufacturer's instructions and then again to track variability caused by noncompliance with directions. It was found that samples where the sample collection stopped 1 minute after the collection swab indicator turns blue were not found to be statistically different from sample collection stopped immediately after the indicator turned blue (p-value: 0.2174). The same was determined for samples collected with the instructions to suck vs does not suck on the collection swab (p-value: 0.7705). Data was analyzed using a two-sided T-test (Table 1).
Samples were obtained from the five subjects during study B to determine variability across laminar flow devices (LFDs) (p-value = 0.9289). Both sets of data were analyzed using ANOVA (single factor), and neither set of data indicated a significant difference across readings (Table 1).
The buffer solution for the collection swabs was evaluated for stability after storage at 4°C. It was found in the five samples evaluated; there was a significant difference in average readings taken between the day of sample collection and 9-10 days after (p-value: 0.0241). Data was analyzed using a one-sided T-test (Table 1).
Correlation with ELISA
Data came from study A. All samples were analyzed first by the cube reader method, and the second analysis was done by a third party (Salimetrics) using an indirect competitive ELISA method. The data was then plotted to determine correlation. The protocol for the Salimetrics ELISA method called for a further dilution of the buffer solution by a factor of five. Data points were plotted as seen in figure 1 and found to have a high correlation (R^2=0.883) (Fig.1).
Figure 1: Cube Reader Correlation with ELISA
Correlation between Cube Reader and ELISA results for salivary IgA quantification. Each data point represents a paired measurement of sIgA concentration (μg/mL) using the Cube Reader and a third-party ELISA assay. The linear regression analysis yielded an R² value of 0.883, indicating a strong positive correlation between the two methods.
Pearson Correlation Coefficient: 0.94, R2 = 0.883, Salimetrics range: 6.525-67.482 µg/mL, Cube Reader range: 33.6-246.2 µg/mL.
Salivary IgA Normal Range Evaluation
Participants of this evaluation provided samples that were plotted in Figure 2 (n=35). 39 participants gave saliva for measurement. Four readings were excluded due to participants eating too close to the time of testing. The data provided a range of measurements for sIgA (5 Number Summary: Minimum: 25 µg/mL, Quartile Q1: 61.5 µg/mL, Median: 113.1 µg/mL, Quartile Q3: 153.2 µg/mL, Maximum: 330.0 µg/mL.
|
Proposed Range Example |
||
|
Low |
25-60 |
µg/mL |
|
Medium |
60-110 |
µg/mL |
|
Medium high |
110-150 |
µg/mL |
|
High |
150-200 |
µg/mL |
|
Very high |
200+ |
µg/mL |
Figure 2: Salivary IgA Normal Range Evaluation
Distribution of salivary IgA levels among healthy participants. This figure displays the frequency of salivary IgA concentrations (μg/mL) measured in 39 individuals as part of the Salivary IgA Normal Range Evaluation. 5 Number Summary: Minimum: 25 µg/mL, Quartile Q1: 61.5 µg/mL, Median: 113.1 µg/mL, Quartile Q3: 153.2 µg/mL, Maximum: 330.0 µg/mL
Discussion
This exploratory study found that the physical components, the cube reader and the LFD, of this method were precise across measurements. LFDs had some variance across different units, although that variance was not significant. Additionally, there is a positive correlation between the ELISA results from Salimetrics and the readings from the cube reader Method. Suggesting that this new method is reasonably accurate for a POC device.
It was also found that the variability between average measurements examining proper incubation time and buffer stability was found to be statistically significant. However, other variations in collection techniques, including sucking vs. not sucking on the collection swab, and continuing saliva collection past the time indicated by the color change on the collector, were not found to have statistically different outcomes. As to be reasonably expected, when proper protocol is not followed, this method can produce less than accurate and less than precise results. Although, the method does have some tolerance for user error and improper collection techniques. However, it is still recommended that potential users of this method follow the instructions provided for the best results. Despite any potential variation due to user error, results could still aid individuals in self-monitoring sIgA levels over time.
Results also gave insight into the impact of biological variability on this point of care method. The average measurements for the inter-day fasted vs fed samples were statistically different. This would indicate users who are tracking sIgA levels over time, should be consistent and take measurements at the same time of day, in a fasted state. This practice is recommended in collection instructions.
The range collected from the salivary IgA normal range evaluation provides a preliminary benchmark to compare IgA levels to what has been observed in other subjects. Because ranges and values vary across methods, it is important to establish an expected range users can reference when monitoring their own sIgA levels using the point of care method of interest in this study. Further research is recommended to more firmly establish a range.
Past research has evaluated this tool in the context of athletic training.7 This study examined this method as it might be used by health-conscious individuals to casually monitor their salivary IgA. The manufacturer of this method has conducted evaluations to determine precision and accuracy.8,9 This study extends research to evaluate the method in a different environment. Results confirm that this method is reasonably reliable and has potential to quantify sIgA conveniently and effectively for individuals wishing to monitor health biomarkers. Additionally, there have been calls to advance point of care testing to leverage saliva as a biofluid of interest.2 This exploratory study takes a small step toward better utilizing saliva and expanding the analytes that can be assessed using point of care methods.
This exploratory study confirms that a novel point of care device can be effective when used to quantify salivary IgA in a non-clinical, non-diagnostic setting. It validates previous work that has evaluated effectiveness and accuracy of this point of care method.7,9 This study also examines the impact of user error and extends application beyond use in athletic training and conditioning setting.7
Rapid POC tests are becoming increasingly available, and there are many current and future applications that can make it easier for individuals to monitor biomarkers with fast and non-invasive techniques. This preliminary data further supports the level of reliability of the new cube reader and displays a high correlation with the current ELISA method for quantifying sIgA.
Traditionally, the ELISA immunoassay has been the primary method of quantifying concentrations of sIgA 4,5. The ELISA method can be expensive, time-consuming, and would require individuals to send a sample to a third-party lab. These are all barriers which could discourage individuals from regularly monitoring this biomarker. Comparatively, the novel point of care method examined in this exploratory study would be more cost effective, accessible, and user friendly.
This preliminary evaluation of this method demonstrates that the cube reader and associated lateral flow devices provide a reasonably accurate and precise method for measuring salivary IgA in non-clinical settings. While proper protocol is important for reliable results, the method shows some tolerance for minor user error and biological variability. Overall, the findings support the potential of this point-of-care approach as a convenient, accessible tool for individuals interested in monitoring sIgA over time.
Conclusion
This exploratory study demonstrates the potential of a novel method for quantifying salivary immunoglobulin A (sIgA) using a point of care device. Researchers found that this method allows for reasonably precise and accurate measurements when quantifying sIgA for non-clinical, self-monitoring purposes. They also determined users need to follow proper testing protocol, but there is some tolerance for user error. Additionally, users should take care when monitoring sIgA levels over time to test at the same time of day, in a fasted state.
This study was limited by sample size. This was an exploratory study intended to collect preliminary research to direct future research projects. It is recommended that future studies expand the participant pool. It was found that when proper protocol is not followed, this method can produce inaccurate and imprecise results, although this is to be expected. It is recommended that users of this method follow the instructions provided for the best results. Despite any potential variation due to user error, results could still aid individuals in self-monitoring sIgA levels over time.
It is recommended that additional investigation and data collection be conducted to create a well-established normal or expected range for this method. Further research should increase the number of participants and evaluate long term patterns in testing and tracking sIgA to strengthen the method’s application in a broader setting. Documenting a standard range for the cube reader method will help to better understand and interpret the results for individuals and clinicians. It would also be advantageous to develop a means to convert the technology of the cube reader into a more accessible form such as a mobile app or computer software. This would further reduce costs and increase accessibility.
It is essential to continue development of methods to accommodate the growing desire for consumers to track and assess health metrics independently. Though not intended for diagnostic use, this method presents a significant advancement in enabling health-conscious individuals to conveniently monitor their sIgA.
Acknowledgements
This research is funded by 4Life Research LLC. This research did not receive any specific grant from funding agencies in the public or not-for-profit sectors.
Limitations
This study has several limitations which should be mentioned. First, small sample sizes were used to analyze impacts and outcomes of the POC device of interest. Second, samples were taken from employee volunteers. Third, participants were not screened for prior illnesses and/or use of medications outside of the study. A further limitation is the lack of sex- and gender-based analyses in the research design of this study.
Declaration of Interest
K.I., D.V. and X.H. were employees of the study sponsor, 4Life Research, LLC (Sandy, UT).
Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.
References
- U.S Food & Drug Admistration. Digital Diagnostics: Over-the-Counter (OTC) and Point-of-Care (POC). FDA Website. Published February 22, 2024. Accessed April 11, 2024. https://www.fda.gov/medical-devices/diagnostic-data-program/digital-diagnostics-over-counter-otc-and-point-care-poc.
- Pittman TW, Decsi DB, Punyadeera C, et al. Saliva-based microfluidic point-of-care diagnostic. Theranostics. 2023;13(3):1091-1108. Published 2023 Jan 31. doi:10.7150/thno.78872.
- Jankowski J, Nijakowski K. Salivary Immunoglobulin a Alterations in Health and Disease: A Bibliometric Analysis of Diagnostic Trends from 2009 to 2024. Antibodies (Basel). 2024;13(4):98. Published 2024 Nov 29. doi: 10.3390/antib13040098.
- Ruschen S, Lemm G, Warnatz H. An ELISA for IgA, IgG and IgM-RF measurement. I. Parameters of the assay. Scand J Rheumatol Suppl. 1988; 75: 32-35. doi:10.3109/03009748809096736.
- Bhavsar D, Sano K, Singh G, et al. An ELISA-Based Method to Measure Mucosal Antibody Responses Against SARS-CoV-2 in Human Saliva. Curr Protoc. 2024; 4(4): e1024. doi: 10.1002/cpz1.1024.
- Morgans R, Owen A, Doran D, et al. Pre-Match Salivary IgA in Soccer Players From the 2014 World Cup Qualifying Campaign. Int J Sports Physiol Perform. 2014; 10. doi: 10.1123/ijspp.2014-0046.
- Springham M, Dunbar J, Jehanli A, et al. Acute immuno-endocrine responses to a mid-season football match in the English Football League Championship. J Strength Cond Res. 2012; 26(10): 2783–2790.
- Han X, Vaughan B, Vollmer D. Safety and efficacy of a multivitamin, multimineral, bovine colostrum–containing supplement: an open-label pilot intervention trial in healthy adult women and men. Altern Ther Health Med. 2023; 29(7): 34‑40. doi:10.1123/ijspp.2023-0046.
- Dunbar J, Hazell G, Jehanli A. Evaluation of a new Point of Care quantitative Cube reader for salivary analysis in Premier League soccer Clubs.