Salivary antibody levels are many fold lower than found in blood serum, Immunoglobulin G (IgG) in the saliva is mainly sourced from leakage from the gingival surface and Immunoglobulin A (IgA) from mucosal membranes.
However, saliva-based tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are minimally invasive compared to nasopharyngeal swabs, also costing considerably less in terms of time and staff commitment for sample collection.
Thus a reliable and convenient detection method utilizing saliva only would be of great benefit during the COVID-19 pandemic.
In a paper recently uploaded to the preprint server medRxiv* by Costantini et al. (September 7th, 2021), a novel enzyme-linked immunosorbent assay (ELISA) is developed that is able to quantify IgG and IgA levels in saliva samples that have been stored for several weeks.
The test is then employed to track the salivary IgG and IgA levels of COVID-19 patients over time, finding a differing immune response between those with mild or severe illness.
How was the IgG and IgA assay developed?
The ELISA developed by the group utilized the pre-fusion form of the SARS-CoV-2 spike protein, which was coated onto the base of a 96-well plate. Samples were added to the plates and incubated for one hour, and any bound antibodies were detected and quantified by the addition of horseradish peroxidize-tagged anti-human IgA or IgG, generating a colorimetric indicator upon the addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) dye.
One hundred eighty-seven participants that had tested positive for COVID-19 by RT-PCR donated saliva samples either once or repeatedly for five weeks. A second group of 98 patients was involved in a longer-term study where several samples were collected over a period as long as possible 203 days. Samples collected before the onset of the COVID-19 pandemic were also utilized as a control and initially used to tune the appropriate concentrations of reagents utilized in order to enhance specificity and limit background noise in the assay.
How did salivary IgG and IgA levels differ?
Of the 118 COVID-19 cases initially tested by the group using the assay, on average around 23 days after diagnosis by RT-PCR, 19.5% were positive for only IgA against the spike protein, 20.3% for IgG only, and 46.6% for both, while 13.6% were negative for any antibodies. When tested on a weekly basis the group note a higher rate of 28.6% for both IgG and IgA in the first week, with IgA then peaking to 62.1% and 68.6% in weeks two and four, respectively, then declining to 0% in weeks nine and ten. In contrast, IgG rose to 80% positivity rate by week three, peaking at 100% by week eight and then remaining positive amongst those tested until at least week ten.
The participants of the long-term study group were categorized based on disease severity, with disparity noted in antibody response between those with mild or severe COVID-19. Patients with mild disease had a 33.3% positivity rate for both IgG and IgA one week after symptom onset, with IgA peaking to 100% positivity at week four and then declining to 0% by week nine, and IgG peaking at 50% at week three, diminishing to 0% by 30 weeks. In patients with severe COVID-19, IgA levels reached 100% positivity slightly earlier, in week three, which slowly declined to baseline by week ten, while IgG peaked at 80% in week five and remained elevated for ten weeks.
Salivary antibody titers closely followed the positivity rate amongst those tested, IgA peaking in weeks 4-5 and declining by weeks 9-10, and IgG peaking slightly later and declining more slowly for the severely ill. Titers and positivity rates were consistently higher amongst the severely ill than the mildly or asymptomatic. The specificity of the assay was further demonstrated against samples known to bear several seasonal alpha- and beta-coronaviruses, to which the response was OD values below 0.06 for both IgG and IgA, compared to 0.841 and 0.742 for these antibodies against equal loads of SARS-CoV-2, respectively.
The assay developed here allows fast and reliable quantification of IgG and IgA against SARS-CoV-2 in saliva, allowing the severity of infection to be inferred. Other reports have demonstrated that salivary IgG antibodies correlate well with serum IgG levels and reportedly linger for several months following disease onset. IgA, however, often becomes undetectable in saliva in 4-6 weeks and frequently only shows a moderate correlation with serum levels. The limit of detection reported for the authors ELISA assay is 1.98 ng/ml for IgA and 0.3 ng/ml for IgG, an improvement over most similar assays that allows samples to be tested several weeks after collection and may provide a suitable alternative where blood tests are not feasible.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
- Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG Veronica P. Costantini, Kenny Nguyen, Zoe Lyski, Shannon Novosad, Ana C. Bardossy, Amanda K. Lyons, Paige Gable, Preeta K. Kutty, Joseph D. Lutgring, Amanda Brunton, Natalie Thornburg, Allison C. Brown, L. Clifford McDonald, William Messer, Jan Vinjé, medRxiv, 2021.09.03.21263078; doi: https://doi.org/10.1101/2021.09.03.21263078, https://www.medrxiv.org/content/10.1101/2021.09.03.21263078v1