Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity

Abstract

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3–97.2%) compared to 91.7% (95% CI 77.5–98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3–100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses

“…[28][29][30] To overcome the challenge associated with low total IgG concentrations in saliva compared to blood some have resorted to concentrating saliva, either with or without additional accounting for salivary total IgG, which resulted in increased assay sensitivity but reduced specificity. 31 Despite the lower assay performance most groups found that blood SARS-CoV-2 IgG levels correlate well with salivary IgG levels 32 , however, such findings are often based on very small sample sizes. 31,33,34 Studies with larger sample sizes typically report less robust correlations between saliva and blood antibody levels.…”

“…31 Despite the lower assay performance most groups found that blood SARS-CoV-2 IgG levels correlate well with salivary IgG levels 32 , however, such findings are often based on very small sample sizes. 31,33,34 Studies with larger sample sizes typically report less robust correlations between saliva and blood antibody levels. 30,35 In the Ambulatory COVID-19 Study cohort nearly all participants had seroconverted by saliva IgG to SARS-CoV-2 two weeks post COVID-19 symptoms onset and SARS-CoV-2 IgG levels were stable for the duration of the study.…”

Advances in Electrochemical Biosensors Based on Nanomaterials for Protein Biomarker Detection in Saliva

“…During the last 2 years, a great number of saliva-based sensor technologies for SARS-CoV-2related proteins, namely viral antigens or SARS-CoV-2 antibodies, have been reported. [5][6][7][8] The positive test for SARS-CoV-2related proteins rapidly identifies the infected individuals in situations where and when the RT-PCR test is not available. The clinical value of saliva-based protein sensors is extended to other infectious disease diagnostics (i.e., rapid tests for malaria), [9] personalized medicine (i.e., routine tests for chronic lung disease or heart disease), [10,11] and other medical conditions affecting millions of people (i.e., on-site tests for physiological stress detection, early-stage detection of cancer, etc.).…”

A booster dose of either BNT162b2 or mRNA-1273 mRNA vaccines induces a robust recall of anti-SARS-CoV-2 spike protein IgG antibodies in saliva

“…Alternatively, oral mucosal fluid (saliva) specimens are non-invasive, enable self-collection, and require less processing, reducing cost 14 . Multiple studies demonstrated that anti-S-protein IgG levels can be robustly measured in saliva specimens with high sensitivity and specificity from infected or vaccinated individuals, even though salivary Ab concentrations are 100-10,000-fold lower than in serum 15,16 . IgG titers for serum and saliva are strongly correlated, indicating they represent a surrogate for anti-S-protein IgG levels in serum 15,16 .…”

“…Multiple studies demonstrated that anti-S-protein IgG levels can be robustly measured in saliva specimens with high sensitivity and specificity from infected or vaccinated individuals, even though salivary Ab concentrations are 100-10,000-fold lower than in serum 15,16 . IgG titers for serum and saliva are strongly correlated, indicating they represent a surrogate for anti-S-protein IgG levels in serum 15,16 . In contrast, anti-S-protein IgA Abs are not reliably detected in saliva, although serum IgA titers increase in response to infection or vaccination and are consistently detected in specimens 16 .…”