Most immunodiagnostic protocols and commercially available kits require serum, plasma, or blood samples. Collecting such samples requires the skills of a phlebotomist. For pathogen detection, particularly in largescale field studies, the collection of noninvasive samples such as nasal swabs, tears, or saliva is undoubtedly an easier option. However, collecting such matrices can present some technical hurdles.
Various factors need to be considered by manufacturers when developing assays to detect unknown analytes reliably and accurately in swabs, saliva, or tears. Many analytes of interest, such as immunoglobulin G antibodies against specific antigenic targets, are found in higher concentrations in blood compared to saliva or nasal passages. To reliably measure low concentrations of analytes in biological specimens, highly sensitive detection methods and procedures are required.
Moreover, matrices such as saliva are more heterogenous compared to blood. Such heterogeneity in saliva is due to factors like age (which causes differences in viscosity), concentrations of protein antibodies, and ion content. Studies have shown that behavioral traits such as smoking status could cause high particle load in saliva. Even chewing gum alters the composition of saliva.1
Standardization of saliva and mucosal samples is critical because the antibody concentration in these samples is affected by an individual’s age, previous mucosal diseases, collection techniques, and the processing and storing of the samples. Whole saliva is easy to collect with or without stimulation. However, antibodies are more diluted in stimulated saliva than in saliva collected without stimulation. Therefore, the method of collecting saliva influences the antibody concentration in the sample.
An accurate measurement of immunoglobulin A in saliva or mucosal samples requires sample standardization first.2 An earlier study reported no correlation of IgG and IgA antibodies against the pathogen Streptococcus mutans between serum levels and that of tear and saliva levels. This suggests remote regulation of ocular and salivary antibody systems,3 rendering tears and saliva unsuitable for pathogen detection.
Moreover, depending on the method of saliva collection, the concentration of the pathogen could be diluted and in turn affect the sensitivity of the tests, be it by direct detection of antigens or through the detection of humoral responses.
Nasal secretions and saliva contain immunoglobulins known as secretory immunoglobulin A (S-IgA) rather than serum-type immunoglobulins (circulating antibodies). For COVID-19, wide-spread attention has been given to virus-neutralizing antibodies. These studies have primarily focused on the circulating antibodies in blood. The role of S-IgA antibodies and whether these can potentially neutralize and inhibit the SARS-CoV-2 virus is still being investigated.4
Immunodiagnostic assays have been developed to detect S-IgA in saliva or mucosal samples from patients infected with SARS-CoV-2 and other infectious diseases, to monitor infection,5-8 or the efficacy of vaccines.2 Most tests are used for the qualitative detection of S-IgA antibodies and have been optimized with a specific type of saliva, nasal sample, and collection device to minimize the matrix effect.
As of October 26, 2021, two qualitative commercial tests for infectious diseases have been approved by the FDA to detect total antibodies (IgA, IgM and IgG) in saliva.9 The FDA granted only one emergency use authorization (EUA) for total antibody detection against SARS-CoV-2 infection using saliva compared to 89 assays using fingerstick or dried whole blood, serum, or plasma.10
Studies have confirmed that saliva can be used for detection of antibodies against infectious pathogens such as SARS-CoV-2. However, the matrix effect is higher in saliva than in serum, plasma, or whole blood. Therefore, developing accurate assays for antibody detection using saliva requires greater optimization and standardization than tests based on blood fluids.
Due to the advantage of noninvasive collection of saliva, further studies are needed to minimize the matrix effect prior to bringing the assays to the market. Saliva as a sample type will be particularly beneficial in the pediatric and elderly populations.
References
1. Hettegger P, Huber J, Paßecker K, et al. 2019. High similarity of IgG antibody profiles in blood and saliva opens opportunities for saliva based serology. PloS one. 14(6):e0218456-e0218456
2. Gianchecchi E, Manenti A, Kistner O, et al. 2019. How to assess the effectiveness of nasal influenza vaccines? Role and measurement of sIgA in mucosal secretions. Influenza Other Respir Viruses. 13(5):429-437. doi: 10.1111/irv.12664
3. Burns CA, Ebersole JL, Allansmith MR. 1982. Immunoglobulin A antibody levels in human tears, saliva, and serum. Infect Immun. 36(3):1019-1022
4. Russell MW, Moldoveanu Z, Ogra PL, Mestecky J. 2020. Mucosal Immunity in COVID-19: A Neglected but Critical Aspect of SARS-CoV-2 Infection. Frontiers in Immunology. 11(3221).
5. Malamud D. 2011. Saliva as a diagnostic fluid. Dent Clin North Am. 55(1):159-178. doi:10.1016/j.cden.2010.08.004
6. Aita A, Basso D, Cattelan AM, et al. 2020. SARS-CoV-2 identification and IgA antibodies in saliva: One sample two tests approach for diagnosis. Clin Chim Acta. 510:717-722. doi:10.1016/j.cca.2020.09.018
7. MacMullan MA, Ibrayeva A, Trettner K, et al. 2020. Sci Rep. 10(1):20818. doi: 10.1038/s41598-020-77555-4
8. Isho B, Abe KT, Zuo M, Jamal AJ, et al. 2020. Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol. 5(52):eabe5511. doi: 10.1126/sciimmunol.abe5511
9. Corstjens PL, Abrams WR, Malamud D. 2016. Saliva and viral infections. Periodontol 2000. 70(1):93-110. doi: 10.1111/prd.12112
Iswariya Venkataraman, PhD, and Maite Sabalza, PhD, scientific affairs, EUROIMMUN US, a PerkinElmer company.
