Immunoassays for Human Saliva Need Validation

Saliva has gained considerable attention as a possible alternate diagnostic fluid to blood because it is easily accessible and convenient to collect.

Commercial enzyme-linked immunosorbent assays (ELISAs) have proven to be a powerful tool for fast and reliable sample analysis, however few ELISAs have been validated for use with saliva, or their validation is often incomplete.

Scientists at Newcastle University (Newcastle upon Tyne, UK) collected from healthy volunteers 5-10 mL of saliva and gingival crevicular fluid (GCF) which is the primary source of inflammatory mediators in saliva. They analyzed and validated commercially available assays for 13 common biomarkers of inflammation and bone resorption, relevant for diseases such as rheumatoid arthritis and periodontitis.

The following ELISAs were assessed for their use with human saliva: C-reactive protein (CRP), hepatocyte growth factor (HGF), interleukin 1β (IL-1β), IL-6, matrix metalloproteinases -3 (MMP-3), MMP-8, MMP-9, Regulated on Activation cytokine (Rantes), tissue inhibitor of metalloproteinases-1 (TIMP-1) (Quantikine; R&D Systems, Abingdon, UK), albumin, hemoglobin (Bethyl Laboratories; Cambridge, UK), collagen telopeptides (Wampole Laboratories; Princeton, NJ, USA) and elastase (Hycult Biotech; Cambridge, UK).

A range of viscosity for the analyzed saliva samples was found which could be graded (subjectively) anywhere from very fluid to highly viscous. Various degrees of viscosity are mainly due to different amounts of large glycoproteins, such as mucins. Handling the highly viscous samples required special attention to pipetting techniques, and in some cases “reverse” pipetting instead of “forward” was considered to improve accuracy and to reduce pipetting errors.

Of all evaluated assays, only three, MMP-8, MMP-9, and TIMP-1 were recommended for use with saliva by the manufacturer. To fit within the range of each standard curve, most assays required a different sample dilution than that recommended for serum samples, which had to be determined empirically. A recovery between 80% and 120% is generally accepted as a good indication that the assay is suitable for use with the tested sample. With the exception of collagen telopeptides, recovery of the analyzed assays was within or very close to this range

The authors concluded that most ELISAs are suitable for analysis of saliva samples, even if this is not specifically recommended by the manufacturer. However, they believe that an independent validation of assays for saliva samples is important, as they have highlighted a number of cases in which validation and quality control analysis for some assays yielded questionable results when using saliva. The study was published in the March 2012 issue of the Journal of Immunological Methods.

Related Links:
Newcastle University
R&D Systems
Bethyl Laboratories