Background/Purpose:

There have been many reports showing that saliva could be a source of biomarkers capable of detecting certain diseases. However, very few studies have been conducted to profile autoantibody isotypes in the saliva of various autoimmune diseases except Sjogren’s syndrome. This study was performed to establish protein microarray for saliva diagnostics in autoimmune diseases and to identify distinct profiles of salivary autoantibody in patients with systemic lupus erythematosus (SLE). 

Methods:

We constructed antigen microarrays and probed healthy saliva spiked with commercial antibodies (Abs) (monoclonal mouse anti-La, anti-Ro52 and anti-Ro60 and polyclonal rabbit anti-U1-70). To investigate whether saliva destroys Abs existing in patients’ serum, we used serum from clinically confirmed SLE and mixed connective tissue disease (MCTD) patients diluted from 250 to 8000 fold with healthy saliva and PBST containing 5% fetal calf serum. Following proof-of-concept experiments, we developed another array with a panel of canonical antigens of SLE as well as cytokines (interferon α-2b, interferon γ, BAFF and CXCL13) to characterize autoantibodies in matched saliva and serum derived from 17 SLE patients and 13 healthy controls. The autoantibody IgG and IgA isotypes were assayed using goat anti-human IgG (AF647 conjugated) and goat anti-human IgA (Cy3 conjugated). The Axon Scanner and GenePix Pro 7.0 were used to determine median fluorescence intensities (MFI) of features and background. Data were analyzed using MultiExperiment Viewer and Significance Analysis of Microarray (SAM) algorithm.

Results:

The dynamic range of detection on the array was 1-10⁴ ng/mL for commercial Abs spiked into saliva. We observed a high degree of specificity and each Ab was specific for its target antigen. In the experiments investigating the effect of saliva on the reactivity of Abs existing in patients’ serum (anti-Ro52 and anti-La in SLE, anti-U1-70 and anti-U1-A in MCTD), saliva made the MFI signals a little bit weaker than buffer, but it was not significantly different. The optimal dilution rate of saliva for protein microarray turned out to be 1:4 to 1:8. Matched saliva and serum samples from 17 SLE patients and healthy controls were incubated with anti-IgG and anti-IgA secondary Abs.  IgG Ab reactivity against specific antigens was found mainly in serum, while IgA Ab reactivity to given antigens was predominant in saliva, suggesting the possibility of isotype switching between saliva and serum. SAM identified 7 antigens including BAFF, Ro60, U1-A and Sm/RNP that were significantly more reactive to IgA Ab in the saliva of SLE patients than in healthy controls (false discovery rate<0.01). The hierarchical clustered heat-map successfully placed SLE patients into close subgroups.

Conclusion:

Protein microarrays facilitate detection of autoantibody in human saliva as well as serum. Saliva profiling revealed that elevated IgA autoantibody reactivity to several targets including BAFF was associated with SLE compared with controls. The noninvasive nature of saliva collection with protein microarray highlights the efficacy of pursuing saliva as a diagnostic medium.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/applications-of-protein-microarray-for-saliva-diagnostics-in-autoimmune-diseases/