Salivary Anti-DNA Autoantibody Testing in Lupus Nephritis Using a Point-of-Care Vertical Flow Test

Background: Lupus nephritis (LN), a serious complication of systemic lupus erythematosus (SLE), typically manifests 3 - 5 years after disease onset and remains a significant cause of morbidity and mortality. Serum anti-DNA is diagnostic of Lupus, and may vary with disease activity in LN. No point-of-care (POC) assay currently exists to identify a patient with lupus using noninvasive samples. Saliva, however, contains established biomarkers such as anti-DNA and anti-nuclear antibodies (ANA), offering an accessible diagnostic medium. Methods: We developed a saliva-based Vertical Flow Assay (VFA) for the rapid detection of LN-associated antibodies. Nitrocellulose membranes were immobilized with dsDNA and HEp-2 cell lysate to capture anti-DNA and ANA, respectively. Biotinylated BSA and APAD were used as positive and negative controls. WHO-standardized anti-DNA was diluted into pooled healthy saliva. A 1:2 dilution yielded optimal sensitivity and specificity. Detection was performed using a biotinylated antibody, and streptavidin-conjugated gold nanoparticles were used as the reporter molecule. Cartridges were photographed and ImageJ was used to quantify spot intensity. Results: A standard curve was generated using spiked saliva with concentrations of anti-DNA and ANA ranging from 0 to 50 IU. The assay demonstrated visible signal intensities correlating with increasing antibody concentrations. ImageJ quantification yielded reproducible standard curves with acceptable coefficients of variation (CVs) across observers. Preliminary testing on patient saliva samples showed positive signal responses, supporting the diagnostic potential of the assay. Conclusion: This saliva-based VFA provides a noninvasive, low-cost, and rapid diagnostic platform for detecting LN biomarkers in under 30 minutes. It holds promise as a POC assay to enhance disease monitoring and access to care for SLE patients. Further validation with a larger patient cohort is underway to refine its sensitivity, specificity, and clinical utility.Figure 1. Vertical flow assay detecting anti-DNA and ANA autoantibodies.