Development and validation of a novel single molecular immunology assay for detection of p-Tau181

BackgroundAlzheimer's disease (AD) presents a major public health challenge. Current diagnostic methods for these disorders are often costly, invasive, and not widely accessible. The recently revised NIA-AA criteria highlight the potential of blood-based biomarkers as a promising non-invasive and cost-effective alternative for diagnosis.ObjectiveTo develop a novel single-molecule immunology assay specifically for the detection of phosphorylated tau at threonine 181 (p-Tau181) protein in blood samples.MethodsA novel single-molecule immunoassay targeting p-Tau181 was developed employing the Lychix Homebrew Kit. This new p-Tau181 assay underwent validation through the analysis of plasma samples from 98 clinically confirmed AD patients and 98 age-matched normal controls (NC), with the aim of assessing its effectiveness in differentiating between these two populations.ResultsInitially, the p-Tau181 immunoassay exhibited high nonspecificity and low discrimination between AD and NC plasma samples, with an AUC of 0.76. Further investigation revealed that false positive signals were caused by enzyme attachment to bead surfaces. By adjusting enzyme concentrations, reaction temperatures, and optimizing the sample diluent, it was ultimately discovered that salt concentration was the key factor in effectively minimizing false signals. With the optimized salt ion concentration, the refined p-Tau181 assay significantly improved its ability to distinguish AD from NC, achieving an AUC of 0.9313. The assay demonstrated improved sensitivity of 84.69% (76.27%-90.50%, 95% CI) and specificity of 87.76% (79.81%-92.85%, 95% CI).ConclusionsThe optimized single-molecule immunoassay p-Tau181 demonstrated significantly improved discrimination between AD and NC populations, underscoring its potential as a valuable diagnostic tool for AD.