Immuno-PCR-based quantification of multiple phosphorylated tau-epitopes linked to Alzheimer's disease

Several lines of evidence suggest that quantification of phosphorylated sites in the tau-protein (phospho-tau) might be favorable for early and specific Alzheimer's disease diagnosis. The typical setup to quantify phosphorylated tau-epitopes relies on a sandwich ELISA with a capture antibody (Ab) recognizing tau independent of its phosphorylation status and a detector Ab binding specifically to a certain phosphorylation site. Besides Ab specificities, major challenges arise from the very low tau-concentrations in cerebrospinal fluid (CSF) ranging from 100 to 2,000 pg/ml. Based on the phosphorylation degree of a given position, which can be below 10%, the corresponding phospho-tau-level might be much lower, especially for multiphosphorylated epitopes studied here. Thus, a novel, highly sensitive, and generally applicable immunoassay is described to quantify tau-versions, which are phosphorylated at pThr212/pSer214/pThr231/pSer235, down to tau-concentrations of 2 pg/ml in CSF.