(M1230-06-37) Development of a Sensitive and Specific Phospho-Tau 217 [Tau (pT217)] Biomarker Assay for Alzheimer’s Disease in Plasma and Serum

Purpose: As the population of older adults continues to increase in the United States and throughout the world, the incidence of Alzheimer’s Disease (AD) is also on the rise. As a result, early detection of AD using simple tests such as blood-based biomarker assays is of increased interest. Although effective treatment strategies are currently limited, early detection will be important for implementing future AD therapies and essential for supporting the testing of new, experimental therapies. Many studies have shown that elevated levels of phosphorylated Tau (pTau) isoforms in an individual’s serum, plasma, and CSF may be useful as early indicators of AD onset. More specifically, Tau phosphorylated at threonine 181 [Tau (pT181)] or threonine 217 [Tau (pT217)] are promising early-stage biomarkers of AD, with Tau (pT217) potentially having more predictive value than Tau (pT181), although this is still under investigation. Due to the relative ease of plasma and serum collection compared with CSF, there is a preference for the use of blood-based matrices for biomarker measurements. However, the detection of Tau (pT217) in plasma and serum is difficult due to the low circulating levels of this protein. Also, the adoption of Tau (pT217) as a biomarker for AD has been hampered by a lack of commercial assays with high sensitivity and specificity. Here, we report the development of a highly specific and sensitive assay for the detection of Tau (pT217) in human serum and plasma.

Methods: Immunization and antibody engineering activities were conducted to produce a number of new clones against Tau (pT217). Each antibody was labeled individually with biotin (to serve as a capture antibody) and TURBO-BOOST (to serve as a detection antibody for the ultrasensitive S-PLEX assay format), and they were tested in various combinations and orientations for their ability to detect Tau (pT217). Antibody pairs were chosen that included both a pan-specific total Tau antibody and an antibody specific for the phosphorylated T217 site. Furthermore, antibodies were tested for specificity against non-phosphorylated Tau protein and the phosphorylated T217 site using a mutated pTau protein in which the threonine was replaced with an alanine at position 217. After a final antibody pair was chosen, the assay was optimized and used to measure Tau (pT217) in serum and plasma from AD patients and healthy donors.

Results: Use of an anti-pTau T217 antibody as the capture and an anti-total Tau antibody as the detect provided assays with the highest sensitivity. During specificity testing of new clones, one anti-pTau T217 antibody outperformed the others. It had significantly lower non-specific signals from non-phosphorylated Tau and mutated A217 Tau than the other clones evaluated. This antibody also exhibited the lowest background signals while maintaining high signals for Tau (pT217). As a result, the final assay design included this particular capture antibody paired with a Tau (total) detection antibody. Plasma and serum samples from
AD patients or healthy controls were tested in the final assay format. Tau (pT217) was found to be elevated in the AD patients, with the majority of healthy controls having values near or below the lower limit of detection for the assay. These results were confirmed using serum samples from a second cohort, and good separation was observed between control and AD donors (n=10 healthy and n=24 AD; p < 0.001).

Conclusion: These data highlight the successful development of an ultrasensitive and highly specific immunoassay for the quantitation of Tau (pT217) in human serum and plasma samples.