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bioRxiv · 10.1101/2023.09.10.557075

Effects of Neighboring Phosphorylation Events on the Affinities of pT181-Tau Antibodies

Min, S.·Mohallem, R.·Aryal, U. K.·Kinzer-Ursem, T. L.·Rochet, J.-C.
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Abstract

A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential Alzheimers disease (AD) biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in neurofibrillary tangles (NFTs) of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibodys cognate antigen, such effects have not been extensively studied. Here, we developed a biolayer interferometry assay to determine the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated. These findings highlight the importance of selecting antibodies that have been thoroughly characterized in terms of affinity and binding specificity, addressing the potential disruptive effects of post-translational modifications in the epitope region, to ensure accurate biomarker quantitation.

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Min, S., Mohallem, R., Aryal, U. K., Kinzer-Ursem, T. L., Rochet, J.-C.. 2023-09-13. Effects of Neighboring Phosphorylation Events on the Affinities of pT181-Tau Antibodies. https://doi.org/10.1101/2023.09.10.557075

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