Evaluation of 6 monoclonal antibodies against Ser129-phosphorylated α-synuclein: Critical role of proteinase K antigen retrieval and superior sensitivity of the D1R1R clone in human skin biopsies

α-Synuclein is an essential component of synucleinopathies including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy (MSA). Misfolded-α-synuclein inclusions that contain high levels of Serine-129 phosphorylated (pS129-α-syn) are key diagnostic markers. Skin biopsies are a promising peripheral tissue for in vivo detection of aggregates using immunofluorescence staining. Several primary antibodies target pS129-α-syn but their diagnostic reliability remains uncertain. Common practice relies on clones EP1536Y and 81A without antigen retrieval; however, recent findings have underscored the need to validate additional methodologies and alternative clones. We compared the diagnostic accuracy of the standard protocol, alongside formic acid and proteinase K (PK) antigen retrieval to evaluate 4 additional monoclonal antibodies (J18, BBF19, pSyn#64, and D1R1R) in a cohort of 43 confirmed synucleinopathy patients (7 with MSA) and 33 healthy controls. The results showed that PK increased the detection rates for EP1536Y, 81A, and D1R1R, with D1R1R outperforming the others in sensitivity. J18, BBF19, and pSyn#64 exhibited insufficient specificity, limiting their clinical applicability. The improved accuracy with PK treatment and the promising performance of D1R1R mark critical advancements for reliable diagnosis, highlighting the importance of optimizing protocols and validating antibodies for dependable detection of pathological aggregates in skin biopsies.