Naturally Occurring Alpha-Synuclein Autoantibodies in Parkinson's Disease: Sources of (Error) Variance in Biomarker Assays

Alpha-synuclein (alpha-Syn) plays a pivotal role in the pathophysiology of Parkinson's disease (PD), which can partly be modulated by innate and adaptive immune functions, and vice versa. Here, naturally occurring alpha-Syn autoantibodies (alpha-Syn-nAbs) may be effective against alpha-Syn pathoetiology and may serve as a PD biomarker. However, serum and cerebrospinal fluid alpha-Syn-nAbs levels still lack consistent evidence as required for a reliable PD biomarker. Serum and cerebrospinal fluid alpha-Syn-nAbs levels of 66 PD patients and 69 healthy controls were assessed using a validated ELISA assay. Moreover, potential sources of error variance including unspecific ELISA background signals, free serum hemoglobin concentrations, alpha-Syn plate coating procedures, and differences in alpha-Syn-nAbs standards, were investigated. PD patients and controls did not differ in serum (p=.49) nor cerebrospinal fluid (p=.29) alpha-Syn-nAbs levels. Interestingly, free serum hemoglobin concentrations were negatively correlated with alpha-Syn-nAbs levels in controls (Spearman rho=-.41, p<.001), but not in PD patients (rho=.16, p=.21). ELISA alpha-Syn plate coating procedures impacted inter-assay variability (same day coating: 8-16%; coating on different days: 16-58%). alpha-Syn-nAbs standards from different purification batches differed regarding optical density measured in ELISAs suggesting differences in alpha-Syn affinity. While alpha-Syn-nAbs levels may represent a potential PD biomarker, several methodological issues have to be considered to increase reproducibility of alpha-Syn-nAbs findings. Further studies using standardized protocols minimizing sources of error variance may be necessary to establish a reliable PD alpha-Syn-nAbs biomarker.