Sensitivity and specificity of phospho‐Ser129 α‐synuclein monoclonal antibodies

Abstract

α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.

Developmental Changes in Dendritic Spine Morphology in the Striatum and Their Alteration in an A53T α-Synuclein Transgenic Mouse Model of Parkinson’s Disease

“…Thereafter, the sections were washed three times for 20 min in 50 m m Tris-buffered saline (TBS; 50 m m Tris-HCl, pH 7.4, and 150 m m NaCl) and incubated in a blocking solution containing 10% normal goat serum (NGS) in TBS for 1 h. The sections were then incubated for 48 h in rabbit monoclonal anti-phosphorylated α-synuclein antibody (phospho-S129, clone EP1536Y, catalog number ab51253; Abcam) at a dilution of 1:1000 in 2% NGS in TBS. The sensitivity and specificity of this antibody was confirmed in a previous study ( Delic et al, 2018 ). Sections were then washed three times for 20 min each with TBS and incubated overnight at 4°C in 1.4 nm nanogold-conjugated goat anti-rabbit secondary antibody (Nanoprobes).…”

Criticality as a measure of developing proteinopathy in engineered human neural networks

“…As has been reported by several other studies investigating alpha-synuclein PFF induced pathology (85)(86)(87)(88)(89)(90), we experienced issues with unequivocal identification of pathological alpha-synuclein aggregates using immunocytochemistry (Fig.S3), even after TritonX-1000 protein extraction, which should leave only insoluble inclusions (42,62,90). Although neural networks from the PFF condition consistently displayed positive immunolabeling of alphasynuclein phosphorylated at S129, unspecific labelling and background staining were also observed in control conditions, rendering the immunoassays inconclusive.…”

The center of olfactory bulb‐seeded α‐synucleinopathy is the limbic system and the ensuing pathology is higher in male than in female mice

“…Sacino and colleagues cautioned that pSer129 antibodies (81A) can cross-react with neurofilament L in white matter tracts (150). Delic et al similarly observed nonspecific bands by Western blotting with all α-synuclein phosphoantibodies, and reported that the rabbit monoclonal anti-pSer129 antibody used here displays the highest sensitivity and specificity of commercially available anti-pSer129 antibodies; there are simply no better markers of α-synucleinopathy to date (47). It is also worth observing that, in those regions where the inclusions were quite dense per field of view (ie, more than one or two), statistically higher numbers of pSer129 + structures were always observed in the fibril-infused mice, confirming that our blinded interpretations of genuine α-synucleinopathic inclusions were indeed correct.…”

“…Monoclonal mouse and rabbit antibodies raised against pSer129 were employed to visualize Lewy‐related pathology, as Ser129 phosphorylation of α‐synuclein is the most frequent posttranslational modification of this protein within Lewy aggregates . Manual counts of the number of pSer129 inclusions were performed on tissue sections stained with the mouse monoclonal antibody, as this antibody was employed before we found that the rabbit monoclonal was even better, as confirmed in a recent study . A blinded observer manually counted the total number of pSer129 + inclusions under 200× magnification (field of view = 447.1 μm × 337.0 μm) in 2–3 sagittal sections per animal.…”