Phosphorylated Alpha-Synuclein in Red Blood Cells as a Potential Diagnostic Biomarker for Multiple System Atrophy: A Pilot Study

Figure 2

Establishment of the ELISA method for pS-α-syn detection. (a) Recombinant human wild type α-syn (WT-α-syn) and Serine129-phosphorylated α-syn (pS-α-syn) were purified and validated by Coomassie blue staining and Western blot. (b) and (c) Purified recombinant WT-α-syn and pS-α-syn were detected by antibodies against WT-α-syn (b) and pS-α-syn (c), respectively. (d) Immunodepletion test for the specificity of pS-α-syn in red blood cell lysates (RBCs). Lane 1: RBC lysates without pre-immunodepleted by the 3D5 antibody; lanes 2–4: RBC lysates pre-immunodepleted by 0.1, 0.33 and 1 μg/ml of the 3D5 antibody. The lysates were analyzed by Western blot using the rabbit anti-pS-α-syn as the primary antibody. (e) Calibration curve for the enzyme-linked immunosorbent assay (ELISA) using the anti-pS-α-syn as the capture antibody and the biotinylated 3D5 anti-WT-α-syn as the detection antibody. Correlations between the standardized protein (WT-α-syn and pS-α-syn) concentrations and their corresponding absorbance values at 405 nm were plotted and the linear equations and correlation coefficients (R2) were calculated.

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