P3-055: Novel high-sensitivity ELISAs for alpha-synuclein quantification in multiple matrices, including human CSF and plasma

The abnormal accumulation of alpha-synuclein (a-syn) is a distinguishing characteristic of many synucleopathies, including PD and DLB. Quantification of CSF and plasma α-syn could be a critical biomarker for these diseases. The utilization of enzyme-linked immunoabsorbent assays (ELISA) to measure α-syn in biological fluids has been published. We sought to develop a robust ELISA assay that utilized commercial antibodies to accurately quantify α-syn in a number of biological matrices. We developed a total of seven different sandwich-type, enzyme-linked immunoabsorbent assays (ELISA) to measure total α-syn levels in unconcentrated CSF, plasma and other matrices. Multiple antibody pairings were used to support the assay development, which encompassed both rabbit polyclonal and murine monoclonal antibodies. The assays took only 2–3 days to be completed in a 96-well format. Cell lysates and culture supernatants were prepared from inducible a-syn cell line expressing with wild-type or the A53T mutant forms of a-syn. Several sandwich ELISAs were established to measure a-syn in multiple matrices. The assay sensitivity was shown to be 1.4 pg/ml and the assays showed consistent and reproducible data for human CSF (a-syn: ∼0.8 ng/ml) and human plasma (a-syn: ∼25 ng/ml). Our values were very comparable with the published data (CSF 1.57ng/ml and plasma 59.5 ng/ml). These assays were used to quantify a-syn levels in inducible (HEK-293) and SH-SY5Y cell lines for expressing either wild-type or A53T mutant a-syn. The data demonstrate that under basal conditions, a-syn is released into the culture medium of both cell lines. The level of a-syn in the culture medium was about 0.2% or 1%, respectively, in the HEK-293 and SH-SY5Y cells relative to the total cell lysate. The data revealed no significant differences in a-syn levels between the WT and A53T cells (either medium or lysate). We have developed a highly sensitive and specific ELISA, which will facilitate drug discovery for Parkinson's disease and other synucleopathies.