DESCRIPTION

alpha-synuclein (α-syn) is a 14-19 kDa phosphoprotein that is primarily localize to the presynaptic terminals of mature neurons, where it is involved in synaptic function and plasticity. a-syn has drawn intense interest ever since the late 1990s, when the first α-synuclein missense mutation was identified as a cause of familial Parkinson's disease (PD). Aggregated and hyperphosphorylated forms of α-syn protein are the pathological hallmark of Lewy body disease, which includes Parkinson's disease (PD), diffuse Lewy body disease (DLBD), and Lewy body variant of Alzheimer's disease (LBV).

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	20	9.43	0.41	4.3
2	20	2.27	0.08	3.5
3	20	0.55	0.02	3.6

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	24	9.61	0.44	4.6
2	24	2.36	0.10	4.2
3	24	0.57	0.05	8.8

RECOVERY

The recovery of human a-Synuclein spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	109	89-125
	1:4	101	94-109

LINEARITY

To assess the linearity of the assay, human plasma samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	89
	Range (%)	85-93
1:8	Average% of Expected	90
	Range (%)	81-105
1:16	Average% of Expected	92
	Range (%)	85-100

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human a-Synuclein produced at Sunlong Medical™.