Objective: To evaluate the use of standards and controls in alpha-synuclein seed amplification assays (ɑ-syn SAAs).

Background: Alpha-synuclein SAAs show great promise for the diagnosis of Parkinson’s disease (PD) and prodromal PD in cerebrospinal fluid (CSF). There is, however, currently no standardised protocol and assay parameters can change between laboratories. Agreeing on a ‘best practice’ set of standards would represent a step forwards for standardisation of the assays and future-proofing.

Method: Alpha-synuclein SAAs were run using 40 mM phosphate buffer (pH 8.2), 20 μM ThT, 0.1 mg/mL alpha-synuclein (rPeptide), 170 mM NaCl, 100 μL reaction buffer, 15 μL sample/ control, 42°C, 6% gain, on a BMG Omega Fluostar plate reader, reading every 15 minutes for 60 hours. Alpha-synuclein pre-formed fibrils (ɑ-syn PFFs) and water were tested as standards and blanks, respectively. All controls/standards had the positivity threshold set as 10 SDs above the average RFU over the first 6 hours from all other data.

Results: When using 15 μL 10ng/uL ɑ-syn PFFs, a consistent fast response was observed and this can therefore be used as a fast ‘synthetic’ positive control, with acceptable levels of variation between batches and plates (n = 15, Fmax = 234,749 (95% CI 214,073 to 255,425), T50 (hours) = 19.9 (95% CI 17.1 to 22.6), average %CV: 34.0 (95% CI 32.4 to 35.7), 100% sensitivity (15/15 positive)). 

Diluting the ɑ-syn PFFs to 0.01 ng/uL ɑ-syn PFFs gave an overall slower response than 10ng/uL ɑ-syn PFFs, again with an acceptable variation between batches and plates (n = 6, Fmax = 241,477 (95% CI 217,715 to 265,239), T50 (hours) = 27.0 (95% CI 22.5 to 31.5), average %CV = 32.8 (95% CI 30.3 to 35.3), 100% sensitivity (6/6 positive)). 

Running a blank sometimes resulted in spontaneous self-aggregation and false positives (n = 20, Fmax = 70,870 (95% CI 35,541 to 106,199), T50 (hours) = 22.4 (95% CI 12.6 to 32.2), average %CV = 98.1 (95% CI 92.2 to 103.9), 60% specificity (12/20 negative)).

Conclusion: It is generally well accepted that CSF (or brain homogenate) from people with PD or controls can serve as reliable positive or negative controls. Looking forward, ɑ-syn PFFs could create a fast-responding positive control, with dilutions potentially used as standards. However, when using water as a blank, spontaneous aggregation may occur and reaction mixtures may need adjustment to prevent this.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/alpha-synuclein-seed-amplification-assay-set-up-quality-control-using-standards-and-controls/