Single molecule array platform

The ADNI project provides multi‐centered data of plasma Aβ_1‐42 and Aβ_1‐40 levels, which vary across laboratories. To avoid experimental factors due to different methods for Aβ protein analysis, we only included the latest samples measured by immunoprecipitation in the Bateman Laboratory.¹¹ Aβ isoforms in human plasma were immunoprecipitated by the anti‐Aβ mid‐domain antibody on the KingFisher (Thermo) automated immunoprecipitation platform, and subsequently handled with Lys‐N protease and liquid chromatography tandem mass spectrometry (LC‐MS/MS).¹¹ Plasma p‐tau181 and NfL data were both provided by the Clinical Neurochemistry Laboratory, University of Gothenburg, Sweden. Through the Single Molecule array (Simoa) technique, plasma p‐tau181 was analyzed by an in‐house assay using a combination of two monoclonal antibodies (Tau12 and AT270).²⁷ Plasma NfL concentrations were generated by an in‐house ultra‐sensitive enzyme‐linked immunosorbent assay on a single molecule array platform (Quanterix Corp) in the Clinical Neurochemistry Laboratory of the University of Gothenburg, Sweden.¹⁹, ²⁸

Serum samples were collected in red-top serum vacutainer tubes (glass, silicone coated, no additives) and left at room temperature for 30–60 minutes to allow the clot to form. The tubes were then centrifuged at 4°C, 2,000 rpm, for 10 minutes, and the serum was collected under sterile conditions and frozen at −80°C until analysis. The levels of sNfL and sGFAP were quantified on a single molecule array platform at Quanterix (Billerica, MA). Samples were measured over 6 runs with reagents from the same lot. Samples from each group were evenly distributed across all plates, and samples from the same patient were measured in consecutive order. All samples were between the lowest and the upper limit of quantification. The average intra-assay coefficient of variation (CV) for duplicate measurements was 4.8% for sNfL and 3.0% for sGFAP. Two quality controls provided with the kit and 2 additional native serum samples were measured at the beginning and at the end of each run. The average CV between begin and end of run was 6.7% for sNfL and 4.5% for sGFAP. The average interassay CV between runs was 5.6% for sNfL and 3.8% for sGFAP.