Simoa homebrew assay development kit

For proof of concept, a prototype assay (supplementary methods) was developed in-house by Amsterdam UMC using a Simoa homebrew assay development kit (Quanterix, MA), utilizing capture antibodies ADx102 (21F12)¹⁴ for Abeta_1-42 or ADx103 (2G3)¹⁴ for Abeta_1-40 and detector antibody ADx101 (3D6)¹⁴ for Abeta₁ (ADx, Ghent, BE). These prototype assays formed the basis for the Amyblood assays that were further developed and upscaled by ADx. The sample diluent formulation and capture antibody conjugation were optimized to improve sensitivity. Reproducibility in %CV was tested on 3 monoclonal antibody lots and on the small batch compared to the upscaled RTU assays (volumes equivalent to 50 assay kits of 96 data points) based on duplicate measurements of the average enzyme per bead (AEB) signal, excluding the blank. Inter- and intra-assay variation were tested in six independent runs by ADx and five runs at Amsterdam UMC based on duplo measurements of two plasma quality control samples. Initial clinical performance of the Abeta_1-42/1–40 ratio was confirmed in 20 CSF Abeta_1-42 positive AD cases and 20 CSF Abeta_1-42 negative controls. Subsequently, the Amyblood RTU kits were shipped to Amsterdam UMC for further assay characterization and clinical validation. The Amyblood reagent preparation and assay set-up are detailed in the Supplementary methods.

Plasma samples stored at −80 °C were thawed and centrifuged at 10,000× g for 5 min. All specimen analyses were conducted using a Simoa HD-X system (Quanterix, Billerica, MA, USA). Prior to the assay, the samples were diluted with the sample diluent provided in each assay kit or the sample diluent in the Homebrew Assay Development Kit (Quanterix). The diluted samples were then applied to plates and assayed individually. The subsequent measurements were performed according to the manufacturer’s instructions provided in the kit (Quanterix).
To measure FABP2, FABP3, FABP5, and FABP7 levels, a custom assay system was developed using the Homebrew Assay. The assay was conducted according to the protocol provided with the Simoa Homebrew Assay Development Kit (#101354, Quanterix). The reagents necessary for the assay were prepared using Pierce EDC (#A35391; Thermo Scientific, Waltham, MA, USA) and EZ-Link NHS-PEG4-Biotin (#A39259; Thermo Scientific). Capture antibodies were coated onto magnetic beads at a concentration of 0.3 mg/mL, whereas detector antibodies were biotinylated at a 1:40 ratio. These customized reagents enabled the specific detection of FABPs in the samples.

During the course of the study, plasma NFL was available for all patients included. Plasma NFL concentrations were measured using an NFL kit (NF light; UmanDiagnostics) and then transferred to an ultrasensitive single‐molecule array platform using a homemade kit (Simoa Homebrew Assay Development Kit; Quanterix Corporation). A 6.7 ng/L was the lower limit of quantification, and 1620.0 ng/L was the upper limit. All measurements fell within the limits of quantification.