A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-β 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

BACKGROUND: Amyloid-β 1-42 peptide (Aβ1-42 ) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aβ1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents.

METHODS: A sensitive digital ELISA for measurement of Aβ1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aβ1-42 in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721.

RESULTS: The prototype assay measured Aβ1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aβ1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ1-42 levels was also observed over the time course of sample collection.

CONCLUSIONS: This digital ELISA has potential utility in clinical applications for quantification of Aβ1-42 in plasma where high sensitivity and precision are required.