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-beta 1-42 peptide (A beta(1-42)) 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 A beta(1-42) 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 A beta(1-42) 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 A beta(1-42) in clinical samples from patients treated with the beta-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. Results: The prototype assay measured A beta(1-42) with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an A beta(1-42) 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 A beta(1-42) in all of the 84 clinical samples tested. A rapid reduction in levels of A beta(1-42) was detected within 1 h after drug treatment, and a dose-dependent decrease of A beta(1-42) levels was also observed over the time course of sample collection. Conclusions: This digital ELISA has potential utility in clinical applications for quantification of A beta(1-42) in plasma where high sensitivity and precision are required.