title: 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.
abstract: 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 Aβ1-42in 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.
  
  
  
  A sensitive digital ELISA for measurement of Aβ1-42using 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 Aβ1-42in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721.
  
  
  
  The prototype assay measured Aβ1-42with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ1-42peptide 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 Aβ1-42in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ1-42was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ1-42levels was also observed over the time course of sample collection.
  
  
  
  This digital ELISA has potential utility in clinical applications for quantification of Aβ1-42in plasma where high sensitivity and precision are required.
authors.affiliation: Quanterix Corporation, Lexington, MA, USA.
authors.forename: Linan
authors.identifier: 0000-0002-3057-5345
authors.initials: L
authors.lastname: Song
authors.affiliation: Eli Lilly and Company, Windlesham, UK. drlachno@lilly.com.
authors.forename: D Richard
authors.initials: DR
authors.lastname: Lachno
authors.affiliation: Quanterix Corporation, Lexington, MA, USA. dhanlon@quanterix.com.
authors.forename: David
authors.initials: D
authors.lastname: Hanlon
authors.affiliation: Quanterix Corporation, Lexington, MA, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Shepro
authors.affiliation: Quanterix Corporation, Lexington, MA, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Jeromin
authors.affiliation: Quanterix Corporation, Lexington, MA, USA.
authors.forename: Dipika
authors.initials: D
authors.lastname: Gemani
authors.affiliation: Lilly Research Laboratories, Indianapolis, IN, USA.
authors.forename: Jayne A
authors.initials: JA
authors.lastname: Talbot
authors.affiliation: Lilly Research Laboratories, Indianapolis, IN, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Racke
authors.affiliation: Lilly Research Laboratories, Indianapolis, IN, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Dage
authors.affiliation: Lilly Research Laboratories, Indianapolis, IN, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Dean
mesh_terms: D000544:Alzheimer Disease / Q000097:blood*; D016229:Amyloid beta-Peptides / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards*; D006801:Humans; D010446:Peptide Fragments / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42)
keywords: Alzheimer’s disease; Aβ1–42; Digital ELISA; Plasma; Therapeutic; Ultrasensitive