Development of an ultrasensitive multiplex assay for simultaneous detection of Aβ1‐42, Aβ1‐40, GFAP and NF‐L in blood

Abstract

AbstractBackgroundPrevious work (1) has described plasma amyloid, GFAP and NF‐L as predictors of Alzheimer’s pathology. Here we build on the Amyblood 1‐42/1‐40 Simoa assay (1,2) where GFAP and NF‐L, as indicators of astrogliosis and neurodegeneration, further added to the prediction for Aβ PET imaging or diagnosis. We report a Simoa multiplex assay that simultaneously measures Aβ1‐42, Aβ1‐40, GFAP and NF‐L in blood.MethodsA 4‐plex immunoassay was designed using Simoa technology. A monoclonal capture antibody specific for each analyte was attached to one of four different fluorescent dye‐encoded beads. Antibodies against Aβ1‐42 and Aβ1‐40 were from ADx (clones 21F12 and 2G3 as capture; 3D6 as detector), NF‐L and GFAP antibodies were as supplied in Quanterix commercial kits. The assay was further evaluated for its analytical performance, including analyte detectability in commercially‐sourced human plasma (n=20) and CSF (n=10) from healthy controls, and for discrimination between AD patient and control plasma (each n=10) from the Alzheimer Center Amsterdam.ResultsAssay calibration ranges were 0.041‐30pg/mL (Aβ1‐42), 0.016‐12pg/mL (Aβ1‐40), 6.86‐5000pg/mL (GFAP), and 0.686‐500pg/mL (NF‐L). Lower Limits of detection were 0.148pg/mL (Aβ1‐42), 0.084pg/mL (Aβ1‐40), 1.66pg/mL (GFAP), 0.157pg/mL (NF‐light). Limits of quantification for assay prototype were 0.381pg/mL (Aβ1‐42), 0.125pg/mL (Aβ1‐40), 4.99pg/mL (GFAP), 0.493pg/mL (NF‐light). In parallelism studies, mean recovery of serially diluted endogenous plasma and CSF (each n=4) was within 80‐120%. Grand mean recovery of expected spike concentration was within 70‐130% (n=4). The assay has 100% detectability for all four analytes in both plasma and CSF samples. NF‐L and GFAP concentrations increased in AD samples (respectively p<0.05 and 0.01) while the Aβ 42/40 ratio decreased (p<0.05) in AD samples versus healthy donors.ConclusionsThis multiplex Simoa assay accurately and specifically measure four biomarkers simultaneously in plasma and CSF, providing a new tool to quantify Aβ1‐42, Aβ1‐40, GFAP and NF‐L. Ongoing studies using a larger cohort of clinical samples will determine the potential clinical utility of this assay.