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

BackgroundAmyloid-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.MethodsA 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.ResultsThe 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.ConclusionsThis digital ELISA has potential utility in clinical applications for quantification of Aβ1–42 in plasma where high sensitivity and precision are required.

A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70

“…Recovery of analyte in spiked human plasma ranged between 57% and 95% of the expected value and was dependent on concentration and varied between samples. This observation corroborates results reported by others—using the same technique—for the measurement of amyloid-β 1–42 peptide in human plasma [ 49 ]. A possible explanation for this difference in recovery in the present study could be the presence of HSP70-specific antibodies, which have been reported in human samples [ 43 , 50 , 51 ].…”

Elevated neurofilament light chain in plasma is associated with reduced right hippocampal and amygdala volume in Alzheimer's patients

“…Now NfL has been considered as an especially promising biomarker for neurodegeneration because it can be measurable in plasma 33 . Single molecule array (SiMoA) digital immunoassay platform can offer heightened sensitivity by miniaturizing the reaction volume 34 , and several biomarkers indicative of peptide fragmentation and neuronal dysfunction can be accurately quantified in peripheral blood. In our study, we used this Simoa technology to compare the plasma NFL concentrations of AD, aMCI and normal elderly people(their gender, age and education are matched), and found the concentration of plasma NfL in AD group were significantly higher(p < 0.05) than that in aMCI group and normal group.…”

miRNAs as biofluid markers for diagnostics of Alzheimer’s disease: recent status and perspectives

“…As of now the most frequently used biomarkers for AD diagnostics are assays for quantification of amyloid beta 42 (Aβ42) or Aβ42/Aβ40; total and phospho-tau in CSF or blood (reviewed in Song et al 2016;Lewczuk et al 2017;Jack et al 2018). Since the senile plaques are made of aggregates mainly consisting of truncated Aβ (Aβ42), development of Aβ42-specific ELISA has improved the specificity of AD diagnostics (Jarrett et al 1993).…”

“…The ultrasensitive ELISA technique for the detection of serine 396 and serine 404 showed 400-fold and about 1300fold higher sensitivity when compared to standard ELISA test (Hu et al 2002). Introduction of digital, single molecule assays based on Quanterix platform was important milestone, since this assay allows to detect tau protein and Aβ42 as low as picogram per millilitre range in the CSF and blood (Song et al 2016;Motamedi et al 2018). Simultaneous determination of tau Figure 1.…”