title: Signal loss due to oligomerization in ELISA analysis of amyloid-beta can be recovered by a novel sample pre-treatment method.
abstract: According to the predominant theories, soluble amyloid-beta (Aβ) aggregates are the principal neurotoxic agents in Alzheimer's disease pathology, making them a popular target for the development of therapeutics and diagnostic markers. One of the most commonly used methods for determining the concentration of Aβ is ELISA. However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates. Therefore, we investigated the effect of aggregation on the ELISA measurement and developed a novel chemical pre-treatment method, designed to disaggregate Aβ peptides, to improve the ELISA measurement of the total Aβ concentration. Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis. In our study we have shown that: •Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA.•Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss.•This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content.
authors.affiliation: Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Janssen
authors.affiliation: Biomolecular & Analytical Mass Spectrometry and Center for Proteomics (CFP-CeProMa), Department of Chemistry, University of Antwerp, Antwerp, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
authors.affiliation: Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium ; Department of Neurology and Memory Clinic, Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken, Antwerp, Belgium ; University of Groningen, University Medical Center Groningen (UMCG), Department of Neurology and Alzheimer Research Center, Groningen, The Netherlands ; Biobank, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.
authors.forename: Peter P
authors.initials: PP
authors.lastname: De Deyn
authors.affiliation: Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.
authors.forename: Debby
authors.initials: D
authors.lastname: Van Dam
keywords: AD, Alzheimers disease; Alzheimer’s disease; Amyloid-beta; Aβ, amyloid-beta; DMSO, dimethyl sulfoxide; ELISA; FA, formic acid; HFIP, hexafluoroisopropanol; Oligomers; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulphate; SP, soluble proteins; Sample pre-treatment; Sample pre-treatment for amyloid-beta ELISA analysis; Steric hindrance; TFA, trifluoroacetic acid; WT, wild-type