Development and Technical Validation of an Immunoassay for the Detection of APP669−711 (Aβ−3−40) in Biological Samples

Language
en
Document Type
Article
Issue Date
2022-10-24
First published
2020-09-08
Issue Year
2020
Authors
Klafki, Hans W.
Rieper, Petra
Matzen, Anja
Zampar, Silvia
Wirths, Oliver
Vogelgsang, Jonathan
Osterloh, Dirk
Rohdenburg, Lara
Oberstein, Timo J.
Jahn, Olaf
Editor
Publisher
MDPI
Abstract

The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.

Journal Title
International Journal of Molecular Sciences
Volume
21
Issue
18
Citation
International Journal of Molecular Sciences 21.18 (2020): 6564. <https://www.mdpi.com/1422-0067/21/18/6564>
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