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Journal Article

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


Jahn,  Olaf
Proteomics, Wiss. Servicegruppen, Max Planck Institute of Experimental Medicine, Max Planck Society;

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Klafki, H. W., Rieper, P., Matzen, A., Zampar, S., Wirths, O., Vogelgsang, J., et al. (2020). Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples. International Journal of Molecular Sciences, 21(18): 6564. doi:10.3390/ijms21186564.

Cite as: https://hdl.handle.net/21.11116/0000-000A-EAC3-2
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. View Full-Text