Development and Technical Validation of an Immunoassay for the Detection of APP(669–711) (Aβ(−3–40)) in Biological Samples

The ratio of amyloid precursor protein (APP)(669–711) (Aβ(−3–40))/Aβ(1–42) 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 Aβ(1–40) 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 Aβ(42)/Aβ(−3–40) and Aβ(42)/Aβ(40) 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 Aβ(42)/Aβ(−3–40) ratio as a novel biomarker candidate for Alzheimer’s disease has been set.