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.
Keywords: Alzheimer’s disease; amyloid β; assay development; assay validation; biomarker.