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

The ratio of amyloid precursor protein (APP)(669-711)(A beta(-3-40))/A beta(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 A beta(-3-x) and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of A beta(-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 A beta immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected A beta(-3-40) with no appreciable cross reactivity with A beta(1-40) or N-terminally truncated A beta variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for A beta(-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 A beta(42)/A beta(-3-40) and A beta(42)/A beta(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 A beta(42)/A beta(-3-40) ratio as a novel biomarker candidate for Alzheimer's disease has been set.