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

The ratio of amyloid precursor protein (APP)669-711 (Aß-3-40)/Aß1-42 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 Aß-3-x and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of Aß-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 Aß immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aß-3-40 with no appreciable cross reactivity with Aß1-40 or N-terminally truncated Aß variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aß-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 Aß42/Aß-3-40 and Aß42/Aß40 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 Aß42/Aß-3-40 ratio as a novel biomarker candidate for Alzheimer's disease has been set.

Solid-Phase Synthesis and Characterization of N-Terminally Elongated Aß-3-x -Peptides.

In addition to the prototypic amyloid-ß (Aß) peptides Aß1-40 and Aß1-42 , several Aß variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aß variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid-phase peptide synthesis of the N-terminally elongated Aß-peptides Aß-3-38 , Aß-3-40 , and Aß-3-42 . Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aß-3-38 and Aß-3-40 are generated by transfected cells even in the presence of a tripartite ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aß peptides starting at Val(-3) can be separated from N-terminally-truncated Aß forms by high-resolution isoelectric-focusing techniques, despite virtually identical isoelectric points. The synthetic Aß variants and the methods presented here are providing tools to advance our understanding of the potential roles of N-terminally elongated Aß variants in Alzheimer's disease.

Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - development of potential agents for the treatment of Alzheimer's disease.

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP).