P4-297: Development of full-length mouse amyloid beta ELISA and determination of endogenous Abeta level in BACE1 knockout mice

Abstract

Transgenic mice expressing mutant human amyloid precursor protein (APP) are widely used in the evaluation of amyloid beta (Abeta)–lowering strategies, such as beta and gamma secretase inhibitors. There is no clear evidence that APP expression is upregulated in sporadic Alzheimer's disease (AD) cases. Therefore, testing therapeutic agents under more physiologically relevant conditions, i.e. in mice with physiological APP expression, is important. Three amino acid residues differ between human and mouse Abeta peptides, and commonly used human Abeta ELISAs do not have optimal sensitivity to mouse Abeta. We aimed to develop a mouse Abeta ELISA, and to determine endogenous Abeta changes in non–transgenic (wild–type) mice. A monoclonal antibody against the N terminus of mouse Abeta was developed using synthetic peptide as an antigen. The antibody was characterized using standard protocols. Using an ELISA developed with this monoclonal antibody, we determined Abeta levels in BACE1 knockout mice. We identified clone 14F1, which recognized both mouse and human Abeta. The epitope is located within amino acid residues 1–4 of Abeta peptide. Clone 14F1 reacted more strongly with mouse Abeta than human Abeta. A sandwich ELISA composed of Abeta 40–specific antibody (clone 1A10) and 14F1 achieved single digit fmol/ml (equivalent to sub pg/ml) sensitivity, which is 1/10th of the endogenous Abeta level in wild–type mouse brain. We determined endogenous mouse Abeta level in BACE1 knockout mice using this ELISA. We determined that there is virtually no Abeta (background level in colorimetric assay) detectable in the brain of homozygous BACE1 knockout mice–/–. In heterozygous mice+/–, brain Abeta level was similar to that of wildtype control mice+/+. We developed a highly sensitive full–length mouse Aβ ELISA, which detected endogenous Abeta in non–transgenic (wild–type) mice. Heterozygous BACE1 gene deletion resulted in minimal Abeta reduction (in contrast to the effect of heterozygous BACE1 gene deletion in mutant APP transgenic mice). Determination of Abeta levels in non–transgenic mice is useful to elucidate the pharmacodynamic effects of secretase modulation; however, disease–modifying effects of therapeutic candidates should be characterized in plaque–forming transgenic mice.