Validation of Various Pan‐ApoE and Isoform‐Specific ApoE Antibodies

作者：Dahlia Siano, Lesley R. Golden, Steven M. MacLean, Gabriela Hernandez, Lance A. Johnson · 发表于：Alzheimer s & Dementia · 年份：2024 · DOI：10.1002/alz.091634 · 研究领域：Alzheimer's disease research and treatments、Biological Research and Disease Studies、Clusterin in disease pathology

BACKGROUND: Apolipoprotein E (ApoE) exists in three protein isoforms: E2, E3, and E4, which differ by only one or two amino acids. These slight differences profoundly effect protein structure and function, allowing each isoform to differentially impact Alzheimer's Disease (AD) risk. Relative to the most common E3 isoform, E4 dramatically increases risk, while E2 confers a substantial decrease in risk. The close similarity between protein isoforms makes it difficult to develop isoform-specific antibodies that are reliable and selective. Here, we aim to validate and optimize a number of common, commercially available ApoE antibodies to determine isoform specificity. METHOD: Control samples included plasma and brain collected from APOE knockout (KO), homozygous E2, E3 and E4 humanized APOE (hAPOE) mice, and human plasma from all 6 possible APOE genotypes. Western blotting was used on plasma and brain homogenates to determine isoform specificity of E2, E3, or E4 antibodies. Commercial antibodies tested included pan-ApoE antibodies from Cell Signaling Technologies (CST) and Abcam, ApoE4 antibodies from Novus and CST, ApoE2 antibodies from CST, and ApoE3 antibodies from Abcam and Novus. Additionally, pan-ApoE antibodies were kindly gifted to us by collaborators (Drs. Lammich and Haass, LMU) and tested. Antibodies were also tested for use in immunohistochemistry (IHC) using hAPOE and APOE KO mouse brain sections (30 uM). RESULT: Despite the ApoE3 antibodies being marketed as isoform...