A Quantitative Assay Selective for Amyloid Oligomer Species Differentiates Cerebrospinal Fluid from Alzheimer's Disease and Age-Matched Normal

Current therapeutic strategies for Alzheimer's disease (AD) target brain amyloid beta (Aβ) reduction. Oligomeric Aβ is thought to be the most toxic Aβ species however analytically validated assays which are quantitative, selective and sensitive enough to measure cerebrospinal fluid (CSF) oligomers have been difficult to develop. An ELISA was developed with these properties, using an Aβ oligomer-specific antibody. Antibody pairs were tested for Aβ oligomer and monomer selectivity using standard, sandwich ELISA methodology on an Envision® chemilluminesence platform. The pair with the best selectivity (1000-fold) and sensitivity (sub-ng/mL) for oligomers, humanized monoclonal antibody 19.3 and mouse monoclonal antibody 82E1, was not sensitive enough to detect endogenous oligomers in human CSF. The 19.3/82E1 assay was migrated to the Erenna® platform, which coupled one antibody to a solution-phase magnetic bead and the other to a cleavable, fluorescent dye. Individual dye molecules were counted as a measure of detected oligomers. CSF from clinically-confirmed AD and age-matched controls was tested in blinded fashion using this assay. Monomeric Aβ40 and Aβ42 levels were also measured by ELISA. Additional studies confirming 19.3 oligomer selectivity included size-exclusion chromatography and one-site competitive ELISA; employing either synthetic oligomers or extracts from APPswe brain at ages of oligomer formation prior to plaque. The oligomer assay had a limit of detection (2x background) for Aβ oligomers at 0.04 pg/mL and a limit of reliable quantification (with CV < 20% over background) of 0.4 pg/mL. In addition, the assay demonstrated 5000-fold selectivity for oligomeric versus monomeric Aβ. The new platform enabled differentiation of CSF between 40 confirmed AD and 32 age-matched controls, with respective mean oligomer signals of 2 pg/mL and 0.56 pg/mL. In addition to cognitive score, the respective CSF Aβ monomer levels confirmed diagnosis as the AD patients had significantly reduced Aβ42, but unchanged Aβ40 versus controls. Aβ oligomers were elevated approximately 4-fold in AD CSF compared to control, consistent with previous reports using relative measures. Future work using larger sample sets will correlate oligomers with other fluid and cognitive AD biomarkers. In addition, the pharmacodynamic response of CSF Aβ oligomers following therapeutic intervention will be examined.