Evaluating amyloid-β oligomers in cerebrospinal fluid as a biomarker for Alzheimer's disease

Abstract

The current study evaluated amyloid-β oligomers (Aβo) in cerebrospinal fluid as a clinical biomarker for Alzheimer's disease (AD). We developed a highly sensitive Aβo ELISA using the same N-terminal monoclonal antibody (82E1) for capture and detection. CSF samples from patients with AD, mild cognitive impairment (MCI), and healthy controls were examined. The assay was specific for oligomerized Aβ with a lower limit of quantification of 200 fg/ml, and the assay signal showed a tight correlation with synthetic Aβo levels. Three clinical materials of well characterized AD patients (n = 199) and cognitively healthy controls (n = 148) from different clinical centers were included, together with a clinical material of patients with MCI (n = 165). Aβo levels were elevated in the all three AD-control comparisons although with a large overlap and a separation from controls that was far from complete. Patients with MCI who later converted to AD had increased Aβo levels on a group level but several samples had undetectable levels. These results indicate that presence of high or measurable Aβo levels in CSF is clearly associated with AD, but the overlap is too large for the test to have any diagnostic potential on its own.

Figure 1. ELISA method for Aβ oligomers in cerebrospinal fluid.

A) Schematic drawing of the principle for the method. Left: The Aβo ELISA is based on the use of the same N-terminal anti-Aβ monoclonal antibody twice. The ELISA plate is coated with 82E1 to capture all forms of Aβ, while biotinylated 82E1 is used for detection. A synthetic Aβ dimer, with two N-termini, is used as standard. Middle: Aβos, with several free N-terminals, are detected in the assay. Right: monomeric Aβ will have their epitopes blocked by the capture antibody and are thus not detected by the detection antibody. B) Example of a typical standard curve from the Aβo assay. The standard curve ranges from 200–102,400 fg/mL. The assay has a lower limit of quantification of 200 fg/mL. C) Measurement of synthetic Aβo formation by the Aβo ELISA. Synthetic Aβ_1–42 was allowed to aggregate into Aβ oligomers. The signal in the Aβo ELISA was compared with a Thioflavin-T (ThT) assay for aggregated Aβ. The Aβo ELISA detects the formation of synthetic Aβo at an earlier stage than the ThT assay, while following the increase of oligomerization in parallel with the ThT assay after 5 hours.

Figure 2. Cerebrospinal fluid Aβ oligomers in independent clinical samples.

A) First AD study (Malmö). Increased CSF levels of Aβo in the AD group (n = 42) compared to the control group (n = 31), p<0.0001. Bars indicate median with interquartile range. B) Second AD study (Piteå and Stockholm). Increased CSF levels of Aβo in the group of patients with AD (n = 51) compared to the control group (n = 22), p<0.001. Bars indicate median with interquartile range. C) MCI study. Increased CSF levels of Aβo in the group of MCI patients who converted to AD during the follow-up period (n = 58) as compared to the control group (n = 62), p<0.01. No significant difference in CSF Aβo between stable MCI (p = 0.059) and controls. Bars indicate median with interquartile range. D) Clinical study on AD with different severity of dementia. Increased CSF levels of Aβo in the group of AD patients with mild (n = 44, p<0.01) and moderate (n = 51, p<0.001) dementia as compared to the control group (n = 33). No significant change was found in the AD group with severe dementia (n = 11) compared to the control group. Bars indicate median with interquartile range.