Anyone who has pored over a patch of clover, trying to locate the elusive four-leaved variety, can relate to the challenge of hunting for Aβ oligomers. Widely thought to be the most toxic form of Aβ and a potentially valuable biomarker for Alzheimer’s disease (AD), oligomers are extremely rare in CSF, making them onerous to detect. In the February 19 Journal of Neuroscience, researchers led by Mary Savage at Merck & Company, West Point, Pennsylvania, detailed a new method to scavenge for them. Not only did the group detect minute amounts of Aβ oligomers in human CSF samples, they reported that they could distinguish AD patients from healthy controls. “It’s one of the most sensitive assays published to date,” said Henrik Zetterberg, University of Gothenburg, Sweden, who was not involved in the work. “Yet another high-quality paper shows that it is possible to measure Aβ oligomers in CSF.”

Alzforum covered the basics of this technique when it was presented at the 2012 Alzheimer’s Association International Conference (see Jul 2012 conference story). Merck scientists and their collaborators developed the 19.3 antibody, which binds preferentially to Aβ oligomers. In a sandwich ELISA, they used 19.3 to capture oligomers, and the Aβ antibody 82E1 to detect them. For maximum sensitivity, the researchers incorporated this ELISA onto a magnetic bead-based platform developed by Singulex, Alameda, California. The company's Erenna system, designed for extremely diluted biomarkers, can detect single molecules at femtogram/mL levels.

The researchers now report that they can detect synthetic oligomers (see Lambert et al., 1998) when as little as 0.09 pg/ml are present. Twice that amount is needed for the assay to be reliably quantitative. The assay detects oligomers over monomers with a 2,500-fold selectivity. Other labs have reported similar sensitivities in their tests for Aβ oligomers. Researchers led by David Brody, Washington University in St. Louis, detected as little as 1.56 pg/ml and could measure amounts above 6.25 pg/ml (see Esparza et al., 2013). A study from Zetterberg’s group used a different ELISA to quantify as little as 0.2 pg/ml (see Hölttä et al., 2013). However, while an ELISA developed in the lab of Dennis Selkoe, Brigham and Women’s Hospital, Boston, was sensitive enough to pick up as little as 6 pg/mL from concentrated human CSF, it detected none of these species. Nonetheless, Selkoe's group found that the human brain was packed with oligomers (see Feb 2013 news story). 

To make sure her group's assay specifically detected oligomers, Savage tested for Aβ in fractions separated by size exclusion chromatography. The 19.3/82E1 ELISA picked up high molecular weight species that corresponded to oligomers in two postmortem brain samples, one from a person with AD, and the other from an age-matched control who died of unrelated causes. The assay detected no signal in fractions that contained only monomers. 

Could the ELISA distinguish AD patients from controls? In one cohort, 12 AD patients had about nine times more oligomers in the CSF, on average, than did 11 controls. In a second cohort of 40 AD patients and 32 controls, that difference was threefold. With a sensitivity of 80 percent and a specificity of 88 percent, the assay differentiated AD and control samples better than measures of Aβ42 levels alone, according to the authors. It also distinguished people with AD from those with amyotrophic lateral sclerosis, Parkinson’s disease, or schizophrenia, all of whom had oligomer levels that matched controls. While oligomers in the CSF did not change with age, they did rise as scores on the Mini-Mental State Exam fell. 

The authors acknowledged that CSF oligomer concentrations among AD and controls overlapped considerably. Savage suggested this may be explained partly by oligomers seeping into the CSF in people with prodromal disease. Likewise, brain amyloid and CSF Aβ42 rise and fall, respectively, in people long before symptoms of dementia are apparent. Oligomers could one day make a good antecedent biomarker, but the field needs more cross-sectional and longitudinal studies, said Savage.

The assay represents a major advance in sensitivity, agreed most scientists interviewed for this article. “It would be great to have even more sensitivity than this,” said Selkoe. Researchers could likely distinguish better between patient and control groups and oligomers might then become a good biomarker for predicting AD, he suggested. Selkoe anticipates that other labs will soon replicate these findings. Zetterberg agreed, and envisioned that even more sensitive tests may one day identify patients who would benefit from oligomer-based treatments, and allow pharmaceutical companies to track the effects of oligomer-busting drugs.

Sylvain Lesné, University of Minnesota, Minneapolis, cautioned that it is hard to know exactly which Aβ species the assay measures, since the researchers did not clearly identify which oligomers from the human CSF were captured. Neither did they present the full characterization of the 19.3 antibody. He said that it is difficult to draw conclusions about binding in brain tissues based on only two human samples. In an email, Brody added that to detect oligomers in unfractionated CSF samples, an ideal assay would have to be more selective relative to monomers. He cautioned that the detergent this group used to keep oligomers from sticking to their storage tubes could, in theory, have induced some oligomerization on its own. Similarly, Zetterberg pointed out that the Aβ-derived diffusible ligands used to validate the assay can sometimes clump together to form fibrils, and that might complicate interpretation of the data. In the future, the researchers may want to stabilize the oligomers, he said. Savage said that she plans to test commercially available stabilized preparations, such as Aβ dimers. She said Merck plans to look at CSF oligomers in patients undergoing treatment with its BACE inhibitor MK-8931

One crucial question will be whether oligomers track with other Aβ-related biomarkers or if they will provide additional information. The CSF, Aβ42, and brain amyloid detected by PET imaging did not correlate with cognitive symptoms, for example. Scientists suggested comparing CSF data with amyloid PET measurements to determine if oligomers can distinguish those PET-positive cases who also have symptoms from those with normal cognition. Brody’s group has demonstrated that oligomer levels in the brain distinguished demented from cognitively normal individuals even when plaque levels were similar.—Gwyneth Dickey Zakaib

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References

News Citations

  1. New Assays for Aβ Oligomers in CSF Claim Femtogram Sensitivity
  2. New Assays for Aβ Oligomers—Spinal Fluid a Miss, Brain Awash

Antibody Citations

  1. Amyloid-β (N-terminal)-biotin (82E1-biotin)

Therapeutics Citations

  1. MK-8931

Paper Citations

  1. . Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53. PubMed.
  2. . Amyloid-β oligomerization in Alzheimer dementia versus high-pathology controls. Ann Neurol. 2013 Jan;73(1):104-19. PubMed.
  3. . Evaluating amyloid-β oligomers in cerebrospinal fluid as a biomarker for Alzheimer's disease. PLoS One. 2013;8(6):e66381. PubMed.

Further Reading

Papers

  1. . Amyloid-β oligomerization in Alzheimer dementia versus high-pathology controls. Ann Neurol. 2013 Jan;73(1):104-19. PubMed.
  2. . The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 2012 Mar;15(3):349-57. PubMed.

Primary Papers

  1. . A sensitive aβ oligomer assay discriminates Alzheimer's and aged control cerebrospinal fluid. J Neurosci. 2014 Feb 19;34(8):2884-97. PubMed.