13 April 2010. Researchers in Japan have developed an assay for amyloid-β (Aβ) oligomers that they suggest might be a useful diagnostic test for Alzheimer disease. The assay seems specific for very high-molecular-weight oligomers. The researchers, led by Takahiko Tokuda, Kyoto Prefectural University of Medicine, claim that their test is not only more sensitive than current methods of detecting monomeric Aβ42, but that their data support a role for large Aβ oligomers in AD pathology. First author Hiroaki Fukumoto and colleagues report that levels of these oligomers are higher in cerebrospinal fluid (CSF) from AD patients compared to CSF from control subjects. “If these findings can be replicated, the present paper represents a breakthrough in the field of AD biomarkers,” suggest Lars Lannfelt and Frida Ekholm Pettersson, Uppsala University, Sweden (see full comment below). The findings appeared in the March 25 FASEB Journal online.
This is not the first test for Aβ oligomers. A similar assay developed by Weiming Xia and colleagues at Harvard Medical School also detects oligomeric species and is similar in design—a single-antibody sandwich ELISA that uses one antibody for both capture and detection. That assay detects primarily low-molecular-weight oligomers (see ARF related news story). It is not clear why the two tests have different oligomeric specificities, though the antibodies are different in each case. Xia and colleagues chose monoclonal antibodies 82EI and 3D6, raised to Aβ amino acids 1-16 and Aβ 1-5, respectively, for their tests, whereas Fukumoto and colleagues used BAN50, also raised against Aβ1-16.
Other novel approaches have been taken to assay oligomers. Researchers led by Bill Klein at Northwestern University used antibodies laced with DNA to amplify, à la the polymerase chain reaction, signals from minuscule amounts of oligomers in biological fluids (see ARF related news story), and an antibody-based biosensor method to detect oligomers (see ARF related news story). Though both methods seem to detect Aβ oligomers in AD patients but not normal controls, they have not yet been approved as diagnostic tests. Aileen Funke and Dieter Willbold at the Forschungszentrum Jülich, near Düsseldorf, Germany, have capitalized on the confocal microscope to detect fluorescing antibodies bound to even a single Aβ oligomer (see ARF related news story).
The Japanese researchers tested their ELISA on Aβ oligomers separated by size-exclusion chromatography. They found that the test failed to detect Aβ monomers, dimers, or small oligomers (up to hexamers), even though these species comprised 99 percent of the total amount of Aβ. The test did detect Aβ oligomers that were 40-200 kDa, with most of them being 45-90 kDa (10-20mers). The assay could detect these species at concentrations down to 1 picomolar.
Turning to biological samples, the researchers used the assay to measure oligomers in human cerebrospinal fluid. They detected more oligomers in samples from people diagnosed with AD (18 patients) or mild cognitive impairment (nine patients), compared to a group of 25 controls, which comprised normal people and people with other neurological conditions, such as peripheral neuropathy. The elevation of high-molecular-weight (HMW) oligomers is in contrast to the well-documented drop in CSF monomeric Aβ42 normally seen in AD. In addition, levels of HMW Aβ oligomers inversely correlated with MMSE scores.
Compared to Aβ42 monomeric analysis, the sensitivity/specificity profile of this ELISA is better, according to the authors. More importantly, a test for HMW oligomers might give a better readout of disease progression and response to treatment. As Lannfelt and Ekholm Pettersson point out, CSF Aβ42 does not correlate with disease severity. That HMW oligomers inversely correlate with MMSE scores “holds promise for foreseeing any bioefficacy of drugs in clinical trials,” suggested Weiming Xia (see full comment below), who is now at Satori Pharmaceuticals Inc., Cambridge, Massachusetts. It might also help researchers figure out whether the known drop of CSF Aβ42 in AD occurs because the peptide is getting trapped in the brain parenchyma, or because it is aggregating in the CSF.
Whether this ELISA truly has the makings of a diagnostic needs further testing. “The biggest challenge in developing this kind of assay is validating that it is actually recognizing just oligomers and not monomers, too,” said Anne Fagan, Washington University, St. Louis. It is also not clear if the test is specific for Aβ oligomers. Some antibodies, such as the X11 antibody raised by Charlie Glabe’s lab at the University of California, Irvine, recognize common conformations adopted by different oligomeric proteins (see ARF related news story). Other researchers pointed out that there is considerable overlap between levels of HMW oligomers found in control and AD/MCI CSF samples, which might limit the clinical usefulness of such a test.—Tom Fagan.
Fukumoto H, Tokuda T, Kasai T, Ishigami N, Hidaka H, Kondo M, Allsop D, Nakagawa M. High-molecular-weight amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J 2010 March 25. Abstract