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Research Brief: New Methods for Aβ Detection, Production
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24 February 2009. So many different Aβs to measure, so little time—and funds. Two papers describe new methods that address these limitations in studies of the peptides found in hallmark plaques ravaging the brains of Alzheimer disease patients. In the first report, researchers led by Dennis Selkoe of Brigham and Women’s Hospital, Boston, describe an enzyme-linked immunosorbent assay (ELISA) specific for Aβ oligomers. Writing in this month’s Archives of Neurology, the researchers use the new assay to show that oligomeric Aβ and monomeric Aβ42 levels are tightly correlated in human plasma and brain. In the second paper, published 27 January in FEBS Journal, Sara Linse and Dominic Walsh at University College Dublin, Ireland, with colleagues there and elsewhere, describe a bacterial expression system for producing large batches of highly pure Aβ peptide quickly and cheaply.
Hungry for quick and reliable biomarkers, AD researchers have tried hard to correlate measurements of plasma Aβ species with disease progression. However, studies of Aβ42 have produced conflicting results. Some have suggested that the peptide holds promise as a blood diagnostic marker (see, e.g., Mayeux et al., 2003), while others have not (see, e.g., Blasko et al., 2006). A recent effort to develop a highly sensitive assay for plasma Aβ also came up empty in this regard, failing to show that plasma Aβ levels could predict sporadic AD (Hansson et al., 2008). Yet another study suggested that a drop in plasma Aβ levels could signal impending dementia in familial AD patients (Ringman et al., 2008). “These often inconsistent reports on the association of plasma Aβ levels with AD may reflect the fact that measurements to date only represent the pools of monomeric Aβ and were measured by different Aβ ELISAs (Fullwood et al., 2006),” write Selkoe and colleagues.
Last year, Selkoe’s group showed that oligomeric forms of Aβ, particularly dimers, are in fact more neurotoxic than plaques (see ARF related news story). However, there were no good methods to specifically detect and quantify these soluble rabblerousers—until now. In the new study, lead author Weiming Xia and colleagues devised an ELISA that used a single monoclonal antibody for both capture and detection of Aβ. This ensured that the assay would only recognize Aβ assemblies containing at least two Aβ molecules. The researchers confirmed the ELISA’s specificity for oligomeric Aβ by putting through a synthetic Aβ peptide that could form reversible disulfide-crosslinked dimers, and checking for its monomeric and dimeric species on silver-stained protein gels. The assay “should be useful to detect and quantify soluble, low-n oligomers in a range of samples, including in postmortem human or APP transgenic mouse brains, in cell culture medium, and in human CSF and plasma,” Selkoe wrote in an e-mail to ARF.
Using this new ELISA and existing assays, Selkoe’s team measured oligomeric and several monomeric Aβ species in plasma samples from AD patients and control subjects. They found that oligomeric Aβ levels were closely linked to Aβ42 monomer levels across all subjects, and that levels of both were higher in the AD samples. They also observed that measurements of each species dropped in parallel over time. The findings suggest that “measuring plasma Aβ and oligomeric Aβ over one to two years or more can reveal a significant reduction in plasma Aβ levels, especially Aβ42 levels, and this finding raises the possibility of a direct relationship of plasma Aβ to brain amyloid formation,” the authors write.
Another way to get a handle on how Aβ peptides contribute to AD is to produce big batches of disease-associated forms of the protein for structural analysis. Such efforts require affordable sources of Aβ peptide. In their FEBS Journal paper, first author Walsh and colleagues offer up a promising approach. Using a bacterial expression system, the researchers churned out hefty supplies of very pure Aβ peptide without using specialized equipment. With this procedure, “even a small lab can make its own highly pure Aβ at minimal cost,” Walsh wrote in an e-mail to ARF. He noted that 30 mg of Aβ40 could be produced in less than a week for about $200, while 30 mg of Aβ42 could be made in that timeframe for about $500. “I think it has the potential to make a big difference to many AD investigators studying Aβ,” he wrote.—Esther Landhuis.
References:
Xia W, Yang T, Shankar G, Smith IM, Shen Y, Walsh DM, Selkoe DJ. A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol. 2009 Feb;66(2):190-9. Abstract
Walsh DM, Thulin E, Minogue AM, Gustavsson N, Pang E, Teplow DB, Linse S. A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide. FEBS J. 2009 Jan 27. Abstract
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Comments on News and Primary Papers |
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Primary Papers: A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide.
Comment by: George Perry (Disclosure)
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Submitted 17 February 2009
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Posted 2 March 2009
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 I recommend this paper
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Comments on Related News |
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Related News: Bad Guys—Aβ Oligomers Live Up to Reputation in Human Studies
Comment by: Sylvain Lesne
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Submitted 7 May 2010
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Posted 7 May 2010
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Two new reports released this week (Villemagne et al., 2010; McDonald et al., 2010) document the prevalence of Aβ dimers in blood and brain samples, respectively, from individuals diagnosed with AD.
The first group used an elegant ProteinChip® array using affinity surfaces coated with various Aβ antibodies including 4G8 or WO2 to measure the levels of species bound to cellular membranes of blood cells in a large human cohort (n = 118). Using this approach, the authors found elevated levels of Aβ monomers and dimers in specimens from AD patients as compared to age-matched controls, though there were large overlaps between clinical groups. They also found that the levels of Aβ dimers strongly correlated with those of monomeric Aβ42. Interestingly, Aβ dimers were not detected when a 40-end specific antibody to Aβ was used as capture agent.
Finally, the authors performed correlation analyses among various clinical and neuroimaging variables, revealing modest but significant correlations between Aβ dimers and cognitive decline. Overall,...
Read more
Two new reports released this week (Villemagne et al., 2010; McDonald et al., 2010) document the prevalence of Aβ dimers in blood and brain samples, respectively, from individuals diagnosed with AD.
The first group used an elegant ProteinChip® array using affinity surfaces coated with various Aβ antibodies including 4G8 or WO2 to measure the levels of species bound to cellular membranes of blood cells in a large human cohort (n = 118). Using this approach, the authors found elevated levels of Aβ monomers and dimers in specimens from AD patients as compared to age-matched controls, though there were large overlaps between clinical groups. They also found that the levels of Aβ dimers strongly correlated with those of monomeric Aβ42. Interestingly, Aβ dimers were not detected when a 40-end specific antibody to Aβ was used as capture agent.
Finally, the authors performed correlation analyses among various clinical and neuroimaging variables, revealing modest but significant correlations between Aβ dimers and cognitive decline. Overall, these findings support the notion that Aβ dimers are elevated in AD compared to healthy controls as first reported by Shankar et al., 2008. However, this new report also documents the presence of Aβ dimers in biological samples from cognitively intact controls; this differs from the aforementioned study. Finally, due to the considerable overlap in the levels of Aβ dimers across tested clinical groups, it is unlikely that solely measuring Aβ dimers will represent a confident diagnostic tool for the prognosis of Alzheimer disease. This is disappointing news.
The second study led by Dominic Walsh’s and Dennis Selkoe’s groups can be viewed as a study extending the findings reported by Shankar and colleagues (2008). Here, McDonald et al. determined the levels of monomeric and dimeric Aβ levels in 43 brain specimens using a combination of immunoprecipitation/Western blotting techniques coupled to infrared detection for enhanced sensitivity. The authors report that soluble Aβ monomers, dimers, trimers, and occasionally tetramers were detected in their cohort. Unfortunately, no other oligomers (including Aβ*56) were observed due to the presence of non-specific bands masking potential oligomeric Aβ assemblies between 30 and 75 kDa. Consistent with their previous findings, Aβ dimers were only detected within the AD group compared to the controls, and their calculated concentration rose sharply in the AD group. One possible explanation for this segregation might be explained by differences in postmortem interval delays (24, 18, and 18 hours for the ND, DNAD, and AD groups, respectively) as well as in apparent age at death among groups (means of 81, 92, and 87.5 years). It would be interesting to see whether these variables have an impact on our biochemical analyses of Aβ oligomers.
Finally, the authors identified an association between the levels of Aβ monomers + dimers and intermediate to high brain amyloid loads. Altogether, these findings suggest that the concentration of brain-soluble Aβ dimers might be related to the extent of amyloid deposition in brain tissues.
Granted that both studies used very different biological samples and reported extremely different segregation profiles between controls and AD groups, blood or brain levels of Aβ dimers do appear elevated in AD.
 View all comments by Sylvain Lesne |
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Related News: Bad Guys—Aβ Oligomers Live Up to Reputation in Human Studies
Comment by: Gerard Roberts
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Submitted 7 May 2010
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Posted 7 May 2010
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I recommend the Primary Papers
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