New Strategy Nets Biomarkers for AD, and More

It met with little enthusiasm when originally proposed, and took more than three years to perfect, but a new strategy for detecting serum antibodies could have important ramifications for Alzheimer’s disease diagnostics. Like many groundbreaking ideas, Thomas Kodadek’s was profoundly simple—use a huge library of synthetic antigens to capture circulating antibodies that are specific to disease states. "Nobody thought this would work," said Kodadek, who is at The Scripps Research Institute, Jupiter, Florida. But as outlined in the January 7 Cell, Kodadek and colleagues have now validated their methodology using a mouse model of multiple sclerosis, and have gone on to demonstrate that it can identify antibodies found only in people with Alzheimer’s. "This has the potential to be a big advance in the AD biomarker field," noted David Holtzman, Washington University, St. Louis, Missouri, in an e-mail to ARF (see full comment below). "It may also be very useful for biomarker identification for other neurodegenerative and neurological disorders."

Fishing for disease-specific antibodies (or antigens) is not a new idea, but as Kodadek and colleagues explain, unmodified native antigens are unlikely to work as bait, because those antigens are also found in non-disease states. It is far more likely that disease-specific antibodies recognize unusually modified natural antigens. But if you don’t know what those antigens are, then how can you find the antibody?

Not such a Catch 22 as it seems. First author Muralidhar Reddy and colleagues used synthetic molecules to make a "shape library," and then used that to capture antibodies in an unbiased fashion. "There has been a mindset that antibodies are only going to bind to their native antigens," said Kodadek. But as the researchers demonstrated, that is not the case.

Reddy and colleagues used an array comprising thousands of peptoids—octamers of glycine with side groups added by way of combinatorial chemistry. To ensure that none of these antigens are normally found in nature, they added the side groups to the glycine nitrogen instead of the α carbon, and they ensured most of the side chains did not resemble any of the 20 natural amino acids. In this way, they generated an array of molecules that are unlikely to be exact matches for native antigens. They used the array to capture antibodies, which they detected using a secondary anti-IgG antibody with a fluorescent tag.

Could this array pull out disease-specific antibodies? "We started with the simplest disease model imaginable," said Kodadek. The researchers tested sera from mice with experimental autoimmune encephalomyelitis (EAE). In this model of multiple sclerosis, mice are immunized with Freund’s complete adjuvant and a peptide derived from myelin oligodendrocyte glycoprotein (Mog). The researchers collected antiserum 38 days later and ran it over an array of more than 4,600 peptoids. Unsurprisingly, serum from the Mog/adjuvant-injected mice contained antibodies that bound to Mog peptide that the scientists had spotted on the array. More importantly, the serum also contained antibodies that bound to three of the peptoids. Those peptoids did not capture antibodies from mice treated only with adjuvant. The same three peptoids also captured antibodies from a second set of EAE mice, giving the test some validity. The peptoids also had some antibody specificity, as they failed to recognize antisera from mice that were immunized with ovalbumin. They gave a hint of how severe disease was, because they captured progressively more antibody up to three weeks after immunization.

But what about disease not induced by an active immunization? This is when the researchers turned to serum from people with Alzheimer’s disease. From an array of more than 15,000 spots, three different peptoids (ADP1 to ADP3) detected higher IgG amounts in serum of six AD patients compared to serum from six age-matched controls. Kristin Martin-Cook at the University of Texas Southwestern Medical Center in Dallas provided the samples. In a subsequent validation test, all three peptoids again detected high IgG levels in sera from 16 separate patients. Sera from 14 of 16 controls did not recognize any of the three peptoids, but sera from two other controls did. These could be false positives, or, as the authors write, these volunteers could have preclinical disease. Because all three peptoids behave similarly, the authors believe that the latter explanation is most parsimonious.

Kodadek told ARF that he is now testing patient samples that were taken longitudinally to see if he can track disease progression by comparing antibody capture to the development of symptoms. Like the Mog peptoids, ADP1 to 3 also seem to be relatively specific, because they did not detect antibodies in sera taken from six Parkinson’s disease patients or six people with systemic lupus erythematosus.

Some news outlets are already touting these findings as a "blood test" for AD, but as Tony Wyss-Coray at Stanford University, California told ARF via e-mail, some of these media reports are over the top. "This is certainly not a blood test at this time," he told ARF. Kodadek and colleagues agree, and are continuing validation studies. A Florida-based diagnostic company, Opko Health Laboratories, where Reddy and several of the coauthors work, has licensed the technology. On Monday, Opko and Bristol-Myers Squibb signed an agreement to pursue AD diagnostics (see company press release). Kodakek, who holds stock options in Opko, told ARF that in second-generation screening, the scientists discovered new antibodies. "The preliminary results look extremely promising," he said.

One burning question that remains is, What constitutes the natural antigen for these antibodies? Kodadek said that he has not started looking into that in earnest. But in principle, if he can capture enough of the antibody, then he could use it to pull down antigens from AD brain extracts and identify the antigens by mass spectroscopy. As Wyss-Coray writes (see full comment below), there is no consistent evidence to suggest that antibodies specific to AD (or other neurodegenerative diseases) exist, though "dozens of isolated studies in the literature have described antibodies with distinct antigen specificity to be overrepresented in AD compared with healthy controls." He did note that the small number of peptoids that bind AD sera in this study could indicate that AD patients possess a highly specific immune response against an unknown antigen. "If true, that would most certainly change the current view of this disease," he wrote.

Reddy MM, Wilson R, Wilson J, Connell S, Gocke A, Hynan L, German D, Kodadek T. Identification of candidate IgG biomarkers for Alzheimer’s disease via combinatorial library screening. Cell 2011 January 7;144:132-142. Abstract

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