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Bioasis Develops MRM-MS Assay for Measurement of Alzheimer's Protein Biomarker

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By Adam Bonislawski

Canadian biotech firm Bioasis this week announced it has completed development of a multiple-reaction mass spectrometry assay to be used in the diagnosis of Alzheimer's disease.

The assay, which uses the stable isotope standards and capture by anti-peptide antibodies, or SISCAPA, technique to measure serum levels of melanotransferrin – a protein that a number of studies have tied to Alzheimer's – marks an example of mass spec-based proteomics' ongoing move into the clinical setting.

The assay is part of Bioasis's Cognitest Alzheimer's diagnostic, for which it hopes to obtain a CE mark next year, CEO Rob Hutchison told ProteoMonitor. The company plans to employ it as a backup to its ELISA version of the melanotransferrin test, he said, using it in cases where the immunoassay is inconclusive.

ELISAs are widely used in clinical proteomics, but, noted Terry Pearson, a professor of biochemistry and microbiology at the University of Victoria and a Bioasis board member, the technology is not without its issues. In particular, he said, interferences from interactions with off-target analytes and autoantibodies can complicate the detection and measurement of target proteins.

With the SISCAPA assay, on the other hand, the researchers "didn't detect any interferences at all," Pearson told ProteoMonitor. "We're picking up only the two surrogate peptides of" melanotransferrin.

It's that lack of interference that makes the assay potentially useful for examining samples that the ELISA assay is unable to clearly classify.

"Typically in the [ELISA] tests that have been run on [melanotransferrin], there has been quite a clear separation between [Alzheimer's] patients and controls. But you get outliers, ones that sort of come in this zone in the middle," Hutchison said. "We think probably the reason is we have some interference in the ELISA. SISCAPA will tell us clearly whether the levels of [melanotransferrin] are elevated, or whether it was just interference in the ELISA."

A similar technique has been used for measuring thyroglobulin, a biomarker for various thyroid cancers, noted Andy Hoofnagle, director of clinical mass spectrometry at the University of Washington's Department of Laboratory Medicine.

Hoofnagle's lab runs a SISCAPA MRM assay for thyroglobulin when the researchers "are worried about the clinical situation not lining up with the results of the [ELISA] assay," he told ProteoMonitor in an e-mail. "It's a very reasonable way to ease into running SISCAPA and other MRM assays in the lab."

The SISCAPA MRM-MS assay could also allow analysis of improperly collected and stored samples, Pearson suggested.

"Because a lot of plasma [samples] are collected inappropriately, you can have protein degradation," he said. "This gives us one more chance to detect the peptides that may have become degraded and therefore aren't susceptible to standard sandwich immunoassays."

MRM mass spec's potential as a clinical tool has so far been tied to its multiplexing ability. As Leigh Anderson, CEO of the Plasma Proteome Institute and founder of the Anderson Forchung Group, which developed the melanotransferrin SISCAPA assay for Bioasis, told ProteoMonitor in October, the "small incremental cost" for adding analytes to an MRM assay makes the case for MRM-MS as a platform for multi-biomarker diagnostics "overwhelming" (PM 10/22/2010).

The technical and economic hurdles involved in using MRM-MS assays for clinical testing have typically made it less appealing as a platform for measuring single analytes, however. The high cost of trypsin needed for protein digestion, along with long assay run times, have hindered its adoption by large clinical reference laboratories.

With that in mind, AFG and Bioasis have been working for several years to streamline the melanotransferrin MRM assay, Pearson said.

"We've settled on some procedures using much less expensive trypsin and it seems to be working really well," he said. "Step two is getting the cost of running mass spec down. That's a matter of automation and learning to clean up our samples so we have quick cycle times. It's inching forward."

The SISCAPA assay can detect melanotransferrin at concentrations as low as 500 femtomoles – roughly equivalent to the sensitivity of immunoassays for the protein, Pearson said. The company is also developing monoclonal antibodies for use in the assay's enrichment step, which, he noted, would improve its sensitivity.

According to Pearson, Bioasis doesn't have figures on the current cost per assay, but he said that he expects the researchers will be able to bring it down to the level of a typical ELISA in the near term. The company runs the assay on Applied Biosystems' API4000 triple quadrupole machines.

Several other Alzheimer's research groups have approached Anderson about designing SISCAPA assays for potential biomarkers, Hutchison said. Anderson declined to comment on this work, telling ProteoMonitor that "those are confidential discussions at this point."

Melanotransferrin's potential as an Alzheimer's biomarker stems from several studies performed over the last decade, including a 2001 trial by University of British Columbia researchers and a 211-subject study in 2001 by South Korean scientists at Chungbuk National University that demonstrated accuracy in the 88 percent to 92 percent range.

Bioasis plans to start running Cognitest in clinical trials early next year and has obtained samples from Fleet Bioprocessing – the London-based biotech firm that built the immunoassay used in Cognitest – as well as from San Diego-based PrecisionMed. Depending on the results of these trials the test could be used as a diagnostic for Alzheimer's, a companion diagnostic, or an early screening device, Hutchison said. The company aims to obtain a CE mark for the test sometime next year, after which it hopes to license it to a large diagnostic provider and prepare a filing for US Food and Drug Administration approval.

In addition to Cognitest, Bioasis is investigating the use of melanotransferrin for the transport of therapeutics aimed at neurological diseases. Because the molecule is able to cross the blood-brain barrier, it could work as a chaperone for delivering drugs to the brain, Hutchison said.

Publicly traded, Bioasis currently has enough cash on hand to fund operations for roughly another 18 months, Hutchison said, but it might raise additional money via a private placement once it finishes clinical validation of Cognitest and submits a CE mark application for the test – likely around April or May of 2011. The company would be looking to raise around $10 million, he said.


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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