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Researchers Develop Mass Spec Reference Method for Measuring Amyloid Beta 42 in Alzheimer's Patients


NEW YORK (GenomeWeb News) – An international consortium of researchers has developed a mass spectrometry-based reference method for standardizing measurements of cerebrospinal fluid amyloid beta 42 levels in Alzheimer's disease research and clinical work.

The method will allow researchers and clinicians to compare CSF Aβ42 measurements made with different assays in different labs, something that is currently not possible due to differences between these assays, said Henrik Zetterberg, a researcher at Sweden's Gothenburg Universityand one of the leaders of the effort.

This, he told GenomeWeb, could help further increase adoption of Aβ42 as an Alzheimer's biomarker and potentially aid in regulatory submissions of tests measuring the analyte.

Along with tau and phospho-tau, Aβ42 is one of the most established protein biomarkers for Alzheimer's and has been shown to be useful for diagnosis of the disease and for early detection. There are a number of commercial assays for the peptide, but while most of these assays are able to effectively distinguish between Alzheimer's patients and controls, they are not standardized against each other.

"So if you go to one clinic and they take a sample and send it to one lab that uses one test, and then you move to another clinic and get another test, you will get different absolute [Aβ42] concentrations," Zetterberg said.

With the established reference method, the researchers can now develop reference materials containing known absolute quantities of Aβ42, and these materials can then be provided to researchers, clinicians, and test vendors for calibrating their tests, making it possible to compare patient measurements made using different tests.

Zetterberg and his colleagues are currently working to generate reference material that will be certified by the International Federation of Clinical Chemistry and kept at the Joint Research Centre Institute for Reference Materials and Measurements in Geel, Belgium. 

The group, he said, has decided to develop three reference materials, representing low, medium, and high levels of Aβ42. In addition to generating the material, they are evaluating such issues as the short-term and long-term stability of the material, and procedures for aliquoting  and storing it, Zetterberg said.

The aim, he said, is to generate enough reference material to serve the industry for 10 to 20 years. Once the material has been generated, it will be provided to kit makers and labs upon request at a not-for-profit price, he added.

Zetterberg noted that while certification of the method and the material is not a substitute for approval of individual Aβ42 assays by regulatory agencies, it will likely help companies win clearance for their assays. For instance, he said, the US Food and Drug Administration has issued a letter of support for such reference material, and while the agency "will judge assays for themselves, [the reference material] will definitely facilitate approval of [Aβ42] tests."

While most Aβ42 tests use some form of immunoassay, Zetterberg and his colleagues turned to mass spectrometry for development of their reference method. Mass spec, he said, was an ideal approach due to its ability to overcome factors contributing to the variability of existing immunoassays.

Specifically, Aβ is a "sticky protein," he said. "It self-aggregates, it interacts with other proteins, with different surfaces. That's basically the basis for why it builds up in humans as we age."

This tendency to aggregate creates problems with epitope masking that affects different immunoassays to different extents, he noted. "And I think that explains a large part of the variation across immunoassay methods."

With mass spec, on the other hand, the researchers are able to denature the sample and prepare it such that they can measure all the Aβ42 present. "We have good spike-in recovery and linearity," Zetterberg said.

"Most of the commercially available assays, they are standardized to measure Aβ [in a way] that gives a good separation between Alzheimer's patients and controls, but they are not measuring all of the Aβ present in a sample, and that is why it was important for us to use the mass spec."

"One could think of other assay formats that might do it," he said. "For instance, if you increased the amount of detergent in an ELISA without destroying the antibodies, then you could liberate a lot more of the Aβ molecules. But Aβ is quite amenable to mass spectrometric analysis, so that was the most straightforward reference method we could think of."

Simplifying the task is the fact that, at just 42 amino acids long, Aβ42 is a small peptide, which means the researchers are able to measure it intact, without the need for trypsin digestion. Due to issues like differences in protein digestion efficiency and differential rates of peptide decay during digestion, digestion has proved one of the biggest challenges to the reproducibility of mass spec-based protein quantitation.

"Digestion has been a big difficulty, but we don't have to include it, which made it a bit easier to use [mass spec] for a reference method," Zetterberg said.

Indeed, he said that he could envision mass spec moving beyond the realm of their reference method and into the clinic more broadly.

"Immunoassays are great in some respects, and they have a long tradition in clinical chemistry, but there are problems with immunoassays, and as mass spectrometers get more and more user friendly, I think the clinical chemistry field will move in the direction of more mass spec-based assays for proteins and fragments of proteins," he said. "Many of these will have to have digestion steps, and that will have to be standardized, but it is not at all science fiction that this method actually may be used in clinical laboratories in the future."