30 May 2012. Testing a widely used cerebrospinal fluid Aβ42 immunoassay, scientists in Cambridge, Massachusetts, proposed modifications that they believe improve its precision and accuracy enough to pick up subtle changes within an AD cohort that would indicate potential therapeutic efficacy in drug trials. The modifications could also help it pass muster with regulatory agencies, the scientists claim. “We have performed the first Good Laboratory Practice (GLP)-equivalent validation of a CSF Aβ42 assay. Our results show that this immunoassay underestimates the amount of Aβ in CSF samples, and that modifications we have introduced improve accuracy,” said Valerie Cullen, who led the study with co-first author Ross Fredenburg and senior investigator Michael Solomon. All three were previously at the biotech company Link Medicine; Cullen is now consulting for Neurophage Pharmaceuticals, Fredenburg works at AstraZeneca, and Solomon has moved to Ember Therapeutics, all in the Boston, Massachusetts, area. Their paper appeared as Open Access May 3 in the American Association of Pharmaceutical Scientists (AAPS) Journal.
CSF Aβ42 drops prior to symptomatic Alzheimer’s dementia, and this decline predicts progression in people with mild cognitive impairment. One of the most extensively used commercial assays for measuring CSF Aβ42 is the INNOTEST Aβ42 ELISA from Innogenetics NV—a Belgian company bought in 2010 by Fujirebio of Tokyo. On the market since 2000, the INNOTEST kit discriminates AD from normal aging and other neurologic conditions (Hulstaert et al., 1999). It has been validated on autopsy-confirmed patient samples (Engelborghs et al., 2008) and has Europe’s CE mark of quality for diagnostic applications. Yet the assay suffers considerable intercenter and interlab variation (Mattsson et al., 2010). So do other CSF biomarker immunodetection methods, and this hinders their use in multisite clinical trials. Furthermore, different assay batches supplied by the manufacturer can give different readings for a given CSF sample (see ARF related news story on Fagan et al., 2011).
Such problems crop up in part because Aβ42 loves to aggregate. Indeed, the same pesky propensity that drives plaque formation within the brain also makes the peptide stick to ELISA collection tubes. Furthermore, Aβ42’s stickiness contributes to matrix effects—a property of the sample that causes faulty measurements for a particular analyte, depending on what else floats in the sample.
In their paper, Cullen, Fredenburg, and colleagues report ways to get around these two issues in the INNOTEST Aβ42 ELISA. To reduce Aβ adherence, the researchers spiked CSF samples with the detergent Tween-20. To deal with matrix interference, they diluted samples sixfold before loading them onto the ELISA plate. The manufacturer's instructions say to use undiluted CSF. However, by doing so, “you actually underestimate the amount of Aβ42 by at least twofold,” Cullen told Alzforum. The researchers determined this by running the ELISA on normal, MCI, and AD CSF samples at concentrations ranging from undiluted to 1:16 dilution.
“This advanced validation of a method for improving the quantification of Aβ1-42 in human CSF is extremely helpful to strengthen the use of CSF biomarkers in future clinical trials and in routine diagnostic settings,” Brit Mollenhauer of Paracelsus-Elena Clinic, Kassel, Germany, wrote in an e-mail to Alzforum (see full comment). “I hope that other assays (e.g., for total tau protein) and platforms for CSF proteins get the same attention and thorough validation.” Mollenhauer and Cullen formerly worked as postdocs in Michael Schlossmacher’s lab at Brigham & Women’s Hospital in Boston.
The authors optimized the ELISA to support a Phase 1b AD trial completed last year. Designed to assess safety and tolerability of LKN-754, a compound related to autophagy developed by Link, the trial monitored CSF Aβ42 pharmacodynamics in response to treatment, Cullen said. She and Fredenburg declined to comment on the trial outcome, but said they would be confident using the optimized methods to stratify patients into study cohorts or to monitor pharmacodynamics of other compounds.
Several scientists expressed concern about the optimizations proposed in the paper. On adding detergent to the CSF samples, Anne Fagan of Washington University School of Medicine, St. Louis, Missouri, wonders if this might affect Aβ42’s native structure in human fluid. “It has been proposed that oligomeric Aβ resides in CSF,” said Fagan, who directs the biomarker core for the international, multicenter Dominantly Inherited Alzheimer Network (DIAN) study. “Could Tween-20 disrupt the oligomeric species, and maybe that’s why you see elevated Aβ42 levels compared to samples without detergent?”
Another question is whether the detergent might influence Aβ42’s immunoreactivity, scientists said. “Detergent molecules will likely associate themselves with what is being measured,” suggested Leslie Shaw of University of Pennsylvania School of Medicine, Philadelphia. “They certainly will be in the matrix of what is being measured. What is the effect of this modified matrix on the immunologic reaction taking place?” Along with UPenn colleague John Trojanowski, Shaw co-directs the Alzheimer’s Disease Neuroimaging Initiative (ADNI) biomarker core.
ADNI uses Innogenetics’ bead-based multiplex AlzBio3 immunoassay—not the plate-based ELISA that Cullen and colleagues optimized. The AlzBio3 system is more suitable for high-throughput analysis because it allows measurement of all three CSF AD biomarkers (Aβ42, total tau, and phospho-tau) in a single well. Prior work has shown diagnostic equivalence between the AlzBio3 and ELISA methods for detecting AD (Olsson et al., 2005; Reijn et al., 2007; Fagan et al., 2011). Cullen and colleagues initially considered validating the AlzBio3 assay; however, when preliminary runs hinted at matrix interference issues for Aβ—which has different chemical properties from tau or phospho-tau—they opted against trying to optimize all three analytes in the same kit. Independent of the current paper, the scientists did examine the Meso Scale Discovery platform, which measures Aβ38, Aβ40, and Aβ42 simultaneously, and hope to publish that data shortly, Cullen said.
Fagan’s group uses both the INNOTEST ELISA and AlzBio3 kits for measuring CSF Aβ42, but has more experience with the former. She and Shaw discussed the possibility of doing a collaborative project testing the reported dilution and/or detergent modifications on samples from people with corresponding amyloid imaging data and clinical follow-up. “Whether we could consider modifying the protocol would depend on the results of that study,” Fagan said. “In our hands, the INNOTEST ELISA as it is currently sold has performed quite well in identifying PIB-positive folks and those who will progress clinically. However, there remain quality control challenges such as lot-to-lot variability in commercial kits, so if we can get a better assay, then we’d certainly consider it, at least for certain projects.”
Sebastiaan Engelborghs, who leads a reference lab for biomarker analysis at the University of Antwerp, Belgium, called the new study “a step forward.” However, he noted that the analysis did not address lot-to-lot and interlab variability. Both are nagging problems under exploration in U.S. and European qualification efforts to render AD biomarker assays more broadly useful and consistent across labs (see ARF related news story and ARF news story). “I would not apply the (modified method) until the findings are reproduced in another independent study,” Engelborghs told Alzforum. Douglas Galasko of the University of California, San Diego, expressed similar concern. “It will be of interest to see whether applying this new methodology changes assay reproducibility (especially between labs), and sensitivity and specificity for AD, compared to the ELISA that follows the Innogenetics instructions,” he wrote in an e-mail to ARF (see full comment below).
The current paper highlights the need for a gold standard method to measure CSF Aβ42, as well as certified reference material for calibrating the various assays for these measurements, suggested Henrik Zetterberg of the University of Gothenburg, Sweden, in an e-mail to Alzforum.
Hugo Vanderstichele, formerly of Innogenetics, helped develop the INNOTEST Aβ42 ELISA. He also emphasized the need for international reference materials, as well as for “regulatory documents describing all necessary requirements for an immunoassay.”
Along with three coworkers, Vanderstichele last year left Innogenetics to co-found a new biotech company called ADx Neurosciences, which focuses on the development of assays for novel biomarkers for dementia diagnosis in Alzheimer’s, Parkinson’s, and others diseases. “The AD diagnostic market is at a critical point in the sense that clear performance requirements for the assays need to be described for in-vitro diagnostics, as well as for pharmaceutical companies,” Vanderstichele wrote (see full comment below). He noted that validating an assay for pharma use is not the same as validating it as an in-vitro diagnostic.
Robert Dean, who oversees Aβ marker methods for Eli Lilly’s AD team, expressed a similar view. “If you are using an assay in a clinical research setting that is more academic, then you don’t have to make a regulatory claim,” he told Alzforum. “But when a pharmaceutical company runs a trial and uses an assay, we are typically expected to validate the assay’s performance—and in the process define sources of pre-analytical, analytical, and post-analytic variability—as well as determine whether the assay is fit for intended use. I think this paper does a nice job addressing these issues.”—Esther Landhuis.
Cullen VC, Fredenburg RA, Evans C, Conliffe PR, Solomon ME. Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ42 in Human Cerebrospinal Fluid. AAPS J. 3 May 2012. Abstract