Disease-Relevant Protein Isomers
Protein variants, such as conformational isomers, post-translational modifications, point mutations, and degradation products, represent a rich resource of diagnostic information that has remained unexploited. These isoforms are normally present at low concentrations, and often cannot be differentiated by antibodies alone.
At GTCbio’s Biomarker Discovery and Development Conference, which will be held next week in San Francisco, scientists will discuss a range of approaches for working with protein biomarkers. For example, Intrinsic Bioprobes is developing mass spectrometric immunoassays (MSIA™) for clinical diagnostics.
“Our goal is to translate the MSIA technology into routine clinical applications,” says Urban Kiernan, Ph.D., director of biomarker discovery. “MSIA occupies a unique niche in diagnostics—quantification of disease-relevant protein isomers. Even if antibodies are unable to separate truncated forms or point mutations of the same protein, the combination of affinity-capture with MS provides a powerful diagnostic tool.”
The Massay® platform supports the fully automated process that begins with the capture of the target analytes from crude biological fluids by specific antibodies. The capture antibodies are immobilized on a proprietary solid support fitted within functional pipette tips. Each antibody is empirically selected for the desired target.
During the sample incubation process, biological fluid is recirculated through the affinity tip for enhanced enrichment of the desired target. The captured analyte is then eluted and directly deposited onto a mass-spec target for MALDI-TOF MS analysis. Integrated robotics enables high-throughput processing of 96 samples at a time.
“Research behind clinically relevant protein isoforms had been lagging until we pioneered a robust way to quantify protein variants,” continues Dr. Kiernan. “Just by screening the normal population, we were able to identify a substantial number of new isoforms. This opens the door to a completely new branch of clinical proteomics.”
One of the company’s early successes came from the analysis of brain natriuretic peptide (BNP), a well-known marker of congestive heart failure. The BNP assay has become one of the most important blood tests in cardiology. And yet, the available antibody-based test is unable to distinguish between circulating BNP forms, some of which are not clinically relevant but add to the total BNP measured.
Massay technology not only detects such low-abundance analytes but enables the unequivocal quantitation of active and inactive forms, ultimately driving the choice of therapeutic intervention. The company has also recently discovered and qualified two isoform biomarkers for type 2 diabetes that have been licensed to Ortho-Clinical Diagnostic.
“This approach makes sense,” comments Lawrence Oliver, Ph.D., professor in the department of laboratory medicine and pathology at the Mayo Clinic. “A number of clinical biomarkers are known to be present in different forms. For example, IL-6 is present in 17 different isomeric forms. This complicates our ability to consolidate the results of various clinical trials measuring the same analyte.”
Dr. Oliver is proposing a study that would re-evaluate the clinical validity of a well-established biomarker, an indicator of increased risk of coronary disease and stroke. Lipoprotein-associated phospholipase A2 (Lp-PLA2) generates pro-inflammatory products as a result of its natural enzyme action and is both a biomarker and a therapeutic target studied in multiple trials of vascular disease prevention.
Most recently, The Lancet published the retrospective analysis of 32 clinical studies with over 79,000 participants. The meta-analysis of this data demonstrated association of Lp-PLA2 activity and mass with risk for coronary disease. However, Dr. Oliver demonstrated that this analyte is not only unstable at room temperature, but also at –20ºC.
In his studies, the values for Lp-PLA2 stored at –20ºC steadily rose by as much as 40%. Such drift was not observed at –70ºC. Freeze-thaw cycles increased values by as much as 18%. Mayo Clinic collaborated with diaDexus, the manufacturer of PLAC® test for Lp-PLA2, on validation of an automated test.
“diaDexus had to overcome significant issues with the reagents,” continues Dr. Oliver. “Their newest test meets analytical validation criteria. But the absolute values of Lp-PLA2 detected by the new version of the test are only at 50% of what was published in the previous 32 studies.”
The contrasting values may be due to the drift under low temperature storage conditions, or due to differential stability of the enzyme in distinct states of the disease, or because the new test differentiates between bound and unbound forms of Lp-PLA2.
Regardless of the cause, the new test may not be measuring the risk component to the same extent as the old one. This means that the prospective data cannot be correlated with any previously collected clinical data. “This very promising biomarker can no longer be supported by the previous body of data,” adds Dr. Oliver. “Whenever a new biomarker is discovered and put to clinical use, the manufacturer has to ensure that with every new iteration of the test, we can continue to trace the results back to the clinical trials used to validate its interpretation.”