Almost 50 years after their inception, immunoassays (IAs) are still the predominant method in biomarker diagnostics. IAs are used to detect pregnancies, infections, allergies, and many other health events in our lives. But for how much longer? Alternative, mass spectrometry (MS)-based techniques are getting off the starting blocks. Biomarker discovery has made great leaps forward since technology improvements in MS were applied. However, still very few new biomarkers are established per year. This may be because many labs use MS for the early steps in biomarker discovery and validation, but go back to IA for development of the final assay and confirmation for publications. Why are we not taking full advantage of MS for biomarker tests, especially the speed of development and the ability for multiplexing?
Immunoassays are widely accepted as the gold standard, as they are easy to use and cover all needs from individual over the counter tests to high throughput clinical analyses. At a closer look, immunoassays have a number of limitations that may be overcome by mass spectrometry (2). Most of these restraints are caused by the most essential part of an IA: antibodies. Generating a selective and sensitive antibody is time consuming. False results may occur if an antibody is not isoform-specific, interferes with autoantibodies or other sample components. Many antibodies are not multiplex-compatible or have issues detecting proteins present at very low or high concentrations. Furthermore, IAs do not detect biomolecules other than proteins, because raising antibodies against e.g. sugars or lipids is nearly impossible. In contrast, a mass spectrometry experiment can be set up fast to detect any kind of molecule, and to discriminate protein isoforms or post-translational modifications. It can be used in multiplex experiments and covers a wide dynamic range.
Another important factor is sample volume needed per assay – several hundred microliters in IA and only a few microliters in MS. Correspondingly, MS enables higher throughput at a lower cost per sample and allows to perform analyses even if very little sample is available – e.g. blood samples from infants or small animals. Last but not least, immunoassays typically include manual steps which lead to high variability – both between assays and individual labs. In direct comparison, accuracy and reproducibility were shown to improve with MS procedures (2).
TIME TO CHANGE?
So is it time to say goodbye to immunoassays? Are novel methods such as MS ready to take over? At least this is what a recent publication (3) from researchers at the Max-Planck Institute of Biochemistry in Martinsried near Munich suggests. The authors were able to develop a robust, streamlined, and high-throughput-compatible MS workflow that quantified up to 300 proteins within only three hours. The resulting plasma proteome profile revealed 45 out of 54 FDA-approved act in plasma biomarkers in a statistically relevant way. Quantification across several orders of magnitude was achieved using isotope-labelled standards. The researchers describe a straightforward sample preparation method that can be automated to run up to several hundred samples per day. At this throughput, a significant database for new biomarker discovery can be created, and large cohort studies can be performed for their evaluation and validation.
Obviously, great progress has been made in MS-based proteomics, and the advantages of mass spectrometry are clear. But what else is needed to dethrone immunoassays? Maybe both methods will co-exist for some time, with newly developed MS-based assays complementing the existing IA-based ones. Or, this might be the time to critically review all tests, and for moving only the relevant ones to new platform technology. To increase acceptance of MS methods, infrastructure and training programs need to be established. More importantly, significantly faster and more robust protocols are needed to match the throughput and reproducibility required in the clinic. With these developments in place, cost savings and improvements in patient care might soon outweigh today’s investments.
(1) Cross, T.G., Hornshaw M.P. (2016) Can LC and LC-MS ever replace immunoassays? Cell Systems 2, 185-195. http://pubman.mpdl.mpg.de/pubman/item/escidoc:2352959:3/component/escidoc:2352964/1-s2.0-S2405471216300722-main.pdf
(2) Travers, S., Martinerie L., Bouvattier, C., Boileau P.Lombes, M., Pussard E. Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method. J Steroid Biochem Mol Biol. (2016) http://www.sciencedirect.com/science/article/pii/S0960076016301777
(3) Geyer, P.E., Kulak, N.A., Pichler, G., Holdt, L.M., Teupser, D., Mann, M. (2016) Plasma Proteome Profiling to Assess Human Health and Disease. Cell Systems 2, 185-195. http://pubman.mpdl.mpg.de/pubman/item/escidoc:2352959:3/component/escidoc:2352964/1-s2.0-S2405471216300722-main.pdf