Biomarkers


closely related forms of specific proteins. Currently, commercial tests are available for more than 200 different proteins (using various methodologies); custom-immunoaffinity assay services for others are provided by a number of specialty-providers.


Mass spectrometry assays: hidden importance of antibodies


While mass spectrometry has been widely used over the years for hypothesis-free detection of pro- tein biomarkers, its application has been impeded by lack of sensitivity and the non-quantitative nature of the tests. More recently, a variant of the technology commonly referred to as peptide MRM (multiple reaction monitoring) is gaining impor- tance as a more quantitative variant for protein biomarker measurements of this platform. Peptide MRM can combine the high selectivity and speci- ficity of MS for the protein of interest with impres- sive quantitative accuracy and dynamic range. Quantitation obtained by this method is based on the peak area for the mass spectra data of the ana- lyte relative to a known quantity of an isotope- labelled standard. The peak area can be used to provide relative quantitation (similar to most immunoaffinity assays) or absolute quantitation (protein concentration)2. Proteins at low abun- dance levels in samples will require a method to enrich for the protein(s) of interest. One such enrichment method is immuno-enrichment with antibodies to the protein or the peptides3, another one is the immune-adsorption-based depletion of the sample of abundant protein species. Thus, even MS is highly dependent on antibody technology. Such is the case of the MRM assay, which uses the stable isotope standards and capture by anti-pep- tide antibodies, or SISCAPA, technique to measure serum levels of melanotransferrin (p97) – a protein that a number of studies have tied to Alzheimer’s – marking a move into clinical setting to employ as a backup to the ELISA version of the in vitro diag- nostics (IVD) melanotransferrin test, in cases where the immunoassay is inconclusive (biOasis website). Indeed, the marriage of the two approaches, as discussed below, is fast emerging as one of the most powerful approaches in biomarker research. Figure 4 shows the potential role of immunoassays and mass spectrometry technology platforms as well as the potential applications/ben- efits derived from each platform.


Immunoaffinity LC-MS/MS assays


One of the major challenges facing the emerging field of protein biomarkers is the fact that many biomedically relevant biomarkers are present at


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very low abundance in human samples. The immunoaffinity LC-MS/MS approach has been specifically devised to address the analytical chal- lenge imposed by the tremendous dynamic range of protein biomarkers, especially in biofluids. For instance, serum or plasma analytes of interest are first enriched in the sample using immuno-based approaches, followed by mass spectrometry-based further characterisation. An example of this approach is the design and validation of an immunoaffinity LC-MS/MS assay for the quantifi- cation of a collagen type II neoepitope peptide in human urine as a biomarker of osteoarthritis (Methods Mol. Biol. 2010; 641: 253-70). Another example is IBI’s mass spectrometric immunoassay platform (MSIA). MSIA relies on a patented pipette immunoenrichment technology that uses a high-throughput, high-binding-capacity microcol- umn activated with antibodies to isolate low- abundance proteins in complex samples. Researchers analyse the isolated proteins via single reaction-monitoring mass spectrometry, which enables them to quantitate protein variants. In the case of parathyroid hormone (PTH), MSIA enabled researchers to identify a number of new protein variants associated with the hormone that may be useful in developing biomarkers for vari- ous skeletal and endocrine diseases (Clinical Chemistry 2010; 56: 281–290).


Steps towards a meaningful protein immunoassay


Antibody availability and quality: A number of public domain initiatives to develop antibodies against all human proteins (eg HUPO Antibody Initiative www.hupo.org; FP7 Program ‘Affomics’) are currently being undertaken. Both immuno- and MS-based protein assay platforms will benefit from these initiatives as they will provide critical assay reagents. Antibodies are essential for immuno-affinity-based assays, where the selectivi- ty of the antibody needs to be as high as possible. Mass spectrometry-based assays use antibodies to enrich a complex sample (eg plasma) for the pro- teins of interest; here, the specificity of the anti- body does not have to be as high since the antibody is not used to characterise/identify the protein of interest. Therefore, a ‘fit-for-purpose’ antibody is viewed based on application and utility.


Analytical accuracy: Analytical accuracy refers to a number of metrological parameters that deter- mine the performance of an assay on the level of technical specification. The term represents a com- posite assessment that comprises both random


Drug Discovery World Winter 2010/11


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