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May 1, 2012 (Vol. 32, No. 9)

Biomarker Discovery Methods Evolving

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    The ProtoPlex™ assay from Life Technologies: A GST-tagged protein is captured by an anti-GST antibody conjugated Luminex bead. Each antigen is coupled to a different spectrally distinguishable bead region, allowing the assay to be multiplexed with up to 80 antigens plus controls. The beads are then incubated with serum, and autoantibodies present in the serum sample bind to the target protein. Autoantibody binding is then detected using an R-PE conjugated secondary antibody.

    Biomarker research has made substantial progress recently beyond the proverbial low-hanging fruit. Emerging technologies and techniques are helping make biomarker detection and application faster, cheaper, and more diverse. This was the focus of talks at GTC’s recent “Biomarker Summit” conference.

    Autoantibodies, which recognize the body’s own antigens, can function as biomarkers not only for autoimmune disorders but also for diseases like cancer, in which atypically high levels of proteins can trigger autoantibody production.

    Both Life Technologies’ ProtoArray® microarray and its ProtoPlex™ Luminex® assay have roots in the company’s successful recombinant expression of over 9,000 full-length human proteins. These proteins are printed on the ProtoArray microarray, which can be used to identify possible differences in autoantibody prevalence among samples from different populations (patients versus healthy controls, for example).

    According to Lisa Freeman-Cook, Ph.D., manager of R&D, researchers consider the ProtoArray microarray a good tool for discovering autoantigen/autoantibody candidates, but have had trouble following up on these candidates with new samples and independent methods. “They were using many different low-throughput techniques—ELISAs, Westerns, immunohistochemistry—and were getting stuck in the workflow,” she said.

    The new ProtoPlex assay, which Dr. Freeman-Cook described as “a multiplexed ELISA on beads,” addresses this bottleneck. A client may select up to 80 candidate protein antigens (chosen from Life Technologies’ library and/or provided by the client) to be included in every well of a 96-well plate; each antigen is distinguishable from the others because it is conjugated to microsphere beads with a unique dye-based “bar code.” Thanks to the multiplex design, as little as 3 microliters of serum is required for testing in triplicate.

    Validation of the ProtoPlex assay has come in part from a study of systemic lupus erythematosus (SLE). Forty-seven possible autoantigens originally identified with the ProtoArray microarray, along with known autoantigens such as Ro52, were tested against new serum samples from 174 women who had SLE, another autoimmune disease, or were healthy. Fifteen of the antigens remained strongly associated with SLE in the new study; each reacted with antibodies from at least 20% of the SLE samples but did not react with control samples.

    “ProtoPlex is an ideal custom validation platform because each customer will be interested in a different subset of proteins from their discovery study,” Dr. Freeman-Cook noted. Life Technologies has anticipated these interests to some extent by scanning customers’ published reports, making a list of potential cancer autoantigens, and conjugating 116 of them to beads for use in the ProtoPlex assay. Increasing use of this assay to discover novel cancer biomarkers thus appears likely, she remarked.

    Ezose Sciences grew out of a Japanese consortium between scientists at Hokkaido University and industry partners such as Shionogi & Co., a mid-sized pharmaceutical company that launched Ezose as a U.S.-based subsidiary in 2009. Its specialty is high-throughput glycomics—the analysis of sugars in biological systems, analogous to genomics and proteomics.

    Ezose considers glycomics a promising platform for biomarker discovery because glycosylation influences the stability, activity, binding, and immunogenicity of many proteins, and because altered glycosylation patterns are seen in numerous diverse diseases.

    Two glycosylation-based biomarkers are already clinically informative, noted Scott Siegel, Ph.D., vp of business development at Ezose. Measurement of CA19-9 (Lewis “a” antigen) sialylation is used to guide pancreatic cancer management, and detection of fucosylated alpha-fetoprotein (AFP-L3) aids in risk assessment and early recognition of hepatocellular carcinoma.

    “So there are actually already glycan biomarkers out there in clinical use,” he summarized. “Think about how many more could be discovered if you actually had a tool to get in there and do the research.”

    Ezose aims to provide such a tool via its GlycanMap® technology. Dr. Siegel indicated that GlycanMap has three key components: (1) bead-based chemoselective glycan enrichment known as Glycoblotting, (2) robotics for automated processing of samples in 96-well plate format, and (3) MALDI-TOF mass spectrometry coupled to bioinformatic analysis.

    In a 2010 collaboration between Ezose and Genentech, GlycanMap’s results were comparable to those of a traditional low-throughput method in which glycans were fluorescently labeled and detected via HPLC. With this validation in hand, Ezose is now applying the GlycanMap platform to biomarker discovery projects such as a diabetes study recently launched in cooperation with Merck.

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