Over 40 marker assays are available to run on the samples. These include markers such as Osteopontin, Mesothelin, Periostin, Endoglin, intestinal Fatty Acid Binding Protein, and FAS-Ligand, some of which have been previously described in the literature. Other proprietary markers are derived from internal discovery efforts and from collaborator programs.
Several of the proprietary markers were discovered in collaborator programs employing novel discovery platforms. These methods include differential proteomics and differential gene expression measurements in ovarian cancer and normal tissue samples. At Biosite, novel high throughput technologies are used to raise antibodies against the markers and to develop immunoassays for application in serum and plasma samples. We have preliminary data on our markers from measurements made in individual serum samples from 72 ovarian cancer patients and 37 normal healthy controls. The ROC AUC numbers exceed 0.70 for five proprietary markers for both serous and non-serous ovarian cancer samples. The ROC AUC numbers exceed 0.75 for two proprietary markers for serous ovarian cancer samples. The ROC AUC numbers exceed 0.75 for four proprietary markers for non-serous ovarian cancer samples. These proprietary markers outperform Osteopontin and Mesothelin with respect to discrimination between ovarian cancer and normal samples. Additional sample sets are required for further verification of the clinical utility of these proprietary markers and how they perform against the current benchmarks in the field of ovarian cancer diagnosis.
Antibody libraries for markers are generated from immunized mice and enriched for high affinity and specificity using phage display methods. The libraries are screened to select monoclonal antibodies to construct assays for the markers.
A multiplexed assay platform employing custom Luminex xMap ™ magnetic beads is used to measure the markers in samples. Different bead types, based on their unique embedded reporter dyes, are assigned to different marker assays. Sandwich ELISAs are used for markers present at picomolar to nanomolar concentrations in samples. The magnetic beads, conjugated to capture antibodies, are incubated with samples. The captured markers are detected with biotinylated antibodies binding to complementary epitopes, using avidinylated phycoerythrin as a secondary label to generate assay signals. Competitive ELISAs are used for markers present at micromolar concentrations in samples. Here, the capture antibodies conjugated to the magnetic beads are incubated with samples that are pre-spiked with biotinylated marker constructs. The markers and their biotinylated counterparts compete for binding to the capture antibodies. The bound biotinylated constructs are labelled with avidinylated phycoerythrin to generate assay signals that are inversely related to the concentrations of the markers in the samples. For both sandwich ELISAs and competitive ELISAs, the final assay signals are read in a Luminex 200 flow cell. One laser measures the assay signals on the beads derived from phycoerythrin and a second laser identifies the unique bead types and hence the corresponding marker assay identities.
The assays described above are conducted in 96-well assay plates. A robotic platform conducts all liquid handling steps and employs a 96-well magnetic separator station to manipulate the beads. Up to 25 marker assays are multiplexed in each 25 µl sample, resulting in nominal sample volume consumption of only 1 µl per marker assay.
There are currently no biomarkers annotated for this protocol.
No datasets are currently associated with this protocol.
Thank you to everyone who helped make the 9th EDRN Scientific Workshop a success. We look forward to seeing everyone at the 28th EDRN Steering Committee Meeting from March 31-April 2, 2015, in Atlanta, GA.
Funding Opportunity Available
Both RFAs for Molecular and Cellular Characterization of Screen-Detected Lesions have been published.