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Multiplexed Proximity Ligation Assays to Profile Putative Plasma Biomarkers Relevant to Pancreatic and Ovarian Cancer

¹ Stanford Genome Technology Center, Bio-X, Stanford University, Stanford, CA; ² Department of Radiation Oncology, Stanford University, Stanford, CA; ³ Departments of Health Research and Policy and Statistics, Stanford University, Stanford, CA;⁴ Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden;

^aAddress correspondence to this author at Olink Bioscience, Dag Hammarskjoldsvag 54A, 75183 Uppsala, Sweden. e-mail simon.fredriksson{at}olink.com.

Background: Sensitive methods are needed for biomarker discovery and validation. We tested one promising technology, multiplex proximity ligation assay (PLA), in a pilot study profiling plasma biomarkers in pancreatic and ovarian cancer.

Methods: We used 4 panels of 6- and 7-plex PLAs to detect biomarkers, with each assay consuming 1 µL plasma and using either matched monoclonal antibody pairs or single batches of polyclonal antibody. Protein analytes were converted to unique DNA amplicons by proximity ligation and subsequently detected by quantitative PCR. We profiled 18 pancreatic cancer cases and 19 controls and 19 ovarian cancer cases and 20 controls for the following proteins: a disintegrin and metalloprotease 8, CA-125, CA 19-9, carboxypeptidase A1, carcinoembryonic antigen, connective tissue growth factor, epidermal growth factor receptor, epithelial cell adhesion molecule, Her2, galectin-1, insulin-like growth factor 2, interleukin-1, interleukin-7, mesothelin, macrophage migration inhibitory factor, osteopontin, secretory leukocyte peptidase inhibitor, tumor necrosis factor , vascular endothelial growth factor, and chitinase 3–like 1. Probes for CA-125 were present in 3 of the multiplex panels. We measured plasma concentrations of the CA-125–mesothelin complex by use of a triple-specific PLA with 2 ligation events among 3 probes.

Results: The assays displayed consistent measurements of CA-125 independent of which other markers were simultaneously detected and showed good correlation with Luminex data. In comparison to literature reports, we achieved expected results for other putative markers.

Conclusion:

Multiplex PLA using either matched monoclonal antibodies or single batches of polyclonal antibody should prove useful for identifying and validating sets of putative disease biomarkers and finding multimarker panels.