title: Multiplexed proximity ligation assays to profile putative plasma biomarkers relevant to pancreatic and ovarian cancer.
abstract: 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 microL 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-1alpha, interleukin-7, mesothelin, macrophage migration inhibitory factor, osteopontin, secretory leukocyte peptidase inhibitor, tumor necrosis factor alpha, 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Stanford Genome Technology Center, Bio-X, Stanford University, Stanford, CA, USA. simon.fredriksson@olink.com
authors.forename: Simon
authors.initials: S
authors.lastname: Fredriksson
authors.forename: Joe
authors.initials: J
authors.lastname: Horecka
authors.forename: Odd Terje
authors.initials: OT
authors.lastname: Brustugun
authors.forename: Joerg
authors.initials: J
authors.lastname: Schlingemann
authors.forename: Albert C
authors.initials: AC
authors.lastname: Koong
authors.forename: Rob
authors.initials: R
authors.lastname: Tibshirani
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood*; D005260:Female; D006801:Humans; D007118:Immunoassay; D008297:Male; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000175:diagnosis*; D010190:Pancreatic Neoplasms / Q000175:diagnosis*; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor