title: Issues on fit-for-purpose validation of a panel of ELISAs for application as biomarkers in clinical trials of anti-Angiogenic drugs.
abstract: BACKGROUND
  Successful introduction of new anticancer agents into the clinic is often hampered by a lack of qualified biomarkers. Studies have been conducted of 17 ELISAs representing a potential panel of pharmacodynamic/predictive biomarkers for drugs targeted to tumour vasculature.
  
  
  METHODS
  The fit-for-purpose approach to method validation was used. Stability studies were performed using recombinant proteins in surrogate matrices, endogenous analytes in healthy volunteer and cancer patient plasma. The impact of platelet depletion was investigated.
  
  
  RESULTS
  Method validation focused on measuring precision and showed that 15 of the 17 assays were within acceptable limits. Stability at -80 degrees C was shown for 3 months with all recombinant proteins in surrogate matrices, whereas under the same conditions instability was observed with KGF in platelet-rich and platelet-depleted plasma, and with PDGF-BB in platelet-depleted plasma from cancer patients. For measurement of extracellular circulating analytes, platelet depletion should be conducted before freezing of plasma to prevent release of PDGF-BB, FGFb and VEGF-A. A protocol was developed to remove >90% platelets from plasma requiring centrifugation at 2000 g for 25 min.
  
  
  CONCLUSIONS
  These studies highlight the need for assay validation and crucial assessment of sample handling issues before commencement of biomarker analysis in clinical trials.
authors.affiliation: Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK.
authors.forename: K
authors.initials: K
authors.lastname: Brookes
authors.forename: J
authors.initials: J
authors.lastname: Cummings
authors.forename: A
authors.initials: A
authors.lastname: Backen
authors.forename: A
authors.initials: A
authors.lastname: Greystoke
authors.forename: T
authors.initials: T
authors.lastname: Ward
authors.forename: G C
authors.initials: GC
authors.lastname: Jayson
authors.forename: C
authors.initials: C
authors.lastname: Dive
mesh_terms: D020533:Angiogenesis Inhibitors / Q000097:blood / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000097:blood; D002986:Clinical Trials as Topic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards*; D006801:Humans; D009369:Neoplasms / Q000097:blood / Q000188:drug therapy*; D055550:Protein Stability; D011786:Quality Control; D011933:Reagent Kits, Diagnostic / Q000592:standards; D013048:Specimen Handling; D054928:Validation Studies as Topic
chemical_list: D020533:Angiogenesis Inhibitors; D014408:Biomarkers, Tumor; D011933:Reagent Kits, Diagnostic
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