title: Accuracy and reproducibility of a multiplex immunoassay platform: a validation study.
abstract: BACKGROUND
  Multiplex immunoassays offer many advantages over singleplex assays for the analysis of multiple analytes in a single sample. We sought to validate a specific multiplex cytokine immunoassay (Human 9-plex cytokine array on the Searchlight® platform by Thermoscientific) prior to use in a large clinical study.
  
  
  METHODS
  We compared spike and recovery of recombinant proteins on the Searchlight® platform to singleplex immunoassays purchased from R&D Systems, measured identical patient samples on the two different platforms, and measured identical patient samples on different days to measure intra- and inter-assay variability.
  
  
  RESULTS
  Assays using the Searchlight® platform had inefficient recovery of spiked recombinant proteins compared to R&D Systems singleplex assays. Assaying identical patients samples on different days on the Searchlight platform had acceptable intra-assay variability (intra-assay coefficient of variation (CV%) range for all analytes of 9.1-13.7) but unacceptably high inter-assay variability (CV% range for all analytes 16.7-119.3) suggesting plate-to plate variability. Similar assays for individual cytokines on the R&D platform had an intra-assay CV% range of 1.6-6.4 and an inter-assay CV% range of 3.8-7.1. Some deficiencies in Searchlight® assay performance may be due to irregularities in spotting of capture antibodies during manufacturing.
  
  
  CONCLUSIONS
  We conclude that the Searchlight® multiplex immunoassay platform would require extensive additional assay optimization prior to widespread clinical research use.
authors.affiliation: Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, United States. Julie.bastarache@vanderbilt.edu
authors.forename: Julie A
authors.initials: JA
authors.lastname: Bastarache
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Koyama
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Wickersham
authors.forename: Daphne B
authors.initials: DB
authors.lastname: Mitchell
authors.forename: Ray L
authors.initials: RL
authors.lastname: Mernaugh
authors.forename: Lorraine B
authors.initials: LB
authors.lastname: Ware
mesh_terms: D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
chemical_list: D016207:Cytokines; D011933:Reagent Kits, Diagnostic