title: An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.
abstract: BACKGROUND
  Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.
  
  
  METHODS
  The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories.
  
  
  RESULTS
  Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty.
  
  
  CONCLUSIONS
  Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.
authors.affiliation: Analytical Science Group, National Physical Laboratory, Teddington, UK. james.noble@npl.co.uk
authors.forename: James E
authors.initials: JE
authors.lastname: Noble
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Eleonora
authors.initials: E
authors.lastname: Cerasoli
authors.forename: Alex E
authors.initials: AE
authors.lastname: Knight
authors.forename: Robert A
authors.initials: RA
authors.lastname: Porter
authors.forename: Elaine
authors.initials: E
authors.lastname: Gray
authors.forename: Chris
authors.initials: C
authors.lastname: Howe
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Hannes
authors.forename: Philippe
authors.initials: P
authors.lastname: Corbisier
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Liqing
authors.initials: L
authors.lastname: Wu
authors.forename: Ilaria
authors.initials: I
authors.lastname: Altieri
authors.forename: Marina
authors.initials: M
authors.lastname: Patriarca
authors.forename: Angelika
authors.initials: A
authors.lastname: Hoffman
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
authors.forename: Bernd
authors.initials: B
authors.lastname: Ebert
authors.forename: Jan
authors.initials: J
authors.lastname: Voigt
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shigeri
authors.forename: Maxim S
authors.initials: MS
authors.lastname: Vonsky
authors.forename: Leonid A
authors.initials: LA
authors.lastname: Konopelko
authors.forename: Adolfas K
authors.initials: AK
authors.lastname: Gaigalas
authors.forename: Marc J A
authors.initials: MJ
authors.lastname: Bailey
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D035501:Uncertainty
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins