title: Human tear analysis with miniaturized multiplex cytokine assay on "wall-less" 96-well plate.
abstract: OBJECTIVE
  Tears are a particularly limited body fluid and commonly used in the diagnosis of patients who have ocular diseases. A popular method for analysis of ocular inflammation in tears uses Luminex® bead multiplex technology to generate valuable multiple cytokine profile outputs with 25-50 µl tear sample volume. We propose a method for measuring tear cytokines with 5 μl tear sample volume and 80% reduced Luminex reagents compared to previous protocols.
  
  
  METHODS
  Using human tears pooled from 1,000 participants, the DA-Bead-based method running at 5-20 µl volume, using manual pipetting, in conjunction with a magnetic Luminex cytokine (four-plex) panel assay in a 96-well format was performed and validated for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β, and IL-6.
  
  
  RESULTS
  Upon use of the DA-Bead method at the 5 μl volume with cytokine standards, the concentrations of each of the four cytokines were found to be linear over a range of 3.5-4 log pg/ml with an intra-assay coefficient of variation (CV) ≤5%, inter-assay %CV ≤10%, and accuracy within the 70-130% range. Upon use of a 5 µl healthy pooled tear sample, cytokine concentrations were detected with a precision intra-assay %CV ˂ 20% for IL-6, IFN-γ, or TNF-α or 30.37% with IL-1β. The inter-assay %CV with tears was ≤20.84% for all cytokines. Tear volumes run at 5 μl on DA-Bead produced a similar cytokine expression profile at a 1-month interval and were highly correlated with the larger 10 μl-based tear sample volume cytokine profile with R(2) = 0.98.
  
  
  CONCLUSIONS
  DA-Bead assay is highly sensitive and reproducible and has a performance profile that is potentially suitable for use in standard clinical scenarios. Considering the use of as little as 5 µl of assay beads and 5 µl sample, this is also likely to reduce the assay cost significantly and ease diagnosis of patients with ocular diseases.
authors.affiliation: Curiox Biosystems, Woodlands Sector, Singapore.
authors.forename: Xavier
authors.initials: X
authors.lastname: Le Guezennec
authors.affiliation: Singhealth Polyclinics, Singapore.
authors.forename: Joanne
authors.initials: J
authors.lastname: Quah
authors.affiliation: Singapore Eye Research Institute, Singapore ; Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
authors.affiliation: Curiox Biosystems, Woodlands Sector, Singapore.
authors.forename: Namyong
authors.initials: N
authors.lastname: Kim
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D053583:Interleukin-1beta / Q000032:analysis*; D015850:Interleukin-6 / Q000032:analysis*; D008163:Luminescent Measurements; D015588:Observer Variation; D011933:Reagent Kits, Diagnostic / Q000592:standards; D015203:Reproducibility of Results; D013666:Tears / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
chemical_list: C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D011933:Reagent Kits, Diagnostic; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma