Human tear analysis with miniaturized multiplex cytokine assay on "wall-less" 96-well plate

Mol Vis. 2015 Oct 13;21:1151-61. eCollection 2015.


Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / instrumentation
  • Biological Assay / methods
  • Biological Assay / standards*
  • Humans
  • Interferon-gamma / analysis*
  • Interleukin-1beta / analysis*
  • Interleukin-6 / analysis*
  • Luminescent Measurements
  • Observer Variation
  • Reagent Kits, Diagnostic / standards
  • Reproducibility of Results
  • Tears / chemistry*
  • Tumor Necrosis Factor-alpha / analysis*


  • IL6 protein, human
  • Interleukin-1beta
  • Interleukin-6
  • Reagent Kits, Diagnostic
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma