Crosstalk in Multiplex ELISA

Crosstalk in chemiluminescent or infrared fluorescent arrays is any phenomenon by which a signal from one spot creates an undesired effect on another. For example, if the signal from one well or microspot is so bright that it causes falsely high background or signal on one or more wells or assays next to it. Crosstalk is different from cross-reactivity in that the former accounts for light effects seen during image capture and analysis, while the latter deals specifically with chemical interferences between systems.

Well-to-Well Crosstalk

Crosstalk between wells is a potential issue in clear-walled microplates. Using microplates with opaque black walls will minimize the amount of light that can travel between wells. Such microplates are used for all non-stripwell Q-PlexTM products.

Spot to Spot Crosstalk

Crosstalk between spots within a well is also a potential issue in any microarray. To minimize the amount of light detected outside spot boundaries:

  1. Ensure that samples are diluted correctly according to the kit protocol.
  2. Ensure that the imager is suitable for use with Q-Plex Technology
    • Q-View Imager Pro
    • Q-View Imager LS
Crosstalk Testing at Quansys Biosciences

A Human Cytokine Screen Kit, consisting of 16 assay spots per well, was run using individual antigens to create signal on one select assay per well. Concentrations in pg/mL were calculated in Q-ViewTM 2.14 using a 5PL regression model with the LLOQ set as the lower limit for each standard curve. Percent crosstalk for each assay spot was calculated as: (spot signal / calibrator high-point signal for that assay) * 100. Values for each assay spot location were then averaged over all 16 test conditions.


Analytes Calibrator Range Units LLD
GMCSF2000 - 2.74 pg/mL 2.30
IL-1α5000 - 6.86 pg/mL 4.20
IL-1β14000 - 19.20 pg/mL 12.41
IL-25000-6.86 pg/mL 3.44
IL-35000 - 6.86 pg/mL 3.18
IL-45000 - 6.86 pg/mL 2.50
IL-65000 - 6.86 pg/mL 2.90
IL-102000 - 2.75 pg/mL 3.00
IL-12p7012000 - 16.46 pg/mL 12.20
IL-1710000 - 13.72 pg/mL 6.20
MCP-13000 - 4.12 pg/mL 3.40
MIP-1α5000 - 6.86 pg/mL 0.61
RANTES3000 - 4.12 pg/mL 2.15
TNFα3000 - 4.12 pg/mL 3.40


The spacing used between spots in the Human Cytokine Screen Q-Plex Kit is sufficient to avoid crosstalk.