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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-Plex™ products.

Spot to Spot Crosstalk

Crosstalk between spots within the same 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-View 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.


Spot Position Analyte Average Crosstalk
Spot 1 IL-1α 0.00%
Spot 2 IL-1β 0.06%
Spot 3 IL-2 0.03%
Spot 4 IL-4 0.01%
Spot 5 IL-5 0.00%
Spot 6 IL-6 0.00%
Spot 7 IL-8 0.00%
Spot 8 IL-10 0.00%
Spot 9 IL-12p70 0.00%
Spot 10 IL-13 0.00%
Spot 11 IL-15 0.00%
Spot 12 IL-17 0.09%
Spot 13 IL-23 0.03%
Spot 14 IFNγ 0.00%
Spot 15 TNFα 0.04%
Spot 16 TNFβ 0.00%