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ELISA troubleshooting

Table of Contents

Hundreds of variables influence ELISA data. Below is a collection of general assay running tips and troubleshooting strategies that you may find helpful when trying to resolve ELISA issues.

For Q-View™ Data Analysis tips, please see https://www.quansysbio.com/support/getting-the-most-from-q-view-software/.

When running a Q-Plex™ Array:

  • DO be exact when setting shaker speed, being off by even 100 RPM can affect results
  • DO dilute all sample types at least 1:2 (50%) with sample diluent (except cell culture media, which can be tested NEAT)
  • DO load all standards and samples into the microplate within 10 minutes of each other
  • DO be exact with incubation times, particularly the SHRP incubation
  • DO be exact when mixing Substrate A and B, being off by even 50 µL can affect results, and mix thoroughly
  • DON’T allow the plate to dry out between steps, particularly between washing SHRP and adding substrate
  • DON’T allow the SHRP, substrate, or IR dye to be exposed to UV light, as this may degrade it

Saturated Signal on Whole Plate

Probable Cause Solution
Longer incubation times than recommended Incubation times and temperatures are optimized by our QC team. Review provided protocol and retest.
Incubation warmer than recommended
Shaker set at higher RPM than recommended

No or Dim Signal Accross Whole Plate

Probable Cause Solution
Incubation times shorter than recommended Incubation times and temperatures are optimized by our QC team. Review provided protocol and retest.
Incubation temperature colder than recommended
Shaker set at lower RPM than recommended
Incorrect imager settings Check to see if exposure time matches protocol recommendation.
SHRP or substrate were exposed to UV light SHRP and substrate are degraded by UV light. New reagents will needed to properly test the kit.

Problems with the Standard Curve

Probable Cause Solution
Saturated signal on standard curve only Calibrator vial was reconstituted with less volume (more concentrated, higher signal) or more volume (less concentrated, lower signal) than recommended.
No or dim signal on standard curve

High Well Background

Probable Cause Solution
Total protein content of sample is high Samples may require a greater dilution factor
Insufficient plate washing or aspiration Ensure that all channels in the automatic plate washer or pipette are functioning properly and uniformly
Sample type is incompatible with assay Several different sample types and preparations have been tested before. Please see what sample types are compatible with the Q-Plex arrays for more information

High Spot Background

Probable Cause Solution
Negative control wells were contaminated Limits of quantification and detection are calculated with the negative control wells in mind. Contamination here can mean sample values at the extremes of the curve may be incalculable.

High Variation Between Replicates

Probable Cause Solution
Plate washing or aspiration is not uniform Best practice would be to retest after ensuring all channels in automatic plate washer or pipette are functioning normally. If automatic plate washer does not remove wash uniformly, excess wash can be removed from the plate by tapping the plate, upside-down, on a paper towel after each wash.
Well-to-well contamination Retest, ensuring no reagent touches plate seal, pipettes, etc. Also, do not reuse the plate seals.
Partial drying between steps Partial drying of the well can lead to abnormal spot morphology. Be sure to load the next reagent immediately after wash steps.
Plate overlay misaligned In Q-View, ensure that the plate overlay properly covers each spot.

Edge Effects

Probable Cause Solution
Uneven temperature around workspace Be careful of any heating or cooling elements near the workspace
Plate seals not used Plate seals can help with temperature regulation and evaporation


Probable Cause Solution
Replicates not loaded within 10 minutes Samples and reagents should be brought to temperature and prepared for loading onto the 96-well plate in advance
Reagents not brought to temperature before use

Blurry Spots

Probable Cause Solution
Imager out of focus Load focus plate and adjust until the picture is clear. If noticed quickly, image can be captured a second time with re-focused imager.

We take great care to ensure that our products are suitable for use with all valid samples. If you have any questions about troubleshooting, or about our products or services, please contact us at 888-QUANSYS (782-6797) or support@quansysbio.com.