ELISA troubleshooting

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 http://www.quansysbio.com/data-analysis-q-view.

When running a Q-Plex™ Array:
Do’s

  • DO be exact when calibrating 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’ts

  • 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
  • DON’T analyze from a color or jpeg image; save grayscale images using a lossless image file type such as Tiff or RAW

Q-Plex Experimental Troubleshooting

ProblemProbable CauseSolution or Action
Saturated signal on…Whole Plate/Standard: Incubation conditions for one or more steps were too long, too warm, or had too high RPMDecrease incubation time, temperature, or RPM if found to be incorrect (instructions for shaker calibration can be found on our website)
Whole plate/Standard: Incorrect imager settings: aperture may be too open, exposures too longAdjust settings, e.g. set aperture (f-stop) to the lowest value possible to a minimum of 2, and reimage immediately
Whole Plate/Standard: Image has been compressedExport using higher resolution/bit depth and lossless file type such as Tiff
Standard only: Standard was reconstituted with less volume than recommended (too concentrated)Rerun, reconstituting with recommended volume
Samples only: Analyte concentrations in the samples may be above the limit of quantification or signal may be a false positive due to heterophilic antibodiesSamples may require a greater dilution. Test them at several dilutions, such as 1:2, 1:20, and 1:200
No or dim signal on…Whole Plate/Standard: Incubation conditions for one or more steps were too short, too cold, or had too low RPMIncrease incubation time, temperature, or RPM if found to be incorrect (instructions for shaker calibration can be found on our website)
Whole plate/Standard: Incorrect imager settings: aperture may be too closed, exposures too short, poor focusAdjust settings, e.g. set aperture (f-stop) to the lowest value possible to a minimum of 2, and reimage immediately
Whole Plate/Standard: Image has been compressedExport using higher resolution/bit depth and lossless file type such as Tiff
Whole Plate: One or more steps of the assay protocol was skipped or performed with expired reagentsCheck kit expiration. Retest with non-expired reagents, ensuring that all reagents are added to the plate at the appropriate times
Whole Plate: The SHRP, substrate, or IR dye was exposed to UV light and has degradedRetest using fresh substrate
Whole Plate (IR): Spots disappear when image is importedEnsure that a grayscale image is imported
Standard Only: Standard was reconstituted with higher volume than recommended (less concentrated)Reconstitute with the volume recommended in the kit manual
Samples Only: Analyte concentrations in the samples may be below the limit of quantification for the assay(s)Samples may require less dilution, Retest using a minimum dilution of 1:2 for serum and plasma, or undiluted for cell culture media and urine. Also inquire about our high sensitivity products/protocols
High well backgroundTotal protein content of samples is too highSamples may require a greater dilution. Test them at several dilutions, such as 1:2, 1:20, and 1:200
Plate washing or aspiration is not sufficientEnsure that all channels in the automatic plate washer or pipette are functioning properly and uniformly
Sample type is incompatible with the assayTry a different sample type sample preparation. Inquire about our sample testing or custom assay development services
High spot backgroundNegative control samples, diluents, or wells were contaminated with positive sampleRetest samples and ensure that no contamination takes place
High variation between replicates, poor precisionPlate washing or aspiration is not uniformMask outliers or retest, ensuring that all channels in the automatic plate washer or pipette are functioning properly and uniformly
Well-to-well contaminationMask outliers or retest, ensuring that no reagent touches plate seals, pipette tips, etc., between uses
Reagents, such as samples or Substrate A and B, were not homogenousMask outliers or retest, ensuring that reagents are well mixed and that particulate matter is removed by centrifugation before addition to the microplate
Saliva contamination may cause certain assays in a well to be falsely dim or brightMask outliers or retest, wear a face mask during all sample handling, pipetting, and washing steps
Partial drying between stepsMask outliers or retest, ensuring that reagents are added immediately after washing; if you cannot load the next reagent right away, leave wash buffer in the wells up to 10 mins
Pipetting or sampling errorsEnsure that pipettes are calibrated, and samples are thoroughly homogenized and particulate matter has been removed by centrifugation
Image has poor resolution or has been overly compressedEnsure that the imager is compatible with the product; don’t analyze from a jpeg, only analyze from grayscale Tiff
Plate overlay in Q-View Software may be misaligned for a well or group of wellsIn Q-View, ensure that all overlay spots are over the correct assay spots
Edge Effects: Outside wells different signal than inner wellsUneven temperature around work spacesEnsure proper incubation conditions are used
Plate cover not affixed to edges leading to reagent evaporationEnsure plate seals are adhered to entire microplate
Drift: Replicates loaded over time differ in signalReplicates were not loaded within 10 minutes, or reagents were not brought to temperature before being loadedSamples should be brought to temperature, prepared for loading in advance, then loaded onto the microplate together, generally within 10 minutes
Additional Troubleshooting for IR Q-Plex Kits
ProblemProbable CauseSolution or Action
Flecks of signal outside of wellsDust, finger prints, etc.Clean the bottom of the plate and the imager glass with 70% ethanol
Spots have dim centersPlate became re-humidified after being driedIf the Licor scanner is not immediately accessible after drying, tightly seal the plate using a plate seal, keep the plate in the dark.
Dim signal for standard curve or whole plateIR dye was exposed to ambient light for too long.Ensure that the IR dye in the vial and on the plate are kept in the dark as much as possible.
All spots disappear when importedImage is not grayscaleEnsure that a grayscale image is imported.
Imaging Troubleshooting
ProblemProbable CauseSolution or action
High variation between replicates, poor precision Image has poor resolution or has been overly compressed Ensure that the imager is compatible with the product; don’t analyze from a jpeg, only analyze from lossless image file types such as Tiff
Blurry SpotsCamera is out of focusRefocus camera and retake image
f-stop may be too lowSet f-stop to a minimum of 2
Rectangular spots or repeating pixel intensity values (e.g. 0 or 65535)Image is saturated or has been compressed Ensure binning/pixel size is as low as possible; Export using higher resolution/bit depth and lossless file type such as Tiff
Standard curves appear flat at one or both ends Images are over/under exposedRe-image using shorter/longer exposure times
Image has been compressedEnsure binning/pixel size is as low as possible; Export using higher resolution/bit depth and lossless file type such as Tiff
Recommended exposure time(s) are insufficient to obtain quality standard curves F-stop or ISO problem Adjust to settings recommended in user manual
Flat-Field correction problemRetake flat-field image (recalibrate the imager)
Auto-Stop Exposure is turned onTurn off Auto-Stop feature
Spots disappear when the image is imported into Q-ViewImage is not grayscale Ensure that a grayscale image is imported

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 INFO@QUANSYSBIO.COM

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