ELISA Troubleshooting Guide

Possible problem

Possible cause or experimental variables

Possible corrective action

Blank well absorbance too high Contaminated reagents or contaminated well Check the storage condition. Repeat with new reagents from the same lot if possible.
  Blank wells with sample and conjugates Use only buffer in blank well.
Positive standard values lower than the stated range Expired reagents or reagents deteioration Check the expiration date and storage conditions.
  Used components from different lots or manufacturers Use only components from the same lot and do not substitute components from different kits or from different suppliers.
  Incubation time too short or incubation temperature too low Follow instruction and check incubation conditions and equipment.
  Incorrect readings Read the results within 5 minutes after the reaction. Check the reader optical pathway and wavelength.
  Signal suppression by serum matrix Prepare the standard curve in 50% serum (diluted in sample diluent). Also, use 50% serum as the blank. To get an accurate backfitted concentration of the unknown sample, dilute the serum sample (unknown) to 50% in sample diluent as well.
Poor Precision Incomplete washing and/or aspiration of the wells Ensure that wash apparatus is working correctly. If washing by multi-channel pipettor, be sure that all channel uptakes are the same.
  Unequal mixing of reagents Ensure adequate mixing.
Edge Effect Experiment performed under uneven temperature Ensure that laboratory temperature is in the 20-26°C range. If possible, perform experiment in a controlled temperature incubator set at 24°C.
  Plate was not covered during incubation steps Ensure that the plate is properly covered during incubation periods.
Drift Effect Uneven air flow in the laboratory where the experiment was carried Ensure that experiment is done under an enclosed area with constant temperature.
  TMB substrate was not at room temperature Ensure that TMB substrate is properly equilibrated to room temperature. To achieve proper equilibration, incubate the substrate (initially stored at 4°C) for at least 30 minutes at room temperature in dark prior to use.
  Long time intervals between addition of reagents to the wells Ensure that reagents were added to all the wells in a short period of time.
Poor Standard Curve Improper dilution of reagents, or contamination of blank buffer Ensure correct dilution steps were taken and that appropriate buffer is used as blank.
  Unequal volume of reagents used Check pipette function and calibration. Ensure that correct pipetting technique is used.
  Incomplete washing of the wells Ensure that wash apparatus is working correctly. If washing by multi-channel pipettor, be sure that all channel uptakes are the same.
  Plate defects or contamination Check plate for scratches and fingerprints.
  Improper curve fitting Perform 4-parameter (logistic-log-model) curve fitting.

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