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ELISA Troubleshooting Guide

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If your ELISA is not working properly, below are some common problems and possible solutions to help you troubleshooting your assay. Our technical support team is also available to assist you via chat or email.

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.

Gerenal ELISA plate

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