Is your ELISA not working? What seems to be the problem? While running an ELISA there are several steps where potential problems can occur and affect the outcome. The most commonly seen problems are listed below with possible solutions.

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Problem: Weak or no signal in ELISA
Possible Cause Solution
Reagents not at room temperature
at start of assay
It is recommended that all reagents be at room temperature before starting the assay. Allow reagents to sit on bench for 15–20 minutes to reach room temperature.
Incorrect storage of components Double check storage conditions on kit label. Most kits need to be stored at 2–8oC.
Expired reagents Confirm expiration dates on all reagents. Do not use reagents that are past the expiration date.
Reagents added/prepared incorrectly Check protocol, ensure reagents were added in the proper order and prepared to correct dilution.
Incorrect dilutions prepared Check pipetting technique—see below—and double check calculations.
Capture antibody didn’t bind to plate
  • If using a ready-to-use kit: Manufactured kits come with plates pre-coated with capture antibody.
  • If coating your own plate with an Antibody Pair Kit: Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Not enough detector antibody used Manufactured kits have optimized protocols. Make sure to follow recommended antibody dilutions. If developing ELISA using an Antibody Pair Kit you may need to optimize the assay. See ELISA Development and Optimization for more information.
Wells scratched with pipette or washing tips
Use caution when dispensing and aspirating into and out of wells. Automated plate washers may need to be calibrated so tips don’t touch bottom of wells.
Plate read at incorrect wavelength Manufactured kits have optimized protocols. Make sure to use recommended wavelength/filter. Ensure plate reader is set accurately for type of substrate being used.

Problem: Too much signal in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.
Longer incubation times than
recommended
Manufactured kits have optimized protocols. Make sure to follow recommended incubation times. If developing ELISA using antibody pairs you may need to optimize the assay. See ELISA Development and Optimization for more information.

Problem: High background in ELISA
Possible Cause Solution
Insufficient washing
Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Substrate exposed to light
prior to use
Ensure substrate is not exposed to light—store in a dark place. Limit exposure to light while running assay.
Longer incubation times than
recommended
Manufactured kits have optimized protocols. Make sure to follow recommended incubation times. If developing ELISA using antibody pairs you may need to optimize the assay. See ELISA Development and Optimization for more information.
Incorrect standard curve
dilutions prepared
Check pipetting technique—see below—and double-check calculations.

Problem: Poor standard curve in ELISA
Possible Cause Solution
Incorrect standard curve
dilutions prepared
Check pipetting technique—see below—and double-check calculations.
Capture antibody didn’t
bind to plate
Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.

Problem: Poor replicate data in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Capture antibody didn’t
bind to plate
Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.

Problem: Inconsistent results assay-to-assay in ELISA
Possible Cause Solution
Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Inconsistent incubation temperature Manufactured kits have optimized protocols. Make sure to follow recommended incubation temperatures. Be aware of fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.

Problem: Edge effects in ELISA
Possible Cause Solution
Uneven temperature
Seal the plate completely with a plate sealer during incubations. If 37oC incubation is indicated make sure plate is in the center of incubator.
Evaporation Seal the plate completely with a plate sealer during incubations.
Stacked plates Avoid stacking plates during incubation.

Tips on pipetting and washing techniques for ELISA

Pipetting technique

  1. Use the correct pipette that is within the range suggested by manufacturer
  2. Confirm tip is firmly seated on the pipette
  3. Confirm there are no air bubbles while pipetting
  4. Change tips between each standard, sample, or reagent
  5. Use different reservoirs for each reagent
  6. Pipette sample into the side of wells to avoid splashing
  7. Always run samples/standards in replicate

Washing procedure

  1. Completely aspirate liquid from all wells by gently lowering an aspiration tip into the bottom of each well.
    Note: Take care not to scratch the inside of the well.
  2. Fill the wells with at least 400 µL of diluted wash buffer
  3. Let soak for 15 to 30 seconds
  4. Aspirate wash buffer from wells
  5. Repeat as directed in protocol (usually 3-4 times)
  6. After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid.
  7. Alternatively, a squirt bottle or automated plate washer may be used. Be sure to follow the above 
Immunoassays guide to protein quantitation

This 64-page guide provided detailed information about different tools for protein quantitation. Download this valuable technical resource that covers technologies useful for cancer and inflammation research, immunology, neurology and more. Learn more about how antibody pairs, ELISA kits, and multiplex kits for the Invitrogen Luminex platform may help advance your research.

Click here to download the Immunoassays guide to protein quantitation

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Recommended reading
  1. John R. Crowther, Methods in Molecular Biology, the ELISA Guidebook. Second Edition. Humana Press, a part of Springer Science + Business Media, LLC 2009.
  2. Butler J.E. The Behavior of Antigens and Antibodies Immobilized on a Solid Phase. In: M.H.V. Van Regenmortel, ed. Structure of Antigens. Boca Raton, FL: CRC Press, 1992: 209-259. Vol.1, 209; CRC Press, Inc.
  3. Lequin, Rudolf M. "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)." Clinical chemistry 51.12 (2005): 2415-2418.