ELISA Troubleshooting Guide

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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Find tips on pipetting and washing techniques for ELISA below

Explore different ELISA troubleshooting issues and potential solutions

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 2–8^oC.
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 37^oC 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.

Signal

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 2–8^oC.
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.

High background

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.

Poor standard curve

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.

Poor replicate data

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.

Assay variability

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.

Edge effects

Problem: Edge effects in ELISA

Possible Cause	Solution
Uneven temperature	Seal the plate completely with a plate sealer during incubations. If 37^oC 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

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

Washing procedure

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.
Fill the wells with at least 400 µL of diluted wash buffer
Let soak for 15 to 30 seconds
Aspirate wash buffer from wells
Repeat as directed in protocol (usually 3–4 times)
After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid.
Alternatively, a squirt bottle or automated plate washer may be used. Be sure to follow the above.

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Explore different ELISA troubleshooting issues and potential solutions

Tips on pipetting and washing techniques for ELISA

Pipetting technique

Washing procedure

Biomarker quantitation assay guide

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