101 ELISA Troubleshooting Tips

Our 101 ELISA troubleshooting tips is designed to help researchers improve and troubleshoot a range of common problems that they may face with their ELISA kits when performing assays. Optimising your ELISA and removing mistakes can dramatically improve results and the sensitivity of your ELISA assays. In this ELISA troubleshooting guide we have detailed the 6 most common areas where researchers encounter problems with their ELISA.

ELISA Troubleshooting Areas

High Signal

High Signal may occur for a number of reasons including insufficient plate washing, not stopping the reaction or adding too much detection reagent. If you have a high signal this can result in false positives and incorrect data.

High Signal Troubleshooting Tips


TMB Substrate Solution was contaminated

Use fresh TMB substrate solution which should be clear and colorless prior to addition to wells. Use a clean V bottom container prior to pipetting substrate solution into wells. Use a clean V bottom container prior to pipetting substrate solution into wells.


Reaction not stopped

Colour will keep developing if the substrate reaction is not stopped.


Plate left too long before reading on the plate reader

Colour will keep developing (though at a slower rate if stop solution has been added)


Contaminants from laboratory glassware

Ensure reagents are fresh and prepared in clean glassware


Substrate incubation carried out in the light

Substrate incubation should be carried out in the dark. Ensure substrate is not exposed to light—store in a dark place. Limit exposure to light while running assay.


Wells are insufficiently washed

Wash wells as per protocol recommendations.


Too much detection reagent added

Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent.


Blocking buffer ineffective (e.g. detection reagent binds blocker; wells not completely blocked)

Try different blocking reagent and/or add blocking reagent to wash buffer.


Salt concentration of incubation/wash buffers

Increasing salt concentrations may reduce non-specific and/or weak off-target interactions.


High antibody concentration

Try different dilutions for optimal results.


Precipitate formed in wells upon substrate addition

Increase dilution factor of sample or decrease concentration of substrate.


Dirty plate

Clean the bottom of the plate.


Incorrect standard curve dilutions

Check your pipetting technique. Calibration of pipettes might be required.


Longer incubation times than

Make sure your incubation times are correct and adhere to the protocol provided with the technical manual.


Plate sealers or reagent reservoirs resused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically.

Reuse of plate sealers may lead to the presence of residual HRP, leading to non- specific colour change of TMB. To avoid this use fresh plate sealer and reagent reservoir for each step.


Contamination of buffers

Always make fresh buffers.

Out of Range

Out of Range issues may arise due to your samples, insufficient washing or incorrect dilution preparation. This can result in a loss of data due to negative or no results.

Out of Range Troubleshooting Tips


Samples contain no or below detectable levels of analyte

If samples are below detectable levels, it may be possible to use high sample volume. Check with technical support for appropriate protocol modifications.


Samples contain analyte concentrations higher than the highest standard point

Samples may require further dilution


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

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.


Substrate solution mixed too early and turned blue

Substrate solution should be mixed and used immediately


Too much streptavidin-HRP

Check dilution, titrate if necessary


Plate sealers or reagent reservoirs resused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically

Use fresh plate sealer and reagent reservoir for each step


Buffers contaminated with metals or HRP

Make fresh buffers

High Variation

Pipette errors, mistakes during sample preparation and insufficient plate agitation may all lead to High Variation. Data with high variation can skew real results and cause inconsistencies in your data.

High Variation Troubleshooting Tips


Multichannel pipette errors

Calibrate the pipettes


Plate washing was not adequate or uniform

Make sure pipette tips are tightly secured. Confirm all reagents are removed complete in all wash steps


Non-homogenous samples

Thoroughly mix samples before pipetting


Samples may have high particular matter

Remove the particulate matter by centrifugation


Insufficient plate agitation

The plate should be agitated during all incubation steps using an ELISA plate shaker at a speed where solutions in wells are within constant motion without splashing


Cross well contamination

When reusing plate sealers check that no reagent has touched the sealer. Care should be taken when using the same pipette tips used for reagent additions. Ensure that pipette tips do not touch the reagents on the plate.


Plates stacked during the incubations

Stacking of plates does not allow even distribution of temperature across the wells of the plates. Avoid stacking.


Pipette inconsistent

Ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid.


Antibody dilutions/reagents are not well mixed

To ensure a consistent concentration across all wells, ensure all reagents and samples are mixed before pipetting onto the plates.


Well allowed to dry out

Ensure lids are left on the plates at all times when incubating. Place a humidifying water tray (bottled clean/sterile water) in the bottom of the incubator.


Bottom of the plate is dirty

Clean the bottom of the plate carefully before re-reading the plates


Bubbles in wells

Ensure no bubbles present prior to reading the plate


Edge effects

Ensure the plate and all reagents are at room temperature



Ensure reagents and samples are stored are correct temperature


Capture antibody didn’t bind to the 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.


Variations in protocols

Adhere to the protocol that comes with your assay


Improper calculations of standard curve

Check calculations, make new standard curve & use internal controls


Buffers contaminated

Use fresh buffers


Well bottom scraped

Avoid contact with the bottom of the well during pipetting. Aim the pipette tip to the side of the well to avoid disrupting the bottom

Background is High

High background may result from inadequate washing steps, cross reactivity of samples or contamination. High background may result in false positive/negative data and affect your results.

Background is High Troubleshooting Tips


Background wells were contaminated

Avoid cross-well contamination by using the sealer appropriately. Use multichannel pipettes without touching the reagents on the plate.


Matrix used has endogenous analyte or interference

Check the matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium).


Insufficient washes

Increase number of washes. Increase soaking time between washes prior to addition of substrate solution.



Detection antibody cross-reacting with coating antibody. Run appropriate controls.


Non-specific binding of antibodies

Ensure a block step is included and a suitable blocking buffer is being used. We recommend using 5 to 10% serum from the same species of the secondary antibody, or bovine serum. Ensure wells are pre-processed to prevent nonspecific attachment Use an affinity purified antibody, preferably pre-absorbed


Concentration of conjugated second antibody too high

Perform dilutions to determine optimal working concentration.


Incorrect assay temperature

Check that the incubation temperature did not exceed 37°C


Inadequate washing

Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer if available. Increase number of washes. Add 30 second soak step in-between washes.


Contaminating enzymes present in sample

Test sample with substrate alone to check for contaminating enzyme activity.


Wells are insufficiently washed

Wash wells are per protocol recommendations.


Contaminated wash buffer

Prepare fresh buffers


Too much detection reagent

Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent


Blocking buffer ineffective

Try different blocking buffer reagent and/or add blocking reagent to wash buffer


Salt concentrations of incubation/wash buffers

Increasing salt concentrations may reduce non-specific and/or weak off target interactions.


Waiting too long to read plate after addition of stop solution

Read plate immediately after adding stop solution


High antibody concentration

Try different dilutions of optimal results


Substrate incubation is carried out in light

Substrate incubations should be carried out in the dark or as recommended by manufacturer.


Precipitate formed in wells upon substrate addition

Increase dilution factor of sample or decrease concentration of substrate


Dirty plate

Clean the bottom of the plate with a wipe

No Signal

No signal in your ELISA assay may occur due to your wash buffer containing azide, failing to add avidin-HRP or your desired target levels being below the detection range of the assay. No signal may mean no results from precious samples. Have a read through the reasons below to avoid these problems.

No Signal Troubleshooting Tips


Incorrect or no detection antibody was added

Add appropriate detection antibody and continue


Avidin-HRP was not added

Add avidin-HRP according to protocol and continue


Substrate solution was not added

Add substrate solution and continue


Wash buffer contains azide

Avoid sodium azide in the wash buffer


Incubation time too short

Incubate samples overnight at 4’C or follow manufacturers guidelines


Target present below detection limits of assay

Decrease dilution factor or concentrate samples


Incompatible sample type

Detection may be reduced or absent in untested samples types. Include a sample that the assay is known to detect the positive control


Recognition of epitope impeded by absorption plate

To enhance the detection of a peptide by direct or indirect ELISA, conjugate peptide to a large carrier protein before coating onto a microtiter plate


Assay buffer incompatibility

Ensure assay buffer is compatible with the target of interest (e.g. enzymatic activity retained, protein interactions retained.)


Not enough detection reagent

Increase concentration of amount of detection reagent following manufacturer guidelines


Sample prepared incorrectly

Ensure proper sample preparation/dilution. Samples may be incompatible with microtiter plate assay format


Insufficient antibody

Try different concentrations/dilutions of antibodies


Incubation temperature is too low

Ensure the incubations are carried out at the correct temperature. All reagents including plate should be at room temperature or as recommended by the manufacturer before proceeding.


Incorrect wavelength

Verify the wavelength and read the plate again


Plate washing is too vigorous

Check the correct pressure in the automatic plate washer. Pipette wash buffer gently if washes are done manually.


Wells dried out

Do not allow wells to become dry once the assay has started. Cover the plate using sealing film or tape for all incubations.


Slow colour development of enzymatic reactions

Prepare substrate solution immediately before use. Ensure the stock solution has not expired and is not contaminated. Allow longer incubation.


Uneven Colour

Ensure all wells are washed correctly, use a ELISA plate washer where possible


Reagents not at room temperature

All reagents should at room temperature from the start of the assay. Room temperature should be reached following 15–20 minutes on the bench.


Expired Reagents

Ensure all reagents used are within date


Assay format not sensitive enough

Switch to a more sensitive assay type (e.g. direct ELISA to sandwich ELISA). Lengthen incubation times or increase temperature. Or change the detection method


Buffer containing FCS used to reconstitute antibodies

Re-evaluate reagents used.

Poor Standard Curve

A poor standard curve will prove unpublishable results if not prepared correctly. Reasons for a poor standard curve include inadequate mixing of reagents, degraded standard or pipetting errors.

No Signal Troubleshooting Tips


Standard was incompletely reconstituted or was incorrectly stored

Reconstitute standard according to the protocol provide and follow storage instructions


Reagents were added to the wells at incorrect concentrations

Check for pipetting errors and correct the reagent volume


Incubations done at incorrect temperature

Follow protocol for storage, incubation and agitation


Wells not completely aspirated

Completely aspirate between steps, use plate wash where possible


Plates stacked during incubation

Keep plates separated


Poor dilution series

Check dilution steps according to protocol


Reagents poorly mixed

Make sure to mix reagents thoroughly


Poor or variable adsorption of reagents to plate

Check choice of coating buffer, usually PBS with a pH of 7.4 or carbonate bicarbonate buffer pH 9.6. Try extending this incubation time or consider using different plates


Standard degraded

Check that standard was stored correctly


Curve doesn’t fit scale

Try plotting use different scales, e.g. log-log, 5 parameter logistic curve fit


Pipetting error

Check pipettes and calibrate


Capture antibody didn’t bind to the plate

Ensure that you are using and ELISA plate, not a tissue culture plate.


Not enough detection antibody

Check dilution, titrate if necessary


Incorrect calculation of standard curve dilution

Check your calculations and make a new curve.


Mixing or Substituting reagents from different kits

Avoid this as it can affect the quality of your assay