The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the ELISA assays.

  1. Weak or No Signal
  2. Saturated Signal
  3. High Background
  4. Low Sensitivity
  5. Poor Standard Curve
  6. Poor Replicate Data
  7. Inconsistent Assay-to-Assay Results
  8. Slow Color Development
  9. Plate Imaging Problem
  1. Weak or No Signal


    Possible Cause Solution
    1 Blocking protein in coating solution
    • Eliminate blocking protein from coating solution
    2 Capture antibody (or antigen) does not bind to plate
    • Use ELISA plate, not tissue culture plate
    • Try longer coating time
    • Increase concentration of coating components
    3 Problem with the standard
    • Use new sample
    • Check that the standard is appropriately handled
    4 Incubation time too short
    • Follow the manufacturer guideline (If the problem persists, try incubating samples at 4°C overnight)
    5 Incubation temperature too low
    • Ensure incubations are done at correct temperature
    • Before proceeding, all reagents, including plate, should be at room temperature or as recommended by the manufacturer
    6 Incompatible sample type
    • Use sample that the assay is known to detect a positive control (Include such control in your experiment)
    7 Incompatible assay buffer
    • Ensure assay buffer is compatible with the target of interest
    8 Target present below detection limit
    • Decrease dilution factor or concentrate samples
    10 Incorrect/Insufficient/No substrate
    • Check the substrate identity
    • Increase concentration or amount of substrate
    • Follow manufacturer guidelines
    11 Incorrect/Insufficient/No antibody
    • Check the antibody identity
    • Repeat the assay with higher antibody concentrations to find the optimal one for your experiment
    12 Antibody stored at 4°C for several weeks or subjected to repeated freeze-thaw cycles
    • Use fresh aliquot of antibody that has been stored at -20°C or below
    13 Incorrect reagents added/ prepared; Missing reagents
    • Check protocol, ensure correct reagents are added in proper order and prepared to correct concentrations (e.g. TMB for HRP-labeled antibodies)
    14 Expired/Contaminated reagents
    • Make and use fresh/uncontaminated reagents
    15 Enzyme inhibitor present
    • Avoid sodium azide in HRP reactions
    • Avoid phosphate in AP reactions
    16 Incorrect storage of components
    • Double check storage conditions on kit level (Most kits need to be stored at 4°C)
    17 Ultra vigorous plate washing
    • Gently pipette wash buffer (manual method)
    • Ensure correct pressure (automatic wash system)
    18 Wells dry out
    • Cover the plate using sealing film or tape for all incubations
    19 Wells scratched with pipette or pipette tips
    • Carefully dispense/aspirate solutions into and out of wells
    20 Plate read at incorrect detection wavelength
    • Use recommended wavelength/filter
    • Ensure plate reader is set correctly for type of substrate used
    21 Slow color development
    • Prepare substrate immediately before use
    • Allow longer incubation
    • Ensure stock solution is unexpired and uncontaminated
    22 Epitope recognition impeded by adsorption to plate
    • Conjugate peptide to large carrier protein before coating onto plate
  2. Saturated Signal


    Possible Cause Solution
    1 High sample concentration
    • Use higher sample dilutions (Determine the optimal dilutions by titration assay)
    2 Excessive substrate
    • Decrease concentration or amount of substrate: Follow manufacturer guidelines (The substrate provided with the ELISA kit might require further dilution)
    3 Substrate color changed before use
    • Make substrate immediately before use
    4 Non-specific antibody binding
    • Try different formulations in coating solutions
    • Ensure wells are pre-processed to prevent non-specific binding
    • Use affinity-purified antibody and preferably one that is pre-adsorbed.
    • Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended)
    5 Incubation time too long
    • Follow the manufacturer guidelines (If the problem persists, try incubating samples at 4°C overnight)
    6 Excess antibody
    • Repeat the assay with lower antibody concentrations to find the optimal one for your experiment
    7 Contaminated buffers with metals or HRP
    • Make and use fresh buffers
    9 Insufficient washing
    • Follow the manufacturer guidelines
    • At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel
    10 Plate sealers not used or re-used
    • During incubations, cover plates with plate sealers.
    • Use a fresh sealer every time the used sealer is removed from the plate
    11 Plate read at incorrect detection wavelength
    • Use recommended wavelength/filter
    • Ensure plate reader is set correctly for type of substrate used
    12 Excess time before plate reading
    • Read your plate within 30 minutes after adding the substrate (If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate)

  3. High Background

    Possible Cause Solution
    1 Insufficient washing
    • Follow the manufacturer guidelines
    • At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel
    2 Ineffective/Contaminated blocking buffer
    • Try higher blocking protein concentration
    • Increase blocking time
    • Use fresh buffer
    3 Excess antibody
    • Repeat the assay with lower antibody concentrations to find the optimal one for your experiment
    4 Excess substrate
    • Decrease concentration or amount of substrate
    • Follow manufacturer guidelines (Note: The substrate provided with the ELISA kit might require further dilution)
    5 Cross reactivity (Detection antibody reacts with coating antibody)
    • Run appropriate controls
    6 Non-specific antibody binding
    • Try different formulations in coating solutions
    • Ensure wells are pre-processed to prevent non-specific binding
    • Use affinity-purified antibody and preferably one that is pre-adsorbed
    • Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended)
    7 Insufficient Tween in buffers
    • Use PBS containing 0.05% Tween
    8 Suboptimal salt concentration in washing buffer
    • Optimize salt concentration as high concentration can reduce non-specific interactions
    9 Incubation temperature too high
    • Optimize incubation temperature for your assay (antibodies bind optimally at very specific temperature)
    10 Reagents were not mixed properly
    • Thoroughly mix all reagents and samples before pipetting solutions into wells
    11 Blanks contaminated with samples
    • Change pipette tips when switching between blanks and samples
    • Put a lid on pates to avoid any spilling between wells
    12 Sample contaminated with enzymes
    • Test samples with substrate alone to check for contaminating enzymes
    13 Contaminated TMB substrate
    • Use a clean container to check that the substrate in not contaminated (TMB substrate should be clear and colorless before adding to wells)
    14 Substrate incubation in light
    • Carry out substrate incubation in dark or follow recommendation from manufacturer
    15 Uneven evaporation of solution from wells during incubation
    • Always incubate with a lid on the plate
    16 Precipitate created in wells upon substrate addition
    • Increase dilution factor of sample or decrease concentration of substrate
    17 Incubation time too long
    • Follow the manufacturer guidelines (If the problem persists, try incubating samples at 4°C overnight)
    18 Incorrect standard curve dilutions
    • Check pipetting techniques
    • Double check calculations
    19 Plates stacked during incubations, leading to uneven temperature distribution
    • Avoid stacking plates
    20 Dirty or defective plates
    • Clean the plate bottom
    21 Unstopped color development
    • Use Stopping solution to prevent over-development
    22 Excess time before plate reading
    • Read your plate within 30 minutes after adding the substrate (If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate)
    • Note: Color continues to develop even after adding the stopping solution (although at a slower rate)
    23 Incorrect plate reading setting
    • Use recommended wavelength/filter
    • Ensure plate reader is set correctly for type of substrate used
  4. Low Sensitivity


    Possible Cause Solution
    1 Assay format not sensitive enough
    • Switch to a more sensitive detection system (e.g. colorimetric to chemiluminescence)
    • Switch to a more sensitive assay type (e.g. direct ELISA to sandwich ELISA)
    • Increase incubation time and/or temperature
    2 Improper storage of ELISA kit
    • Store all reagents as recommended
    • Note: All reagents may not have identical storage requirements
    3 Insufficient target
    • Reduce sample dilution or concentrate sample
    4 Inactive substrate
    • Ensure reporter enzyme has the expected activity
    5 Poor target adsorption to wells
    • Covalently link target to wells
    6 Insufficient substrate
    • Increase concentration or amount of substrate
    7 Incompatible sample type
    • Use a sample that the assay is known to detect a positive control
    • Include positive control in your experiment
    8 Interfering ingredients in buffers and sample
    • Check reagents for any interfering chemicals, e.g. sodium azide in antibodies inhibit HRP enzyme; EDTA used as anti-coagulant for plasma collection inhibits enzymatic reactions
    9 Mixing or substituting reagents from different kits
    • Avoid mixing components from different kits
    10 Incorrect plate reading setting
    • Use recommended wavelength/filter
    • Ensure plate reader is set correctly for type of substrate used
  5. Poor Standard Curve


    Possible Cause Solution
    1 Improper standard solution
    • Confirm dilutions are done correctly
    • Make new standard curve as appropriate
    2 Standard improperly reconstituted
    • Briefly spin vial before opening
    • Inspect for undissolved material after reconstituting
    3 Standard degraded
    • Store and handle standard as recommended
    • Prepare standards no more than two hours before use
    4 Improper curve fitting
    • Try plotting using different scales, e.g. log-log, 5-parameter logistic curve fit
    5 Pipetting error
    • Use calibrated pipettes and proper pipetting technique
    6 Insufficient washing
    • Follow the manufacturer guidelines
    • At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel
    7 Poorly mixed reagents
    • Thoroughly mix reagents
    8 Poor/variable adsorption of reagents to plate
    • Extend incubation time
    • Check coating buffer
    • Use a different plate as appropriate
    • Check homogeneity of samples
    9 Plates stacked during incubation
    • Keep plates separated if not using rotating plates
    10 Dirty or defective plates
    • Clean the plate bottom
  6. Poor Replicate Data


    Possible Cause Solution
    1 Bubble in wells
    • Ensure no bubbles are present prior to reading plate
    2 Insufficient washing of wells
    • Carefully wash wells
    • Follow recommended protocols
    • Check that all ports of the plate washer are unobstructed
    3 Incomplete reagent mixing
    • Ensure all reagents are mixed thoroughly
    4 Inconsistent pipetting
    • Use calibrated pipettes and proper pipetting techniques
    • Use a fresh sealer every time the used sealer is removed from the plate
    5 Inconsistent sample prep or storage
    • Ensure consistent sample prep and optimal sample storage conditions (e.g. minimize freeze/thaw cycles)
    6 Particulates in samples
    • Remove the particulates by centrifugation
    7 Plate sealers not used or re-used
    • During incubations, cover plates with plate sealers
    • Use a fresh sealer every time the used sealer is removed from the plate
    8 Cross-well contamination
    • Ensure plate sealers and pipette tips are not contaminated with reagents
    9 Edge effect (higher or lower OD in peripheral wells than in central wells)
    • Ensure plates and reagents are kept at room temperature before pipetting into wells unless otherwise instructed
    • During incubation, seal the plate completely with a plate sealer and avoid stacking plates
  7. Inconsistent Assay-to-Assay Results


    Possible Cause Solution
    1 Insufficient washing of wells
    • Carefully wash wells
    • Follow recommended protocols
    • Check that all ports of the plate washer are unobstructed
    2 Variation in incubation temperature
    • Adhere to recommended incubation temperature
    • Avoid incubating plates in area where environmental conditions vary
    3 Variation in protocol
    • Adhere to the same protocol from run to run
    4 Plate sealers not used or re-used
    • During incubations, cover plates with plate sealers
    • Use a fresh sealer every time the used sealer is removed from the plate
    5 Incorrect dilutions
    • Confirm dilutions are done correctly for standard solutions, etc
    • Make new standard curve as appropriate
    6 Contaminated buffers
    • Make and use fresh buffers
    7 Plates stacked during incubation
    • Keep plates separated if not using rotating plates
  8. Slow Color Development


    Possible Cause Solution
    1 Substrates too old, contaminated or used at incorrect pH
    • Make and use fresh substrates at correct pH: they should be prepared immediately before use
    2 Expired/Contaminated solutions
    • Make and use fresh reagents
    3 Incorrect incubation temperature
    • Ensure plates and reagents are kept at room temperature before pipetting into wells unless otherwise instructed
    • During incubation, seal the plate completely with a plate sealer and avoid stacking plates
    4 Low antibody concentration
    • Repeat the assay with higher antibody concentrations to find the optimal one for your experiment
    5 Low substrate concentration
    • Add more substrate to the wells
    • Make substrate no more than one hour before use
    • Note: Typical ELISA sensitivity is ~0.1 pg/mL with exact value depends on antibody used.
  9. Plate Imaging Problem


    Possible Cause Solution
    1 Oversaturated image after acquisition
    • Use full resolution image to analyze results (Do not use jpeg or other compressed formats)
    2 Blurry spots in images
    • Re-focus your camera before taking a new image
    3 Repeated pixel values or rectangular spots
    • Use lower bin size, higher image resolution and/or lossless file type
    4 Flat standard in images
    • Reduce acquisition time