ELISA Technical Resources

Picokine ELISA Troubleshooting

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.

How to Troubleshoot ELISA

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.

If you do not see the issues you are having featured in this page, please contact us at [email protected] and we will help you resolve your specific trouble.

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Weak or No Signal

Weak or no color development in an ELISA assay can indicate that the target protein is present in minute quantities in the sample, if at all. It can also mean that there is something wrong with the assay or the reagents that prevent efficient detection. If your control reactions indicate that an error is causing your poor results, use this troubleshooting guide to diagnose and resolve your ELISA weak signal.

There are three main causes of weak signals in ELISA

  • Antibody/epitope reaction problems
  • Insufficient reporter enzyme activity
  • Plate related errors
S.No. 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
  • Use Boster pre-coated ELISA kits
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 Loss of binding activity because antibody was 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
  • Avoid repeated freeze-thaw cycles
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 Excessive 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
23 Primary antibody concentration too low
  • Increase primary antibody concentration
  • Incubate for longer
24 Detection reagent old, contaminated, or wrong pH
  • Use fresh substrate at the correct pH

Saturated Signal

If your ELISA signal is too high, the results of the experiment can become unusable. Saturated signal can cause wells to appear uniformly reactive, or cause the standard curve to become unusable. Before you repeat your ELISA experiment, we've provided some troubleshooting tips to identify possible sources of the saturated signal error as well as solutions to solve it.

S.No. 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 (e.g. the substrate provided with the ELISA kit might require further dilution).
3 Substrate color changed before use
  • Make substrate immediately before use.
4 Nonspecific antibody binding
  • Try different formulations of coating solutions.
  • Ensure wells are pre-processed to prevent nonspecific binding.
  • Use affinity-purified antibody and preferably one that is pre-adsorbed.
  • Use Boster antibodies guaranteed to only react with their targets.
  • Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended).
5 Incubation time too long
  • Reduce incubation time. Follow the manufacturer guidelines. If the problem persists, try incubating samples at 4°C overnight.
6 Excessive antibody concentration
  • 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.
8 Insufficient washing
  • Follow the manufacturer guidelines.
  • At the end of each washing step, thoroughly drain the plate by flicking the plate over a sink and patting the plate on a paper towel.
9 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.
10 Plate read at incorrect detection wavelength
  • Use recommended wavelength/filter.
  • Ensure plate reader is set up correctly for type of substrate used.
11 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.

High Background

S.No. 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. Run negative control to determine cross-reactivity.
  • Cross-adsorb your antibody against cross-reactive targets
  • Use Boster antibodies guaranteed to bind only their specified targets
6 Non-specific antibody binding
  • Try different formulations of 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
  • Wash plates before coating
  • Clean the plate bottom
  • Use Boster pre-coated ELISA plates
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

Low Sensitivity

S.No. 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).
  • Use Boster's high-sensitivity ELISA kits.
  • Increase incubation time and/or temperature.
2 Improper storage of ELISA kit
  • Store all reagents as recommended by the manufacturer.
  • 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. You can run a control to verify reporter enzyme reacts with the substrate.
5 Poor target adsorption to wells
  • Covalently link target to wells.
6 Insufficient substrate
  • Increase concentration or amount of substrate.
7 Incompatible sample type
  • Include positive control in your experiment.
  • Use sample types (e.g. serum vs. cell extract) that have been verified to work as positive controls.
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 the type of substrate used.

Poor Standard Curve

S.No. 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

Poor Replicate Data

S.No. 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

Inconsistent Assay-to-Assay Results

S.No. 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

Slow Color Development

S.No. 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.

Plate Imaging Problem

S.No. 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

High Well-to-Well Variation

S.No. Possible Cause Solution
1 Plates stacked during incubation
  • Do not stack plates to ensure even temperature distribution.
2 Plates warmed unevenly before use
  • Allow plate temperature to stabilize before use to avoid edge effects.
3 Bubbles in wells
  • Check for bubbles before incubations.
4 Uneven washing
  • Wash all wells uniformly.
  • Check plate washer for obstructed parts.
5 Incomplete reagent mixing
  • Make sure all reagents are homogenous before use.
6 Inconsistent storage.
  • Make sure all samples and reagents are stored under uniform conditions.

Matrix Effect

The matrix effect occurs when the target antigen interacts with matrix components in plasma or serum samples. These matrix components can be endogenous biological components such as phospholipids, carbohydrates, and metabolites. Matrix components can reduce the binding of the antibody to the target protein, or non-specifically bind the antibody and generate weak or noisy results.

S.No. Possible Cause Solution
1 Matrix effect
  • Centrifuge the sample. Centrifugation can separate matrix components from soluble antigens, reducing the concentration of the components and the matrix effect on results.
  • Increase dilution. Increasing the dilution factor 2-5 fold will reduce matrix component binding and mitigate the matrix effect. Note: when diluting the samples remember to use the same diluents as used for the standard curve.