ELISA Guide; Part 4: Troubleshooting

ELISA Troubleshooting

Several common issues can occur when running an ELISA. These are detailed in the following table, along with suggested solutions.

Capture antigen or antibody may not have adhered to the microplate Check the binding capacity of the microplate according to the manufacturer’s description
Try using a different coating buffer
Increase the incubation time for plate coating
Concentration of analyte-specific antibodies may be too low Increase the concentration of the capture antibody and/or the analyte-specific detection antibody
Increase the incubation time for analyte-specific antibody binding
Analyte-specific detection antibody and secondary antibody may be incompatible Confirm that the host species of the analyte-specific detection antibody is compatible with the secondary antibody (for example, if the analyte-specific detection antibody was raised in rabbit, an anti-rabbit secondary antibody is required for detection)
Capture and detection antibodies may be competing for the same epitope (sandwich ELISA) Confirm that the capture and detection antibodies recognize different epitopes
Switch to using a matched antibody pair if available
Consider using a different ELISA format (e.g. try using an indirect ELISA that requires only one analyte-specific antibody rather than a sandwich ELISA)
Fluorophore-conjugated antibodies may have been compromised by exposure to light Ensure fluorophore-conjugated antibodies are stored correctly
Protect fluorophore-conjugated antibodies from light when adding them to, and incubating them in, the microplate wells
If only the standard wells (and not the sample wells) are affected, the standard may have degraded Try using a fresh vial of standard
Check that the standard has been prepared and stored correctly
Azide (often added to antibody storage buffers as a preservative ) may be inhibiting HRP activity Ensure sufficient washing to remove any residual traces of azide
Sample material may contain only low levels of the target analyte Obtain more concentrated samples
Spike samples with a known amount of analyte to check the sample matrix is not a source of interference
ELISA kits / kit components may have been stored incorrectly Check the manufacturer’s instructions for storage
Plate may have been read at an incorrect wavelength Check the reader settings are compatible with the chosen detection method
Washing may be inadequate Increase the number and/or duration of wash steps
Try adding detergent (e.g. 0.01-0.1% Tween-20) to wash buffers
Blocking may be insufficient Try using a more concentrated blocking solution
Increase the incubation time for blocking
Switch to using a different blocking buffer
Sample may be too concentrated Try diluting the sample
Antibody concentration(s) may be too high Decrease the concentration of the capture antibody, analyte-specific detection antibody, or secondary antibody
Decrease the antibody incubation time
Colorimetric substrates may have been prepared too early Always prepare substrates such as TMB immediately prior to use to avoid unwanted color development
Microplates may have sat around after the addition of stop solution (colorimetric detection) Read colorimetric assays as soon as the stop solution has been added
Consumables such as pipette tips, reservoirs or buffers may have introduced contamination Use fresh plasticware for each step
Prepare fresh buffers for each assay
Incubation times may have been too long Always follow the protocol and be consistent with reagent additions and timing
Plates may have been coated unevenly Ensure all solutions are thoroughly mixed before coating the plates
Seal plates after adding the coating solution to prevent evaporation; such effects can especially be noticeable in edge wells
Check pipettes have been calibrated and are performing as expected
Washing may be inadequate Increase the number and/or duration of wash steps
Confirm wells are fully emptied between washes
Wells may contain bubbles Centrifuge microplates briefly prior to reading
Plate seals may be a source of cross-contamination Always use fresh plate seals between incubations
Reagents may have degraded Prepare fresh reagents (including buffers) for each assay
Check that the standard has been prepared and stored correctly
Samples may have been handled incorrectly Always store and handle samples with care and avoid repeat freeze-thawing
Assay conditions may be inconsistent Ensure all protocol steps are performed reproducibly
Always run ELISAs under stable environmental conditions
The plate reader may be misaligned Read the plate, then rotate it by 180o and read again; if the effect remains in the same position, the reader may need to be repaired by a qualified service engineer
Solutions may be cold Ensure all solutions are at room temperature upon addition to the microplate unless otherwise stated in the protocol
Delays may have occurred during reagent addition Prepare suitable quantities of reagents (including dead volumes) for the assay to avoid running out part-way across a plate
Volumes may be uneven across the microplate Seal plates between reagent additions to prevent evaporation
Only use calibrated pipettes
Plates may have cross-contaminated one another Avoid stacking plates during incubations


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