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Difficulty in obtaining signal for ELISA

This can range from no to very low or inconsistent signal when signal is expected.

How you've handled your reagents, set-up your assay and detection system can all affect the signal you obtain.

Problems with signal from assay set up

Possible causes


Target or antibody adsorbs poorly to the plate.

  • Try and increase adsorption to the plate through pre-treatment of wells.

  • You can also try plates that offer ‘enhanced binding’.

Recognition of epitope impeded by adsorption to plate (direct or indirect ELISA).

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

The protein of interest isn't present.

  • Run a positive control.

  • Check the scientific literature to see if the protein is expected in your sample.

Not enough antibody is bound to the protein.

  • Add a higher concentration of primary antibody.

  • Incubate the sample for longer with the antibody (eg overnight).

Problems with detection systems

Possible causes


Detection is not sensitive enough.

  • Consider switching to a more sensitive detection system, for example from colorimetric to fluorescence, or from direct to indirect detection.

  • Signal amplification, eg biotinylation, may be used to further enhance the signal.

Incorrect filter settings used for detection.

  • Ensure plate reader / instrument is set to read the correct absorbance wavelength or excitation/emission wavelengths for fluorescent detection.

Slow development of colorimetric reaction.

Some colorimetric reactions develop slowly. 

  • If you read the plate too early, the reaction may not be complete so you might miss some of the signal.

  • Try reading the signal using ‘kinetic mode’ to measure the signal over an extended period of time.

Problems caused by incompatibility issues

Possible causes


The primary antibody and the secondary antibody are not compatible.

  • Make sure you use a secondary antibody that was raised against the primary antibody species.

  • Make sure that the isotypes of the primary and secondary are compatible.

The buffer may be incompatible with the detection method.

Some buffers contain reagents that may interfere with detection. For example, sodium azide is an inhibitor of HRP, so is unsuitable for use with HRP-conjugated antibodies.

  • Check your buffers don't contain any incompatible reagents, and change the buffer if needed.

Sample type may be incompatible.

  • Check that the ELISA kit you're using is compatible with your sample type (eg cell lysate).

Mixing components from different kits.

Each kit is designed to work in a given application under specific conditions, so mixing components may result in an assay that doesn't work as expected.

  • Avoid mixing components.

Incorrect storage and handling of reagents causing issues

Possible causes


Overuse of antibodies has reduced their effectiveness.

Effective antibody concentration is lowered after each use. Therefore, it is best to avoid using antibodies across experiments whenever possible.

  • Make sure you use fresh primary and secondary antibodies for each experiment.

Antibodies, reagents or amplification kits may have lost activity due to improper storage and handling.

Check and follow the storage instructions for your products on the datasheet.

  • Avoid excessive freezing / thawing.

  • Store and handle fluorophores and fluorophore-conjugated antibodies in the dark, and minimize light exposure by wrapping the vial in foil.

  • Run a positive control.

The incubation temperature may be too low.

Check the manufacturer’s guidelines for specific advice.

  • Ensure all steps are carried out at the correct temperature.

  • All reagents, including the plate, should usually be left to equilibrate to room temperature before proceeding.

Too much washing between steps.

Washing with a buffer between steps is necessary, but washing too aggressively can sometimes remove detection reagents.

  • Reduce the duration and / or number of washing steps.

  • Make sure you wash with gentle pressure if using a pipette. If available, automatic wash systems can be set to apply more gentle pressure.

The wash or incubation buffer is contaminated with bacteria.

  • Use fresh, sterile buffer (eg our sterile PBS).