ELISA troubleshooting tips

Possible causes

Solution

Standard solution has not been diluted correctly.

Confirm dilutions are made correctly.

Standard improperly reconstituted.

Briefly spin the vial before opening; inspect for undissolved material after reconstituting.

Standard may be degraded.

Store and handle the standard as recommended.

Pipetting error.

Use calibrated pipettes and proper pipetting technique.

Possible causes

Solutions

You may need a different curve fitting model.

You should always follow manufacturer's instructions in the first instance. However, if the curve-fit doesn't seem to work, try plotting using different models.

Possible causes

Solution

Bubbles in wells

Ensure no bubbles are present prior to reading the plate. You can try to remove bubbles by gently pipetting up and down.

You can avoid the risk of bubbles forming by practicing proper pipetting technique.

Wells not washed equally/thoroughly

Check that all ports of the plate washer are unobstructed. Wash wells as recommended.

Automatic wash systems can help improve control of washing steps.

Inconsistent pipetting.

Use calibrated pipettes and proper pipetting technique

Ensure all reagents are mixed thoroughly by gently pipetting up and down.

Inconsistent sample preparation or storage.

Ensure all samples are stored and prepared consistently, and according to recommendations.

Edge effects due to incorrect handling of plates.

Edge effects occur in ELISA when different wells are exposed to slight variations in temperature and humidity. The outer (or ‘edge’) wells are usually the first to respond to any change in the environment, causing them to dry out or evaporate before the inner wells.

In some cases, the edge effect is clearly visible. Try looking at the plate from the side – if the buffer level is lower on the outside wells, some of the buffer has dried out.

To prevent wells drying out, cover all wells with sealing film or tape.

You should also ensure the plate and all reagents are at left to equilibrate to room temperature before incubation. Don’t use plates straight from the fridge; the inner wells need more time to reach room temperature.

Possible causes

Solution

Target or antibody adsorbs poorly to

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 detection of a peptide by direct or indirect ELISA, conjugate peptide to a large carrier protein before coating 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).

Possible causes

Solutions

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.

Possible causes

Solutions

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.

Buffer may be incompatible with 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. Mixing components should generally be avoided.

Possible causes

Solutions

Overuse of antibodies has reduced their effectiveness.

Make sure you use fresh primary and secondary antibodies for each experiment; the effective concentration of antibody is lowered after each use.

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

Check 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.

Incubation temperature may be too low

Ensure all steps are carried out at the correct temperature. Check manufacturer’s guidelines for specific advice.

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

Too much washing between steps

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

Reduce the duration and / or number of washing steps.

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

The wash or incubation buffer is

contaminated with bacteria

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

Possible causes

Solutions

The secondary antibody may be binding

non-specifically

Run a control without any primary antibody.

Make sure you use a secondary antibody raised in a different species to your sample.

Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.

Ensure the wells have been pre-processed to prevent non-specific attachment.

Primary antibody concentration may be

too high.

Dilute the antibody further to its optimal concentration.

Blocking of non-specific binding may be insufficient

Increase the blocking incubation period and consider changing the blocking agent. We recommend 5-10% normal serum of the same species as the detection antibody.

Too much substrate (if using enzyme-

conjugated antibody)

Dilute the substrate and reduce substrate incubation time.

Signal amplification may be too high (if using a signal amplification technique)

Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation).

Possible cause

Solution

Not enough washing between steps

Residual unbound antibodies remaining between steps can produce a false positive signal.

Wash the wells extensively in buffer between all steps.

Increase the washing time.

Precipitate has formed in wells upon substrate addition.

Check for any visible signs of precipitation in the wells.

Decrease the concentration of the substrate

Waiting too long to read plate after adding stop solution.

Read the plate immediately after adding the stop solution.

If possible, measure at different time points from the moment the stop solution is added to the wells.

The plate may be contaminated or dirty.

Clean the plate using the manufacturer's instructions.

Consider using a new plate if you’re still experiencing issues.