ELISA Troubleshooting Guide

This protocol is intended as a guide only.

ELISA Troubleshooting Guide

Researchers can encounter a variety of problems when running an ELISA. This troubleshooting guide provides potential sources of common problems that researchers encounter when running or developing an ELISA. The steps we recommend for correcting these issues are outlined to help get you on the path to running a successful ELISA. For further assistance, please contact our technical service department.

• No Signal or Weak Signal

• High Uniform Background

• High Variability Between Replicates

• Poor Dynamic Range Between Signal and Background

• Edge and Drift Effects

• Samples Reading Too High, but the Standard Curve Looks Fine

• Green Color Develops upon Addition of Stop Solution when using Streptavidin-HRP

No Signal or Weak Signal

Reagents added in the incorrect order, or prepared incorrectly

• Repeat the experiment, closely following the protocol for solution preparation and the order of addition.

Antibody concentration is too low

• Increase the concentration of the primary or secondary antibody (titrations may be helpful). Antibody concentration kits can be used to increase the primary antibody concentration.

• Increase the incubation time to overnight at 4 °C.

Primary and secondary antibodies are not compatible

• Ensure the secondary antibody was raised against the species in which the primary was raised (e.g. if the primary antibody was raised in mouse, then use an anti-mouse secondary antibody).

Capture antibody (for sandwich ELISA) or antigen (for indirect ELISA) did not adhere to the plate

• Use a plate validated for ELISAs, not a tissue culture plate. Verify that the binding capacity of the plate is suitable for antigen or antibody binding.

• Increase the duration of the coating step to overnight at 4 °C.

Capture and detection antibodies recognize the same epitope (for sandwich ELISA)

• Verify that both antibodies recognize distinct epitopes. Use a different capture and detection antibody pair and try a validated matched pair, if available.

• If there’s limited availability of primary antibodies that recognize the antigen of interest, try a different type of ELISA. An indirect or competitive ELISA requires only on primary antibody.

Standard has gone bad (if there is a signal in the sample wells)

• Verify that the standard was prepared according to the instructions. If lyophilized standard is used, centrifuge prior to reconstitution.

• Use a new vial. The standard may have degraded if used beyond its expiration date or if left at room temperature for an extended period of time.

Biological sample is not in the detectable range

• Perform a serial dilution of the sample and start with a more concentrated sample for testing.

• Spike the sample with a known concentration of the antigen to check for potential interference.

Sample matrix is masking detection

• Dilute samples at least 1:2 in appropriate diluent, or preferably, do a series of dilutions to look at recovery.

Coated plates have gone bad

• Coat new plates.

Buffer(s) contain azide

• Sodium azide (often used to store primary antibodies) inhibits HRP activity. Ensure sufficient washing to remove the presence of sodium azide or use sodium azide-free buffers.

High Uniform Background

Insufficient washing or blocking

• Increase the number and/or duration of washes. Add a 30 second soak step in between washes.

• Increase the blocking time and/or concentration of the blocker (e.g. BSA, Casein, or gelatin). Protein blockers bind to unoccupied sites on the ELISA plate and prevent non-specific binding.

• Add detergents to wash buffers to reduce non-specific binding. Tween-20, a non-ionic detergent is typically used at a concentration between 0.01 - 0.1%.

Antibody concentration is too high

• Decrease the concentration of the primary or secondary antibody (titrations may be helpful).

Substrate solution prepared too early and turned blue (for colorimetric detection using a specific substrate such as TMB)

• Substrate solution should be mixed immediately before adding it to the plate.

Delay in reading the plate (for colorimetric detection)

• The plate should be read immediately after the addition of the stop solution.

HRP reagent is too concentrated

• Check the final concentration and adjust the titration accordingly.

Reservoirs, plate sealers, pipette tips, or buffer(s) contaminated with HRP

• Trace amounts of HRP may remain in reused plastics and turn substrate solutions, such as those containing TMB, blue. Use fresh plastics for each step.

• Prepare fresh buffers.

High Variability Between Replicates

Within the Same Experiment

Insufficient mixing of solutions or uneven plate coating

• Repeat the experiment and ensure each solution is thoroughly mixed before adding it to the ELISA plate.

• Verify that there isn’t a pipette error. Add an equal volume of coating solution to each well.

• Use a plate sealer to avoid evaporation during the coating step.

• If using a capture antibody at a concentration significantly below the amount needed to saturate all binding sites in the well, it may be necessary to increase the duration of the coating step to achieve binding equilibrium.

Inadequate washing of the wells

• Make sure no residual solution remains in the wells between wash steps.

• Increase the number and/or duration of the washes. If using an automatic plate washer, check that all the ports are clean and free of obstructions. Add a 30 second soak step and rotate the plate halfway through the wash.

Contaminated buffer or pipette tips

• Prepare fresh buffers.

• Use fresh tips when dispensing solutions or avoid carry-over contamination.

Plate contains bubbles, affecting the optical reading

• Centrifuge the plate prior to reading.

Between Multiple Experiments

Solutions may be old or contaminated

• Prepare fresh solutions for each experiment. Precipitates may be present in the buffer, or the buffering capacity of the solution may be inadequate.

Biological samples are not prepared the same

• Use the same experimental treatment and ELISA buffers for samples.

• Limit freeze-thaws.

• Add the same amount of sample and at the same dilution.

Variations in incubation temperature and/or time

• Use the recommended incubation times and temperatures. Make sure to adhere to the same protocol from run to run.

• Avoid incubating plates in areas where environmental conditions vary.

Insufficient washing of the wells

• Make sure no residual solution remains in the wells between wash steps.

Plate sealer reused

• Re-using a plate sealer can result in the presence of residual HRP which will turn the TMB blue. Make sure to use a fresh plate sealer for each step of the protocol.

Improper calculation of standard curve dilutions

• Check your calculations and make a new standard curve if necessary. Use internal controls.

Poor Dynamic Range between Signal and Background

Concentration of HRP is too low

• Check dilution, titrate if necessary.

Reading plate using incorrect wavelength

• Check filters/reader. Use the wavelengths provided in the protocol for the chromogen substrate or fluorochrome.

Insufficient development times (for colorimetric detection)

• Increase substrate solution incubation time.

Improper calculation of standard curve dilutions

• Check calculations, create a new standard curve.

Capture antibody did not bind well to the plate

• Use a plate validated for ELISAs, not a tissue culture plate. Dilute in PBS without additional protein.

Detection antibody is too dilute

• Check dilution, titrate if necessary.

Edge and Drift Effects

Check your instrument

• Verify that the plat is flat in the plate reader and read it again.

• Flip the plate 180° and read it again. If the edge/drift effect is still observed in the same position, even though the plate is flipped, then the instrument may need to be repaired. If the edge/drift effect is still observed but it is now in the position that corresponds with your flipped plate, then one of the issue below may be the cause.

Uneven laboratory temperature

• Avoid incubating plates in areas where environmental conditions vary.

• Use a plate sealer to avoid evaporation.

Solutions not at room temperature

• Ensure that all solutions are at room temperature before pipetting into the wells, unless the antibody datasheet indicates otherwise.

Considerable interval of time elapsed during the addition of a solution

• Assay set-up should be continuous. All samples and standards should be prepared prior to starting the assay.

• Reduce the assay time. This is particularly important when adding the chromogen substrate, which is a time-sensitive step.

Solution volume differs between wells

• Use plate sealers or low evaporation lid for long incubation periods to prevent evaporation.

• Ensure your pipette is dispensing equivalent volumes to each well. Calibrate your pipette if necessary.