ELISA Troubleshooting

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ELISA Protocols, Troubleshooting, & Scientific Resources



This troubleshooting guide provides potential sources and solutions for common issues observed with ready-to-use ELISA kits or when developing a new ELISA.


No Signal or Weak Signal

Reagents added in incorrect order, or prepared incorrectly

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

Antibody concentration is too low

  • Increase the concentration of the primary or secondary antibody (titrations may be helpful). Novus Antibody Concentration Kits can be used to increase the primary antibody concentration.
  • Increase the incubation time to 4°C overnight.

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 primary 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 4°C overnight.

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 one primary antibody.

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

  • Verify that 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.

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
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High Uniform Background

Insufficient washing or blocking

  • Increase the number and/or duration of 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 plate (for colorimetric detection)

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

HRP reagent is too concentrated

  • Check 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.  
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High Variability Between Replicates

Within the same experiment

    Insufficient mixing of solutions or uneven plate coating

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

    • Make sure no residual solution remains in wells between wash steps.
    • Increase the number and/or duration of washes.

    Contaminated buffer or pipette tips

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

    Plate contains bubbles, affecting optical reading

    • Centrifuge plate prior to reading.

Between multiple experiments

    Solutions may be old

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

    Biologicals 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 temperature and periods.
    • Avoid incubating plates in areas where environmental conditions vary.
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Poor Dynamic Range between Signal and Background

Concentration of HRP is too low

  • Check dilution, and 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 dilution of standard curve

  • Check calculations, create a new standard curve.

Detection Antibody is too dilute

  • Check dilution, titrate if necessary.
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Edge and Drift Effects

Check your instrument

  • Verify that the plate is flat in the plate reader and read it again.
  • Flip the plate 180 degrees 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 now in the position that corresponds with your flipped plate, then one of the issues 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

  • Reduce assay time. This is particularly important when adding the chromogen substrate, which is a time-sensitive step.
  • Assay set-up should be continuous. All samples and standards should be prepared prior to starting the assay.

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.
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