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Troubleshooting of Indirect ELISA

This troubleshooting guide provides potential sources and solutions for common issues observed in indirect ELISA. Protocol of Indirect ELISA is available for you.

Note: The conditions described here may not pertain to every indirect ELISA assay because specific requirements are different for individual. Be sure to check your product insert for specifications.

Contents

Poor Standard Curve

Cause Solution
Improper standard solution Make sure dilutions are made correctly.
Standard improperly mixed Briefly spin vial before opening.
Standard degraded Store and handle standard as recommended.
Curve doesn't fit scale Try plotting using different scales.
Pipetting error Use calibrated pipettes and proper pipetting technique.

Weak or No Signal

Cause Solution
Incubation time is too short Incubate samples overnight at 4°C or strictly follow the manufacturer guidelines.
Target present below detection limits of assay Decrease dilution factor or concentrate samples.
Assay set up incorrectly Review protocol and set up control group.
Antigen not coated properly Longer coating times, different coating buffers.
Recognition of epitope impeded by adsorption to plate Conjugate peptide to a large carrier protein before coating.
Not enough detection reagent Increase concentration of the secondary antibody.
Not enough primary antibody Increase concentration of the primary antibody.
Incubation temperature too low
  • All reagents should be at ambient temperature prior to use.
  • Ensure the incubation steps are carried out at the correct temperature.
Incorrect wavelength Verify the wavelength and read plate again.
Plate washings too vigorous
  • At least 4 times after incubation.
  • Pipette wash buffer gently if washes are done manually.
Wells dried out
  • Do not allow wells to become dry.
  • Cover the plate using sealing film or tape for all incubations.
Slow color development
  • Prepare substrate solution immediately before use.
  • Ensure the stock solution has not expired and is not contaminated.
  • Allow longer incubation time.
  • Ensure plates and reagents are kept at room temperature.
Antibody quality Use a fresh aliquot of antibody that has been stored at -20°C or below.
Improper buffer Sodium azide inhibits HRP activity.

Too Much Signal

Cause Solution
Insufficient washing
  • Use appropriate washing procedure.
  • Invert plate on absorbent tissue at the end of each washing step.
Plate sealers not used or reused Use a fresh sealer each time the plate is opened.
Incorrect dilutions prepared Follow the manufacturer guidelines
Longer incubation times Follow recommended incubation times.
Excess time before plate reading
  • Read your plate within 30 minutes after adding the substrate.
  • Add stop solution after sufficient color is developed.

High Background

Cause Solution
Insufficient washing
  • Use appropriate washing procedure.
  • Invert plate on absorbent tissue at the end of each washing step.
Contaminated wash buffer Prepare fresh water buffer.
Too much primary antibody/detection reagent
  • Ensure the reagent has been diluted properly.
  • Decrease the recommended concentration of detection reagent.
Blocking buffer ineffective
  • Try different blocking reagent.
  • Add blocking reagent into wash buffer.
Salt concentration of incubation/wash buffers Increasing salt concentrations to reduce non-specific/or weak off-target interactions.
Excess time before plate reading
  • Read your plate within 30 minutes after adding the substrate.
  • Add stop solution after sufficient color is developed.
Non-specific binding of antibody
  • Use suitable blocking buffers.
  • Using the affinity-purified antibody.
  • Ensure wells are pre-processed to prevent non-specific attachment.
Substrate incubation carried out in light Substrate incubations should be carried out as recommended.
Precipitate formed in wells upon substrate addition Increase dilution factor of sample or decrease concentration of substrate.
Reagents were not mixed properly Make sure all reagents and samples have been thoroughly mixed.

Low Sensitivity

Cause Solution
Insufficient target Concentrate target or reduce dilution.
Inactive detection reagent Ensure reporter enzyme or fluorescent is effective.
Plate reader settings incorrect Verify wavelengths.
Assay format Switch to a more sensitive ELISA type and detection system.
Interfering buffers or sample ingredients Check reagents for any interfering chemicals.
Mixing reagents from different batches Avoid mixing components from different batches.

Edge and Drift effects

Cause Solution
Uneven temperature
  • Use a plate sealer.
  • If 37°C incubation is indicated, make sure plate is in the center of incubator.
  • Avoid incubating plates in areas where environmental conditions vary.
Solutions not at room temperature Ensure that all solutions are at room temperature before using.
Evaporation Seal the plate completely with a plate sealer during incubations.
Stacked plates Avoid stacking plates during incubation.
Considerable interval of time elapsed during the addition of a solution
  • All samples and standards should be prepared prior to starting the assay.
  • Reduce assay time, particularly important when adding the chromogen substrate.

Matrix Effect

ELISA quantification of plasma and serum sometimes encounters problems which are caused by the matrix effect. The matrix effect can arise from a number of matrix components including, but not limited to: interaction between endogenous biological components such as carbohydrates, phospholipids, and endogenous metabolites or an interaction between the analyte of interest and the matrix, such as covalent binding to plasma proteins. This results in erroneous sample readings. Simply diluting the samples by 2 or 5 folds reduces the matrix effect, when diluting the samples remember to use the same diluent as used for standard curve.

Poor Replicate Data

Cause Solution
Insufficient washing
  • Use appropriate washing procedure.
  • Increasing duration of soak steps may also help.
Plate sealers not used or reused
  • Use plate sealers.
  • Use a fresh sealer each time the plate is opened.
Incomplete reagent mixing Ensure all reagents are mixed thoroughly.
Inconsistent pipetting Use calibrated pipettes and proper pipetting techniques. If a multi-channel pipette is used, ensure that all channels deliver the same volume.
Inconsistent sample preparation or storage Ensure consistent sample prep and optimal sample storage conditions.
Cross-well contamination Ensure plate sealers and pipette tips are not contaminated with reagents.

Large Coefficient of Variation (CV)

Cause Solution
Bubbles in wells Ensure no bubbles are present prior to reading plate.
Wells not washed equally/thoroughly Check that all ports of the plate washer are unobstructed.
Incomplete reagent mixing Ensure all reagents are mixed thoroughly.
Inconsistent pipetting Use calibrated pipettes and proper technique to ensure accurate pipetting.
Edge effects Ensure the plate and all reagents are at room temperature.
Inconsistent sample preparation or storage Ensure consistent sample preparation and optimal sample storage conditions.

Inconsistent Results Assay-to-assay in ELISA

1. Within the same experiment

Cause Solution
Insufficient washing
  • Use appropriate washing procedure.
  • Increasing duration of soak steps may also help.
Inconsistent incubation temperature
  • Make sure to follow recommended incubation temperatures.
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.

2. Between multiple experiments

Cause Solution
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.

Poor Dynamic Range between Signal and Background

Cause Solution
Concentration of detection antibody is too low Check dilution and titrate if necessary.
Incorrect wavelength Use the wavelengths provided in the protocol.
Insufficient development times 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.

Tips

1. Use the correct pipette.

2. Confirm tip is firmly seated on the pipette.

3. Confirm there are no air bubbles while pipetting.

4. Change tips between each standard, sample, or reagent.

5. Use different reservoirs for each reagent.

6. Pipette sample into the side of wells to avoid splashing.

7. Always run samples/standards in replicate.

1. Completely aspirate liquid from all wells by gently lowering an aspiration tip into the bottom of each well.

Note: Take care not to scratch the inside of the well.

2. Fill the wells with at least 400 µL of diluted wash buffer.

3. Let soak for 15 to 30 s.

4. Aspirate wash buffer from wells.

5. Repeat as directed in protocol (usually 3-4 times).

6. After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid.

7. Alternatively, an automated plate washer may be used.

Although each of the common cause and corresponding solutions are listed above, several problems may occur simultaneously in the actual process. Thus, solutions need to be addressed from multiple perspectives based on the actual situation. For further help and expert advice, contact our scientist staffed technical support department.

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