Troubleshooting of Sandwich ELISA with Direct Detection

The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the sandwich ELISA with direct detection. Protocol of Sandwich ELISA with Direct Detection is available for you.

Contents

Problem 1: Poor Standard Curve
Problem 2: Positive Results in Negative Control
Problem 3: Weak or No Signal
Problem 4: Too Much Signal
Problem 5: High Background
Problem 6: Low Sensitivity
Problem 7: Edge and Drift Effect
Problem 8: Matrix Effect
Problem 9: Poor Replicate Data
Problem 10: Large Coefficient of Variation (CV)
Problem 11: Inconsistent Results Assay-to-assay in ELISA
Problem 12: Poor Dynamic Range between Signal and Background
Tips

Poor Standard Curve

Cause	Solution
Improper curve fitting	Try plotting using different scales such as log-log, 5 parameter logistic curve fit.
Pipetting error	Use calibrated pipettes and proper pipetting technique.
Poorly mixed reagents	Thoroughly mix reagents.
Poor/variable adsorption of reagents to plate	Extend incubation time. Check coating buffer. Use a different plate as appropriate.
The standard stock solution has been improperly prepared	Dilute the lyophilized standard with the suggested diluent buffer as is suggested.
The standard stock solution has been improperly diluted	Check your calculation for the dilution.
Freezing/thawing of the standard	Use fresh standard.
The reagents are used beyond expiration date	Use fresh reagents.
Use of non-calibrated external recombinant protein preparation as standard	Calibrate against reference preparation standard.

Positive Results in Negative Control

Cause	Solution
The reagents are contaminated	Use fresh reagents and pipette carefully.
Sandwich ELISA – The detection antibody is detecting the capture antibody	Check if the correct capture antibody and detection antibody have been used and that they will not detect each other.
Insufficient washing of plates	Ensure that wells are washed adequately by filling the wells with wash buffer. Make sure that all residual antibody solutions are removed before washing.
Too much antibody used leading to non-specific binding	Check the amount of antibody suggested. Try using less antibody.

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 or used incorrect reagents	Review protocol and repeat assay using a positive control.
Antibody not coated properly	Longer coating times, different coating buffers, higher coating concentration.
Poor antibody labeling efficiency	Strictly follow the biotin labeling instructions.
Assay buffer compatibility	Ensure assay buffer is compatible with target of interest.
Not enough detection reagent	Optimize antibody concentrations for your assay. Use fresh reagents at the correct pH.
Sample prepared incorrectly	Ensure proper sample preparation and dilution.
Incubation temperature too low	All reagents including plate 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 once the assay has started. 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. 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.
Enzyme inhibitor present	Avoid sodium azide in HRP reactions. Avoid phosphate in AP reactions.

Too Much Signal

Cause	Solution
Insufficient washing	Use appropriate washing procedure. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
High antigen concentration	Use higher antigen dilutions.
Plate sealers not used or reused	During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Incorrect dilutions prepared	Check pipetting technique and double-check calculations.
Longer incubation times than recommended	Manufactured guide has optimized protocols. Make sure to follow recommended incubation times.
Excess time before plate reading	Read your plate within 30 minutes after adding the substrate. If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate.

High Background

Cause	Solution
Insufficient washing	Wash wells as recommendations.
Contaminated wash buffer	Prepare fresh water buffer.
Suboptimal salt concentration in washing buffer	Optimize salt concentration as high concentration can reduce non-specific and/or weak off-target interaction.
Too much detection reagent	Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent.
Blocking buffer ineffective	Try different blocking reagent and/or add blocking reagent to wash buffer.
Plate left too long before reading	Read plate immediately after adding stop solution.
Non-specific binding of antibody	Use suitable blocking buffers. Using an 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 in the dark or as recommended by manufacturer.
Precipitate formed in wells upon substrate addition	Increase dilution factor of sample or decrease concentration of substrate.
Dirty plate	Clean the plate bottom.
Pipetting errors	Calibrate pipets so that they dispense the correct volumes.
Reagents were not mixed properly	Thoroughly mix all reagents and samples before pipetting solutions into wells.
Excess time before plate reading	Read your plate within 30 minutes after adding the substrate. If the reading is not performed within this time frame, add the stop solution after sufficient color is developed in the plate.

Low Sensitivity

Cause	Solution
Inactive detection reagent	Ensure reporter enzyme/fluorescent has the expected activity.
Plate reader settings incorrect	Ensure plate reader is set to read the correct absorbance wavelength or excitation/emission wavelengths for fluorescent detection.
Assay format not sensitive enough	Switch to a more sensitive detection system (such as colorimetry to chemiluminescence/ fluorescence). Lengthen incubation times or increase temperature.
Not enough substrate	Add more substrate.
Interfering buffers or sample ingredients	Check reagents for any interfering chemicals. For example, sodium azide in antibodies inhibit HRP enzyme and EDTA used as anticoagulant for plasma collection inhibits enzymatic reactions.
Mixing or substituting reagents from different batches	Avoid mixing components from different batches.

Edge and Drift effects

Cause	Solution
Uneven temperature	Seal the plate completely with a plate sealer during incubations. 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 pipetting into the wells.
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 occasionally 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 phospholipids, carbohydrates and endogenous metabolites or an interaction between the analyte of interest and the matrix, for instance 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 of wells	Use appropriate washing procedure. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Bubble in wells	Ensure no bubbles are present prior to reading plate.
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 preparation and optimal sample storage conditions.
Cross-well contamination	Ensure plate sealers and pipette tips are not contaminated with reagents.
Plate sealers not used or reused	During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.

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 (e.g. minimize freeze/thaw cycles).

Inconsistent Results Assay-to-assay in ELISA

Within the same experiment

Cause	Solution
Insufficient washing	Use appropriate washing procedure. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid.
Inconsistent incubation temperature	Make sure to follow recommended incubation temperatures. Be aware of fluctuations in temperature due to environmental conditions.
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

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
Reading plate using incorrect wavelength	Check filters/reader. 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

Pipetting technique

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.

Washing procedure

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 seconds.
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.