Troubleshooting Tips for ELISA

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for the ELISA protocol.

ELISA is a widely employed immunoassay. Typically, it is used to quantify antibodies or antigens in biological samples. Creative Biolabs summarizes the common problems in ELISA assays and solutions. Also, we provide antibody customization services for ELISA assays.

Case 1: Standard Curve Fitting

A well-behaved standard curve ensures that you can reliably determine the concentration of your sample.

NOTE: As long as the curve has a good fit, as measured by the regression coefficient (R²). As long as the R² 0.9, the standard curve can be used with confidence.

Common problems encountered when setting up standard curves include:

Problems related to standard solutions

Possible causes	Solutions
The standard solution is not properly diluted.	Confirm that dilutions are made correctly.
Improper reorganization of standards.	Spin briefly before opening the vial; check for undissolved material after reconstitution.
The standard may be degraded.	Store and handle the standard as recommended.
Pipetting error.	Use calibrated pipettes and proper pipetting techniques.

The curve fitting model does not work on the data

Possible causes	Solutions
You may need a different curve-fitting model.	You should first follow the manufacturer's instructions. However, if curve fitting does not seem to work, try plotting using a different model.

Case 2: Inconsistent Results and High Coefficient of Variation

The coefficient of variation (CV) indicates how much the signal varies between runs. It is the ratio of the standard deviation to the mean. The value of the CV should be <20%.

Possible causes	Solutions
Bubbles in wells	Make sure no air bubbles are present before reading the plate. You can try to eliminate the air bubbles by gently pipetting up and down. You can avoid the risk of forming air bubbles by practicing proper pipetting techniques.
No equal/thorough cleaning of wells	Check that all ports on the plate washer are clear and unobstructed. Clean the wells as recommended.
Inconsistent pipetting	Use calibrated pipettes and proper pipetting techniques. Ensure that all reagents are well mixed by gently pipetting up and down.
Inconsistent sample preparation or storage	Ensure that all samples are stored and prepared consistently and in accordance with recommendations.
Edge effects due to incorrect handling of plates	In ELISA, edge effects occur when different wells are exposed to slight changes in temperature and humidity. The outer (or "edge") pores are usually the first to respond to any changes in the environment, causing them to dry out or evaporate before the inner pores. In some cases, edge effects are clearly visible. Try looking at the plate from the side—if the buffer level is low in the outside wells, then some of the buffer has dried out. To prevent the wells from drying out, cover all the wells with sealing film or tape. You should also make sure to leave the plates and all reagents to equilibrate to room temperature before incubating. Do not use the plates directly from the refrigerator; the inner wells need more time to reach room temperature.

Case 3: Difficulty Obtaining Signals

Problems related to assay set-up

Possible causes	Solutions
Poor adsorption of the target or antibody on the plate	Try to increase the adsorption on the plate by pre-treating the pores. You can also try to provide "enhanced binding" to the plate.
Recognition of epitopes is hindered by adsorption to the plate (direct or indirect ELISA).	To improve the detection of peptides by direct or indirect ELISA, peptides are bound to large carrier proteins prior to coating microtiter plates.
The protein of interest is not present.	Run a positive control. Check the scientific literature to see if you have the expected protein in your sample.
Not enough antibodies are bound to the protein.	Add a higher concentration of primary antibody. Incubate the sample with the antibody for a longer period of time (e.g., overnight).

Problems related to detection systems

Possible causes	Solutions
Not sensitive enough to detect	Consider switching to a more sensitive detection system, such as from colorimetric to fluorescence or from direct to indirect detection. Signal amplification, such as biotinylation, may be used to further enhance the signal.
The filter setting used for the test is incorrect.	Ensure that the plate reader/instrument is set to read the correct excitation/emission wavelength for absorbance or fluorescence detection.
The colorimetric method is slow to develop a response.	Some colorimetric reactions develop slowly. If you read the plate too early, the reaction may not be complete yet, so you may miss some signals. Try reading the signal in "kinetic mode" to measure the signal over a longer period of time.

Problems related to compatibility

Possible causes	Solutions
Primary and secondary antibodies are not compatible.	Make sure that the secondary antibody you use is specific to the primary antibody species. Make sure that the isotypes of the primary and secondary antibodies are the same. Specific primary antibodies can be prepared according to the experiment.
Buffers may not be compatible with the assay.	Some buffers contain reagents that may interfere with the assay. For example, sodium azide is an inhibitor of HRP, so it is not suitable for use with HRP-bound antibodies. Check if your buffer contains any incompatible reagents and change the buffer if needed.
Sample types may not be compatible.	Check that the ELISA kit you are using is compatible with your sample type (e.g., cell lysate).
Mix components from different suites.	Each kit is designed for application under specific conditions, so mixing components may result in assay results that are not as expected. Mixing components should generally be avoided.

Problems related to incorrect storage and handling of reagents

Possible causes	Solutions
The overuse of antibodies reduces their effectiveness.	Ensure that fresh primary and secondary antibodies are used for each experiment; the effective concentration of antibodies will decrease after each use.
Antibodies, reagents, or amplification kits may become inactive due to improper storage and handling.	Check the product storage instructions on the data sheet. Avoid excessive freezing/thawing. Store and handle fluorophore and fluorophore-conjugated antibodies in the dark, wrapping vials in aluminum foil to reduce light exposure. Run a positive control.
The incubation temperature may be too low.	Ensure that all steps are performed at the correct temperature. Check the manufacturer's guidelines for specific recommendations. All reagents, including plates, should normally be left to equilibrate at room temperature prior to proceeding.
Too much washing between steps	Washing with buffer between the two steps is necessary, but sometimes washing too vigorously can cause the loss of assay reagents. Reduce the time and/or number of wash steps. If using a pipette, be sure to wash at gentle pressure. If available, set the automated wash system to apply gentler pressure.
Washing or incubation buffer contaminated with bacteria	Use a fresh, sterile buffer.

Case 4: High Background

Problems related to assay set up

Possible causes	Solutions
Secondary antibodies may bind non-specifically.	The antibody is further diluted to the optimal concentration.
The primary antibody concentration may be too high.	Check the product storage instructions on the data sheet. Avoid excessive freezing/thawing. Store and handle fluorophore and fluorophore-conjugated antibodies in the dark, wrapping vials in aluminum foil to reduce light exposure. Run a positive control.
Blockade of nonspecific binding may not be sufficient.	Increase the blocking incubation time and consider changing the blocking agent. We recommend 5–10% of normal serum of the same species as the tested antibody.
Too much substrate (if using enzyme-conjugated antibodies)	Dilute the substrate and reduce the incubation time of the substrate.
Signal amplification may be too high (if signal amplification techniques are used).	Reduce the amount of signal amplification (e.g., if biotinylation is used, reduce biotin attachment to the secondary antibody).

Problems related to wells

Possible causes	Solutions
Not enough washing between steps	Residual unbound antibodies between the two steps can produce false- positive signals. Wash the wells extensively with a buffer between all steps. Increase the washing time.
After adding the substrate, a precipitate formed in the well.	Check for visible signs of precipitation in the wells. Reduce the concentration of the substrate.
Waiting too long to read the plate after adding the stop solution.	Read the plate immediately after the addition of the stop solution. If possible, take measurements at different time points from the moment the stop solution is added to the well.
The plate may be contaminated or soiled.	Clean the tablet using the manufacturer's instructions. If you are still experiencing problems, consider using a new tablet.

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