ELISA is one of the most widely used immunoassay techniques, being quick, easy, and relatively inexpensive to perform. But, like any immunoassay, it can go wrong. In this article, we highlight some common ELISA problems and suggest ways of overcoming them.
It has been 50 years since ELISA was first described in the literature as a means of quantifying rabbit IgG. Since then, it has evolved into a plate-based methodology suitable for both multiplexed applications and high-throughput screening—and its popularity shows no sign of waning. According to Miranda Lewis, Ph.D., Scientific Communications Manager at Jackson ImmunoResearch, the benefits of ELISA include exceptional sensitivity and robustness, provided it is configured using high-quality reagents and appropriate controls. “A main advantage of ELISA over techniques such as Western blot is that it allows researchers to measure analyte concentrations, even when target biomolecules are present at only low levels in complex samples,” she says. “However, for accurate results, it is critical to use validated reagents for every step of the ELISA workflow and to source reliable controls.”
Ask any researcher to describe an issue they have encountered when running an ELISA and you’ll likely find high background to be top of the list. “High background is often due to poor blocking,” comments Dr. Tobias Polifke, Co-founder and Managing Director at CANDOR Bioscience, “which can result from using an inefficient blocking reagent such as BSA. While the widespread use of BSA for blocking stems from the fact that it was the first purified protein available in large amounts during the last century, modern alternatives offer significant improvements. For example, reagents have been developed that more rapidly provide a denser layer on the microplate surface, greatly reducing the risk of non-specific binding effects.” Other sources of high background include insufficient washing and poor antibody performance, with the latter additionally being a cause of weak signal—another frequent ELISA complaint. “Weak signal can occur when capture and detection antibodies compete for the same epitope during sandwich ELISA, or when analyte-specific antibodies have low affinity for the target,” comments Lewis. Yet, she cautions that while it can be instinctive to blame antibody reagents for sub-optimal ELISA performance, many other factors could be at play.
When ELISA goes wrong, diagnosing and fixing any issues can seem daunting. But whether you’re experiencing high background, weak signal, or poor reproducibility, other researchers will have faced similar problems before and can usually offer help. The following table lists some common ELISA complaints and includes suggested solutions to help get your assay back on track.
Switch to using a different blocking reagent or a source of BSA that is certified as being IgG- and protease-free
Increase the concentration of the blocking reagent
Extend the incubation time for blocking
Increase the number, volume, and/or duration of wash steps
Include a low concentration of detergent (e.g., 0.01–0.1% Tween-20) in wash buffers
Check that wells are fully emptied between washes
Consider automating the wash step rather than performing it manually
Serially dilute the sample to identify a more suitable concentration
Titrate antibody reagents to determine appropriate concentrations
Decrease the duration of antibody incubation steps
Switch to using a different antibody diluent
Run a control that contains no primary antibody
Consider using cross-adsorbed secondary antibodies
Use an affinity discriminating diluent to avoid low-affinity binding effects
Prepare colorimetric substrates (e.g., TMB, OPD, or pNPP) immediately prior to use
Switch to using stable colorimetric substrates (commercially available from various sources)
Read colorimetric assays as soon as the stop solution has been added (unless otherwise directed by the manufacturer
Standardize and document incubation times for substrate, stop solution, and time until read-out
Prepare fresh buffers for every assay or store buffers short-term at 4oC if appropriate, bringing them to room temperature before use
Use fresh plasticware (e.g., pipet tips, reservoirs) for every step
Optimize incubation times during assay development, then stick to the protocol
Confirm antibody reagents are validated to detect the target in the selected sample type and species
Check that antibodies are validated for the ELISA application
Switch to using alternative antibodies
Check that the microplate is validated for the ELISA application and confirm its binding capacity from the manufacturer’s description
Switch to using a different coating buffer
Increase the incubation time and/or temperature of the coating step
Increase the duration of antibody incubation steps
Check that the secondary antibody recognizes the detection antibody (e.g., an anti-rabbit secondary antibody should be paired with a detection antibody raised in a rabbit host)
Confirm that the capture and detection antibodies recognize different epitopes (refer to antibody manufacturers’ datasheets)
Replace both analyte-specific antibodies with a matched antibody pair if available
Consider using a different ELISA format (e.g. switch from a sandwich ELISA to a direct ELISA)
Bring all solutions to room temperature before use unless otherwise stated in the protocol
Ensure buffers do not contain azide
Perform sufficient washing to remove any residual traces of azide introduced with the detection antibody (azide is often used as an antibody preservative)
Confirm the sample choice is appropriate (refer to sites such as UniProt, PAXdb, or proteinatlas.org, and to antibody manufacturers’ datasheets for information about protein expression)
Obtain more concentrated samples
Spike samples with a known amount of analyte to check the sample matrix is not interfering with detection
Refer to the manufacturer’s instructions for storage
Avoid unnecessary freeze-thaw cycles (prepare aliquots for freezing, if appropriate)
Check that the microplate reader supports the chosen readout
Switch from using direct detection (with labeled analyte-specific antibodies) to indirect detection (using labeled secondary antibodies)
Consider using a more sensitive readout (e.g., switch from using colorimetric detection to using enhanced chemiluminescence)
Perform signal amplification (e.g., using biotinylated secondary antibodies and labeled streptavidin reagents)
Ensure the coating solution is thoroughly mixed
Seal plates during the coating step to prevent evaporation
Confirm that pipets have been calibrated
Use a coating stabilizer immediately after the coating process (even if plates will not be stored for a long time prior to use) to reduce well-to-well inconsistencies
Include detergent (e.g., 0.01–0.1% Tween-20) in wash buffers
Pulse centrifuge microplates gently before reading
Improve pipetting technique
Consider automating reagent addition
Use fresh plate seals between incubations
Always run ELISAs under stable environmental conditions (temperature, incubation times, air humidity, avoiding exposure to direct sunlight)
Adhere to the protocol
Perform comparative studies each time a new batch of reagent is introduced
Keep samples on ice and avoid repeat freeze-thawing
Prepare fresh reagents for every assay
Check that standards and controls have been prepared and stored correctly
Ensure plate seals are applied correctly to prevent evaporation
Add reagents to microplates as quickly as possible, avoiding delays
Prepare suitable quantities of reagents for each run, remembering to account for dead volumes
Use multichannel pipettes whenever possible and standardize the pipetting method
Avoid incubating plates where environmental conditions can vary (e.g., near equipment that generates heat, beneath air vents, in direct sunlight)
Rotate the plate by 180o and re-read to determine whether the effect remains in the same position; call a service engineer if this is the case
Avoid stacking plates during incubations