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Troubleshooting Tips for Enzyme-Linked Immunosorbent Assay (ELISA)

General tips to consider when performing an ELISA:

  • Increase antigen availability by building scaffold complex (1st streptavidin 4C overnight, then biotinylated antigen for 15min at RT).
  • Ensure proper positive and negative controls are performed.
  • Increased washing will decrease possible cross-reactivity between your reagents.
  • Use a detection strategy that will not cross-react with your sample.
  • Performing a western blot can confirm the activity of the primary antibody. Remember to include positive controls.

Positive signal seen in negative control wells:

  • This may indicate a contamination of ELISA reagents or the samples themselves. Make sure your samples are not splashing into one another. Use known fresh reagents and perform ELIISA steps carefully.
  • If performing a sandwich ELISA - detection antibody may be binding to plating/coating antibody.
  • Check that all wells are being washed thoroughly with correct buffer during and after wash steps.
  • Ensure that you are using recommended amount of plating/coating and detection antibody. Try using less of either one to reduce signal.

High background seen across entire plate:

  • Detection antibody used in wrong amount/concentration or left on too long. Check that antibodies are being used in the recommended amount/concentration. Add stop solution when desirable level of signal is seen.
  • Check that substrate solution and stop solution are fresh. Fresh stop solution should be clear (yellow = contamination).
  • Make sure that stop solution has been added. Color will continue to develop if stop solution is not added.
  • Do not leave plate too long before reading. Color will continue to develop even after stop solution is added (stop solution slows color formation).
  • Ensure that all laboratory glasswares are clean and sterile.
  • Substrate incubation should be performed in the dark. Do not perform in the light. This will affect signal formation.
  • Check that incubators/thermometers are accurate and working. High temperatures can affect antibody binding kinetics. Incubation temperatures may need optimization.
  • Detection antibody is binding non-specifically. Check to make sure the recommended blocking step with an approved blocking buffer is included. When possible, use pre-absorbed, affinity purified antibodies.
  • See also suggestions under "Positive signal seen in negative control wells" section above

Low signal seen:

  • Absence or low level of target protein expressed. Check that your target protein is expressed in your samples. Increase amount of sample used or change to more sensitive assay. Check that signal from positive control is within assay limits.
  • Too little antibody used. Check that the recommended amount/concentration of each antibody is used. This may require optimization.
  • Substrate solution is not fresh or improperly prepared. Prepare substrate solution immediately before each use. Ensure all solutions are prepared correctly, have not expired and have been stored properly.
  • Too short of an incubation time. Ensure you are following recommended incubation times. This may require optimization.
  • Antibody incubation temperatures are too low. Lower than optimum incubation temperatures can affect antibody binding kinetics. Check that incubators/thermometers are accurate and working. This may require some optimization. Ensure all reagents are at room temperature before beginning.
  • Stop solution was not added. Stop solution slows and stabilizes signal formation.

High signal seen in samples or positive controls, or signal does not decrease over a dilution range:

  • The concentration/amount of samples or positive controls is too high and out of range of the assay. Dilute samples and controls to within assay range.

Atypical signals seen across plate:

  • Confirm that plates are always on level surface to ensure even reagent and temperature distribution.
  • Ensure you are carefully using calibrated pipettes for all pipetting. Duplicates will ensure final signal accuracy.
  • Carefully mix all solutions/reagents before using.
  • If recommended, cover plates with proper lids during incubations. Do not let wells dry out. Maintain appropriate incubator humidity levels.
  • Make sure all wells are being washed correctly and uniformly.
  • Keep bottom of plate clean of any possible contaminants that might affect spectrophotometer.

Signal color developing too slowly:

  • Confirm plates and reagents are at room temperature before beginning.
  • Make sure you are preparing substrate solutions immediately before use. Ensure that all solutions are prepared correctly, have not expired, have been stored properly and are being used at the recommended amounts/concentrations.
  • The presence of contaminants like sodium azide and peroxidase can affect the substrate reaction.

Duplicate/Triplicate wells not yielding similar results:

  • Insufficient washing: If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash
  • Make sure you are using an ELISA plate and not a tissue culture plate
  • Do not reuse plate sealers
  • Your buffers may be contaminated. Remake them.

Poor assay to assay variability:

  • This may be insufficient washing. If using an automatic plate washer, check that all ports are clean and free of obstructions
  • Adhere to the recommended incubation temperature range for a given assay. Avoid environments where temperatures vary
  • Follow the same protocol from assay to assay
  • Use fresh plate sealers for every assay
  • Check your standard curve dilution calculations and make a new standard curve
  • Use internal controls
  • Remake fresh buffers

If edge effects are being experienced, try:

  • Use fresh plate sealers
  • Avoid incubating plates in environments where temperatures vary

If drift effects are being experienced, try:

  • Making sure that all reagents are at room temperatures before pipetting into the wells unless otherwise instructed by the assay literature
  • Always perform an assay continuosly; have all standards and samples ready to go when protocol instructs you to use them