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High background in ELISA

Usually caused by issues in assay set-up or the wells in your plate.

Issues from assay set-up

Possible causes


The secondary antibody may be binding non-specifically.

  • Run a control without any primary antibody.

  • Make sure you use a secondary antibody raised in a different species to your sample.

  • Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.

  • Ensure the wells have been pre-processed to prevent non-specific attachment.

Primary antibody concentration may be too high.

  • Dilute the antibody further to its optimal concentration. This can be found on the antibody datasheet or through titration experiments.

Blocking of non-specific binding may be insufficient.

  • Increase the blocking incubation period and consider changing the blocking agent. We recommend 5-10% normal serum of the same species as the detection antibody.

If using an enzyme-conjugated antibody, too much substrate is present.

  • Dilute the substrate and reduce substrate incubation time.

If using a signal amplification technique, signal amplification may be too high.

  • Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation).

Issues with wells

Possible causes


Not enough washing between steps.

Residual unbound antibodies remaining between steps can produce a false positive signal.

  • Wash the wells extensively in buffer between all steps.

  • Increase the washing time.

Precipitate has formed in wells upon substrate addition.

  • Check for any visible signs of precipitation in the wells.

  • Decrease the concentration of the substrate.

Waiting too long to read plate after adding stop solution.

  • Read the plate immediately after adding the stop solution.

  • If possible, measure at different time points from the moment the stop solution is added to the wells.

The plate may be contaminated or dirty.

  • Clean the plate using the manufacturer's instructions.

  • Consider using a new plate if you’re still experiencing issues.