High background in ELISA

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

What issues can arise relating to assay set up?

Possible issues

Solution

The secondary antibody may be binding non-specifically.

Run a control without any primary antibody.
Use a secondary antibody raised in a different species than your sample.
Try a secondary antibody that has been pre-adsorbed against the Ig of the species of the samples.
Change the blocking buffer and/or blocking time.

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.

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

Reduce the amount of signal amplification (eg, decrease the biotin to antibody ratio when conjugating the secondary antibody).

Possible issues

Solution

Not enough washing between steps.

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

Precipitate has formed in wells upon substrate addition.

Incubating for too long with the substrate.

Decrease substrate incubation time.
Read plate kinetically during substrate incubation (for colorimetric, read at 600-620nm and add Stop Solution when OD ~1).

Waiting too long to read plate after adding stop solution.

Read the plate immediately (within 5-10 minutes) after adding the stop solution to avoid signal drift.

The plate may be contaminated or dirty.