Following our previous post on how to improve results obtained in ELISA, let’s focus today on one specific point, which is reducing background.
ELISA has many advantages, but one of the drawbacks is that, since we cannot “see” how the reaction works (in contrast to other optical-based technologies such as antibody arrays or Q-plex), high final Abs values may come from a specific signal… or be due to background.
Usually, incorporating sufficient controls in the ELISA plate will allow users to discriminate real positives from false positives (e.g. if you are using cell culture supernatant with FBS % over 1 %, it might be wise to include a medium-only control). FBS contains cytokines that can cross-react with antibodies, even if targeted to different species, in about 10 % of the cases (based on our experience at the Biomarkers team at tebu-bio).
Anyway, if you suspect that you are obtaining a high background in your ELISA, and would like to improve it for future experiments, be sure to follow these guidelines (thanks to Daniel at Raybiotech, Inc. for helpful tips & tricks!).
Tip #1 – less is more
Too much HRP-Streptavidin may cause background. To avoid this, briefly centrifuge the vial of HRP-streptavidin concentrate in your ELISA kit, and pipette up and down to mix gently before using, since precipitation may form during storage.
Tip #2 – let me insist… less is more
Too much detection antibody may also contribute to high background. Make sure of correct dilution fold of both the biotinylated antibody in your ELISA kit, and HRP-Streptavidin. Both should be diluted following the manufacturer’s instructions.
Tip #3 – the shorter, the better
Long incubation times in some steps (i.e. overnight sample/standard incubation) can cause high background.
Tip #4 – be clean
The wash steps may introduce background: if the plate is insufficiently washed, or if the wash buffer itself was contaminated. Wash buffer must be removed completely from the wells after each wash. We recommend using an automated plate washer or multi-channel pipettor. Squirt bottles are not advised.
Still, if the high background persists after checking all these steps, try reducing the amount of HRP-streptavidin (by increasing the dilution by 1.5 or 2-fold).
Obviously, all these tips apply if you have found high background in your ELISA experiment. For detection of markers present in very low concentrations in your sample, you might wish to use the tips and contradict them in order to increase signal. Nevertheless, it is somehow tricky. Your wish to increase signal may lead to an increased… but non-specific signal.
At the Biomarkers team at tebu-bio, we are glad to help many researchers all over Europe to find the best solutions suited to their specific research model or cohort study!
Experiencing problems with ELISA? Leave your questions or comments below!
