FAQ

Question

I get a high background in my sandwich ELISA which is detected by a peroxidase labelled secondary antibody. What can be the reason and can LowCross-Buffer® help?

Answer

It’s important to know if the secondary antibody was pre-adsorbed against other antibodies of other mammalian species. Secondary antibodies against rabbit IgG can also crossreact with high affinity with mouse IgG or goat IgG unless purified against antibodies of these species. The reason is the high sequence homology of mammalian antibodies. Secondary antibodies are often not purified (pre-adsorbed) and so crossreact with high affinity. In this case LowCross-Buffer® does not help. The secondary antibody has to be exchanged with a pre-adsorbed antibody which does not crossreact with the capture antibody. For example the company Biotrend (Cologne, Germany) offers many pre-adsorbed antibodies which can avoid these cross reactivities.