Hi,
Hope you can help.
Currently been running some sandwich ELISA assays where I coat with anti-His, block and then capture His-tag protein and then sera and then anti-bovine monoclonal secondary (I do not have a specific antibody for the protein you see). When I don't coat the wells with anti-His, there is little binding as expected but without protein or sera I seem to get non-specific binding. Which we think is the Histidine residues of the secondary interfering. Anybody had this issue that could perhaps provide some insight into reducing this non-specific binding?
Thank you in advance
Unless the secondary antibody has multiple consecutive His residues, it is unlikely to bind tightly enough to the anti-His antibody combining site to cause background. It's more likely that there is non-specific binding of the secondary antibody to the anti-His antibody at other locations. Presumably the anti-His antibody is not the same species that the secondary antibody recognizes, but there may still be a slight degree of recognition. You might be able to eliminate this by preadsorbing the secondary antibody with immunoglobulin from the same species as the anti-His antibody.
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