Unwanted background staining can arise from nonspecific binding of antibodies, particularly prevalent in polyclonal antibodies. Another reason is because the secondary antibody might have a high or moderate binding affinity for the same or similar epitopes on unintended antigens. It can also be caused by endogenous enzymes and from the failure to block endogenous biotin before introducing the avidin–biotin–enzyme complex. Additionally, elevated levels of the primary antibody can enhance interactions between the primary antibody and non-target epitopes, leading to heightened nonspecific binding and background staining.