I am very confused by my results but have repeated them several times with very consistent results. Essentially, I have a sandwich ELISA using biotinylated detection antibody. I coat the plate with capture antibody overnight (anti-HPV 18 E7). The next day I block with 3% BSA, then apply HeLa cell Lysate, then apply biotinylated detection antibody. Finally I apply streptavidin-HRP. As a negative control I have a set of wells with capture antibody, cell lysate, no detection antibody, and streptavidin-HRP. Without the detection antibody, I still get the same signal! It's specific too -- if I don't use cell lysate or use another type of cell lysate, there is no signal with just streptavidin-HRP. It is only present when there is antigen, but doesn't depend on the presence of detection antibody. How can this be??