The microtiter plate is coated with antigen for detection of antibody. Antibodies are added. After that if i add biotinylated antigen (same as coated), then strepavidin HRPO, substrate. I have observed backgruond in known positive and known negative samples. what could be the reason? One basic question is "where would the biotinylated antigen bind on antibody if specific region is already bound to coated antigen".