Sensitivity issues have been of concern with the streptavidin-based detection systems, resulting in high degrees of background with certain tissues. The need for biotinylated primary or secondary antibodies and blocking steps add time and expense to histological assays. The development of a biotin-free detection system removes these concerns and allows for a potentially cheaper, more sensitive assay. Using recently developed hydrazidothiol heterobifunctional agents and a novel thiol protection strategy, a successful conjugate was synthesized with a site-specific attachment through the Fc region of the antibody. This relatively small, well defined conjugate imparts detection sensitive enough to identify single gene copies, exhibiting no background staining. In both IHC and ISH staining protocols, the developed antibody – alkaline phosphatase conjugate outperforms the amplified streptavidin – alkaline phosphatase conjugate in terms of both sensitivity and background.