Effect of alkaline phosphatase fusion on performance in IHC. A. scFv-APs demonstrate improved sensitivity in IHC. HER2 clones are more sensitive when expressed in pSANG14-3F than pSANG10-3F Monovalent G98A, with an affinity of 320 nM, gave weakly positive staining of the antigen in breast cancer tissue. Whereas, bivalent G98A gave staining equivalent to the monomeric scFv clone C6.5, which has a 20-fold higher affinity. Bivalency does not visually improve C6.5 staining. Detection was by anti-FLAG antibody coupled to biotin with the addition of streptavidin-horse-radish peroxidase and a tyramine signal amplification step. B. Image analysis was used to quantify the staining intensities of all five scFvs as both monomeric scFv and bivalent scFv-AP.