The AT-tailing method is a labelling technique that utilises
oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an
oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an
oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base
oligo(dA-dT)-conjugated
antibodies (
IgG-ATs) were prepared in advance by conjugating
maleimide-activated
oligo(dA-dT) to
IgG via free sulfhydryl residues that had been introduced on the surface of
IgG using Traut's
reagent. Following the reaction with the target
antigen and the
IgG-AT,
oligo(dA-dT) was elongated by DeltaTth
DNA polymerase in the presence of dATP,
dTTP and biotinylated dUTP, consequently labelling the
antigen-antibody complex with a large amount of
biotin. To initially evaluate the immunoAT method, the presence or absence of
prion protein (PrP(sc)) was determined in
formalin-fixed and
paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for
bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing
IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.